Sébastien Le Laz

Audrey Le Goaziou

Ghislaine Henneke
email: ghenneke@ifremer.fr

Structure-Specific Nuclease Activities of Pyrococcus abyssi RNase HII

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INTRODUCTION

DNA replication in all living organisms takes place concurrently on two separate strands. The lagging strand consists of multiple discontinuous segments called Okazaki fragments, whereas the leading strand comprises one large continuous segment. Production of each individual lagging strand by DNA polymerase is primed by a short stretch of RNA. Later on, these RNA primers are eliminated and the resulting gap is filled with deoxyribonucleotides prior to ligation. Priming and DNA elongation at the replication fork involve a set of specialized polymerising enzymes which differ from replicative DNA polymerases, and one another, to correct erroneously inserted nucleotides. In archaeal cells, the priming complex lacks proofreading 3'-5' exonuclease activity [START_REF] Lao-Sirieix | The heterodimeric primase of the hyperthermophilic archaeon Sulfolobus solfataricus possesses DNA and RNA primase, polymerase and 3'-terminal nucleotidyl transferase activities[END_REF][START_REF] Breton | The heterodimeric primase from the euryarchaeon Pyrococcus abyssi: a multifunctional enzyme for initiation and repair?[END_REF] present in the replicative DNA polymerases B and D [START_REF] Cann | A heterodimeric DNA polymerase: evidence that members of Euryarchaeota possess a distinct DNA polymerase[END_REF][START_REF] Gueguen | Characterization of two DNA polymerases from the hyperthermophilic euryarchaeon Pyrococcus abyssi[END_REF]. Consequently, mismatches in the vicinity of the RNA-DNA junction could arise in replicating cells, as already observed in eukaryotes [START_REF] Sheaff | Misincorporation of nucleotides by calf thymus DNA primase and elongation of primers containing multiple noncognate nucleotides by DNA polymerase alpha[END_REF][START_REF] Thomas | Fidelity of mammalian DNA replication and replicative DNA polymerases[END_REF]. Similarly, single ribonucleotides incorporated during DNA replication [START_REF] Mcelhinny | Abundant ribonucleotide incorporation into DNA by yeast replicative polymerases[END_REF][START_REF] Rumbaugh | Creation and removal of embedded ribonucleotides in chromosomal DNA during mammalian Okazaki fragment processing[END_REF] or by external agents [START_REF] Sonntag | Radiation-induced degradation of the sugar in model compounds and in DNA[END_REF] would represent another source of erroneous nucleotides. Persistence of residual RNA during DNA replication would be detrimental for the cells, suggesting that a combination of specific and efficient nucleolytic processes is absolutely required to preserve DNA integrity.

Ribonucleases H (RNase H) are enzymes which degrade the RNA portion of RNA/DNA or RNA-DNA/DNA duplexes [START_REF] Stein | Enzyme from calf thymus degrading the RNA moiety of DNA-RNA Hybrids: effect on DNA-dependent RNA polymerase[END_REF]. RNases H are classified into two major families, type 1 and type 2, based on amino acid sequence [START_REF] Ohtani | Molecular diversities of RNases H[END_REF]. The type 1 family includes bacterial RNase HI, mammalian RNase HII, the RNase H domain of reverse transcriptase and archaeal RNase HI and the type 2 contains bacterial RNase HII and RNase HIII, mammalian RNase HI and archaeal RNase HII [START_REF] Ohtani | Molecular diversities of RNases H[END_REF]. While type 2 RNase H enzymes are universally conserved in the three domains of life, their physiological role remains elusive. Much less is known about the type 2 family compared to the type 1 RNase H enzymes. The multiplicity of RNases H within a single cell complicates the situation, although presumable roles in DNA replication, DNA repair and transcription have been assigned as recently reviewed [START_REF] Cerritelli | Ribonuclease H: the enzymes in eukaryotes[END_REF][START_REF] Tadokoro | Ribonuclease H: molecular diversities, substrate binding domains, and catalytic mechanism of the prokaryotic enzymes[END_REF]. In archaea, structural and biochemical characterization of type 2 RNases H [START_REF] Chai | Archaeoglobus fulgidus RNase HII in DNA replication: enzymological functions and activity regulation via metal cofactors[END_REF][START_REF] Chapados | Structural biochemistry of a type 2 RNase H: RNA primer recognition and removal during DNA replication[END_REF][START_REF] Haruki | Cleavage of a DNA-RNA-DNA/DNA chimeric substrate containing a single ribonucleotide at the DNA-RNA junction with prokaryotic RNases HII[END_REF][START_REF] Lai | Crystal structure of archaeal RNase HII: a homologue of human major RNase H[END_REF][START_REF] Meslet-Cladiere | A novel proteomic approach identifies new interaction partners for proliferating cell nuclear antigen[END_REF][START_REF] Muroya | Catalytic center of an archaeal type 2 ribonuclease H as revealed by X-ray crystallographic and mutational analyses[END_REF] suggested they can initiate RNA removal from DNA duplexes, based on their ability to specifically cleave 5' to the junctional ribonucleotide. However, despite this information, the physiological role of type 2 RNases H still remains elusive. They could be involved in the completion of either leading or lagging strands or both. Additional biochemical experiments with catalytic intermediates should provide valuable knowledge on the participation of these archaeal cellular enzymes at the replication fork and in DNA repair.

To investigate this question, we have designed a set of RNA/DNA duplex, cognate RNA-DNA/DNA duplex [START_REF] Matsunaga | Identification of short 'eukaryotic' Okazaki fragments synthesized from a prokaryotic replication origin[END_REF], a single ribonucleotide embedded in a DNA duplex (DNA-1RNA-DNA/DNA) as substrates, and employed type 2 RNase H from the hyperthermophilic deepsea euryarchaeon Pyrococcus abyssi (Pab), PabRNase HII. Since a single rnh gene exists in the genome of P. abyssi [START_REF] Cohen | An integrated analysis of the genome of the hyperthermophilic archaeon Pyrococcus abyssi[END_REF], RNase HII is likely the key enzyme involved in RNA elimination in this organism. Thus, PabRNase HII can be considered as the representative of type 2 RNase H.

Here, we analysed the cleavage specificity of PabRNase HII for substrates with Okazaki fragment-like structure. We also tested PabRNase HII activity on Okazaki fragment-like substrates in the presence of mismatched base pair in order to assess the molecular mechanism of recognition of the RNA-DNA junction and the subsequent cleavage specificity.

In addition, we examined whether PabRNase HII can incise the DNA backbone on the 5'-side of a single ribonucleotide embedded in a DNA duplex. Our data provide substantial evidences that the single RNase H in P. abyssi has a dual role in maintenance of genome integrity. The results from this study are further discussed to define potent roles of type 2 RNase H from P. abyssi in the resolution of RNA fragments at the replication fork and in the repair of single 90 embedded ribonucleotides.

MATERIALS AND METHODS

Nucleic acid substrates.

Gel-purified oligonucleotides for preparing the substrates for RNase HII assays were purchased from Eurogentec (Belgium) and their sequences are listed in Table 1. Fluorescent labelling at the 5'-end was performed with the 5' End Tag kit labelling system from Vector Laboratories (California). Free fluorescent dyes were removed on MicroSpinTM G-25 columns. For some experiments, 5'-end-or 3'-end-fluorescent labelled oligonucleotides were chemically synthesized and HPLC-purified by Eurogentec (Belgium). To generate the substrates for the RNase HII assays, the appropriate oligonucleotides were mixed in 1:1 molar ratio in 20 mM Tris-HCl (pH 7.4), 150 mM NaCl, heated to 75°C and slowly cooled to room temperature.

Cloning, production and purification of PabRNase HII.

The gene encoding PabRNase HII (PAB0352) was cloned into the pQE-80L expression vector (Qiagen). PabRNase HII was overexpressed in E. coli strain BL21-CodonPlus-RIL strain (Stratagene) as a histidine-tagged protein and purified to near homogeneity via Ni-NTA beads (Qiagen) and S200 gel filtration using fast protein liquid chromatography (GE Healthcare) as previously described [START_REF] Meslet-Cladiere | A novel proteomic approach identifies new interaction partners for proliferating cell nuclear antigen[END_REF]. Protein integrity was analyzed by MALDI-TOF analyses (Innova Proteomics, France). PabRNase HII purity was controlled by SDS-PAGE gradient gel (4-20 %) electrophoresis (Thermo Scientific).

Amino acid sequence alignments and secondary structure.

Amino acid sequence alignments have been constructed by ClustalW2 (available at www.ebi.ac.uk/clustalW2/). Secondary structure elements calculated with the program ESPript 2.2 (available at http://espript.ibcp.fr/ESPript/ESPript/) refer to the structure of TkoKOD1RNase HII (PDB: 1IO2).

Assays for RNase HII activity.

Assays to monitor cleavage by PabRNase HII were performed in RNase HII buffer (10 µl) containing: 50 mM Tris-HCl (pH 8), 5 mM dithiothreitol, 5 mM MgCl 2 and 50 nM of DNA substrates. Enzymes were diluted from concentrated stocks in 20 mM Tris-HCl (pH 7.5), 20 % glycerol prior to usage. Enzyme concentrations for a typical reaction ranged from 4 to 400 nM, unless otherwise specified. After addition of PabRNase HII, reactions were incubated at 60°C for 30 minutes and stopped on ice with 15 µl of stop buffer (98 % formamide, 10 mM EDTA). Samples were heated at 95°C for 5 minutes. A base hydrolysis ladder was prepared by incubation of the labelled RNA-DNA strand (10 µM) with snake venom phosphodiesterase I (0.018 units) for 10 minutes at 37°C. Product analysis was carried out by electrophoresis on 15 % denaturing polyacrylamide gels. After visualisation with a Mode Imager Typhoon 9400 (GE Healthcare), quantification of the results was performed using ImageQuant 5.2 software. In all cases, the percentage of substrate hydrolysis was determined by the products / (products + substrate) ratio, allowing a correction for loading errors and a comparison of cleavage efficiency irrespective of the different products generated.

To analyse divalent cations or pH dependence, RNase HII assays were carried out with 50 nM of PabRNase HII and 50 nM of the S1 substrate at 60°C for 30 min. Data are the average of triplicate measurements and are shown with standard deviations (SD).

To determine the kinetic parameters, steady-state kinetic reactions were carried out in the same conditions as described above by using substrate 1 at concentrations ranging from 0.03 to 3 µM. Initial velocity experiments were monitored as a function of time with 60 nM of PabRNase HII at 60°C such that the rate of converted substrate did not exceed 20 % of the total. Velocity measurements were reported as the amount of hydrolysed substrate (µM) over time (min). The observed rates of converted substrates with PabRNase HII were firstly determined from Lineweaver-Burk plots. The data were fit by nonlinear regression using the Marquardt-Levenberg algorithm (EnzFitter 2.0, BioSoft) to the Michaelis-Menten equation.

RESULTS

Archaeal RNase HII homologues

PabRNase HII showed amino acid sequence similarities of 75.2 % with TkoRNase HII, and 65 % with AfuRNase HII. Analysis of protein primary structures and related secondary structures outlined subtle differences between the three proteins (Fig. 1A). On the one hand, PabRNase HII and AfuRNase HII are isoelectric at basic pH (isoelectric point values of 9 and 7.6, respectively), whereas TkoRNase HII is isoeletric at acidic pH (isoelectric point of 5.5).

On the other hand, PabRNase HII, TkoRNase HII and AfuRNase HII exhibit conserved secondary structure elements, with the exception that the α9-helix is incomplete in AfuRNase.

This secondary structure element is important for TkoRNase HII to bind the substrate [START_REF] Muroya | Catalytic center of an archaeal type 2 ribonuclease H as revealed by X-ray crystallographic and mutational analyses[END_REF].

Structural alignments resulted in the identification of conserved active site residues (Asp7, Glu8, Asp105 and Asp135) in PabRNase HII, suggesting a similar catalytic mechanism between the three enzymes.

Enzymatic properties of P. abyssi RNase HII

His-tagged PabRNase HII was overproduced in E. coli and purified to give a single band on SDS-PAGE (Fig. 1B, lane 2). Reactions were carried out at 60 °C, the optimum temperature for PabRNase HII activity [START_REF] Meslet-Cladiere | A novel proteomic approach identifies new interaction partners for proliferating cell nuclear antigen[END_REF]. PabRNase HII was assayed under different pH and ionic conditions, varying both the nature and the concentration of divalent cations, according to the general procedure described in Materials and methods using the RNA-DNA/DNA substrate (S1). The optimum pH for its activity was observed between pH 8.0 and 8.5 (Fig. 1C). However, at pH 7.5 and 9, the enzyme retained about 60 % of the activity measured at pH 8.

PabRNase HII exhibited enzymatic activity in the presence of MgCl 2 , MnCl 2 and CoCl 2 (Fig. 1D). While PabRNase HII activity was entirely dependent on the presence of a divalent cation, the enzyme was not active in the presence of NiCl 2 , FeCl 2 , ZnCl 2 , CaCl 2 or CuSO 4 .

The most preferred metal ion for PabRNase HII was MgCl 2 but the MnCl 2 and CoCl 2 could substitute for the MgCl 2 with reduced cleavage activity. As shown in Fig. 1D, the metal concentrations which gave the highest enzymatic activity were 5 mM for MgCl 2, and 2 mM for MnCl 2 and CoCl 2 . Substrate hydrolysis in the presence of 5 mM for MgCl 2 was 1.5-and 3.2-fold higher than those determined at 2 mM for MnCl 2 and CoCl 2 , respectively.

The kinetic parameters of PabRNase HII were determined in the presence of RNA-DNA/DNA substrate (S1) and 5 mM MgCl 2. The results are summarized in Table 2 and compared to that of type 2 RNase H archaeal homologue from Archaeoglobus fulgidus (AfuRNase HII) as already described (4). Interestingly, AfuRNase HII showed stronger substrate binding affinity than PabRNase HII, as attested by a 10-fold lower K m value, while the catalytic rate constants of the two enzymes were similar (Table 2). As a consequence, PabRNase HII displayed a lower catalytic efficiency on RNA-DNA/DNA substrate as compared to AfuRNase HII (Table 2).

Ribonuclease activity of P. abyssi RNase HII

Firstly, we examined whether PabRNase HII could cleave the RNA strand of a RNA/DNA duplex (S11). As shown in Fig. 2, different 5'-terminal RNA products accumulated depending on the enzyme concentration, indicating that PabRNase HII exhibited endoribonuclease activity. Because control assays without enzyme showed background degradation of the RNA primer (Fig. 2A, lane 2), they were subtracted from cleavage products signal. Less than 19 % intact RNA was present upon incubation with 100 nM PabRNase HII for 30 min at 60°C (Fig. 2A, lane 6). Multiple cleavage sites were detected (Fig. 2A, lanes 2-8) and comparative analysis of products with those from the snake venom phosphodiesterase digest of 5'-end labelled 32 nucleotide (nt) RNA ladder (Fig. 2A, lane 1) pointed out main cleavage events (6-, 8-, 9-, 12-, 13-and 17-nt). Moreover, further processing of short RNA fragments could be observed by increasing enzyme concentrations. It is important to note that PabRNase HII used in this study did not exhibit nuclease activity on single-stranded RNA (Fig. 2B). All together, these results provided evidence that PabRNase HII acts as an endoribonuclease on RNA/DNA duplexes.

Structure-specific cleavage activities in P. abyssi RNase HII

The absence of cleavage specificity of RNA/DNA duplexes prompted us to look for digestion of other relevant physiological substrates. We hypothesize that PabRNase HII participates in the mechanism of RNA primer removal, an activity which can occur once at the leading strand or much more frequently at the lagging strand. Therefore, we explored the cleavage specificity of PabRNase HII for substrates with Okazaki fragment-like intermediates.

PabRNase HII specifically cleaves RNA-initiated DNA segments fully annealed to a DNA template

We initially began to examine whether PabRNase HII could hydrolyse a cognate doublestranded Okazaki fragment [START_REF] Matsunaga | Identification of short 'eukaryotic' Okazaki fragments synthesized from a prokaryotic replication origin[END_REF]. A single strand composed of 12 nucleotides of RNA (RNA12nt) followed by 18 nucleotides of DNA (DNA18nt), fluorescently labelled at the 5'end, was annealed to a complementary 30 nucleotides DNA template to form the S1 substrate as shown in Fig. 3A. When this substrate was incubated in the presence of increasing PabRNase HII amounts, a major product appeared (Fig. 3B, lanes 2-5). This oligomer was shown to correspond to 11 nucleotides RNA by migration with respect to a snake venom phosphodiesterase-generated digest of RNA12ntDNA18nt (Fig. 3B, lane 1). In addition to this main cleavage site, minor cleavage sites characteristic of non-specific nuclease activity were also found throughout the length of the RNA (Fig. 3B, lanes 2-5). Over time, PabRNase HII activity released the same oligomer which was basically free of any additional shorter fragments, suggesting that this product was not transiently formed and prevailed during the reaction (data not shown). Interestingly, PabRNase HII did not hydrolyse single-stranded RNA12ntDNA18nt (Fig. 3B, lanes 6-9) indicating that cleavage is dependent on the heteroduplex structure. It is of note that the pale band at ∼9-nt present at relatively constant levels did not correspond to a specific cleavage product (Fig. 3B, lanes 2-9). Absence of specific cleavage was also observed with substrates lacking the complementary DNA template to either the RNA12nt or the DNA18nt sequence (data not shown). While a fully annealed RNA12ntDNA18nt/DNA is definitely required to detect cleavage specificity, PabRNase HII did not hydrolyse the complementary DNA template (Fig. 3B, lanes 10-13).

Shorter bands were not due to cleavage activity since they were detectable in all lanes with equal intensities even in the absence of enzyme. These data demonstrated that PabRNase HII can act endonucleolytically on initiator RNA and displays a specific cleavage activity dependent on the heteroduplex structure.

PabRNase HII specifically cleaves the fully annealed RNA strand of Okazaki fragmentgapped intermediates but not a 5'-RNA flap

Both at the leading and lagging strands, sequential enzymatic steps are thought to be part of the RNA primer elimination mechanism in P .abyssi. As a consequence, diverse structural Okazaki fragment-like substrates would arise. Therefore, we examined whether structural intermediates (S4, S5 and S6) which can be captured during the process (outlined in Fig. 4A) substrates. Importantly, we demonstrated that PabRNase HII cleaves the RNA initiator fully annealed to the complementary DNA template independently of the size of the gap. In addition, we provided evidence that a 5'-RNA flap is not an appropriate substrate for PabRNase HII, suggesting the requirement of additional enzymes to fully ensure the removal of Okazaki fragment intermediates at the lagging strand.

PabRNase HII specifically cuts the RNA-DNA/DNA when the RNA is completely annealed to the DNA template

The above results indicated that PabRNase HII specifically cleaves the RNA in an RNA-DNA/DNA duplex one ribonucleotide upstream of the RNA-DNA junction. Based on this observation, we attempted to gain further information about the structure-specific recognition of the RNA-DNA junction. We predicted that mismatches located either downstream or upstream of the site of cleavage would alter the structure of the junction and prevent PabRNase HII from recognising and cutting the substrate. Such substrates, which can be created during priming and DNA synthesis in eukaryotes [START_REF] Sheaff | Misincorporation of nucleotides by calf thymus DNA primase and elongation of primers containing multiple noncognate nucleotides by DNA polymerase alpha[END_REF][START_REF] Thomas | Fidelity of mammalian DNA replication and replicative DNA polymerases[END_REF], could also be relevant in P. abyssi cells. In particular, the priming heterodimeric polymerase in P. abyssi, Pabp46/41 complex, does not possess 3'-5' exonucleolytic activity and can consequently misincorporate nucleotides, creating mismatched base pairs at or near the RNA-DNA junction.

The complementary DNA template was designed to produce specific mismatches with the RNA12ntDNA18nt strand (Fig. 5A). When the mismatch was the deoxynucleotide downstream of the site of cleavage, PabRNase HII efficiently cleaved the S7 substrate and cut at one site into the RNA segment, leaving a monoribonucleotide attached to the DNA18nt strand (Fig. 5B, lanes 2-5). Cleavage efficiencies were still comparable to those of model Okazaki fragments described above. This result seems to point out that a deoxynucleotide mismatched Okazaki fragment does not affect recognition and specific cleavage by PabRNase HII. We next considered that ribonucleotide mismatches positioned downstream (Fig. 5A, S8 substrate) or upstream (Fig. 5A, S9 substrate) of the cutting site would be crucial for directing the cleavage specificity of PabRNase HII. Interestingly, the presence of the ribonucleotide just downstream of the cutting site induced random endonucleolytic cleavage with predominant products (Fig. 5B, lanes 6-9 and Fig. 5C, S8 substrate) and the percent of hydrolysed products was equivalent to that of Okazaki fragment-like substrates. When the ribonucleotide mismatch was positioned upstream of the site of cleavage, random endonucleolytic activity was enhanced but cleavage efficiencies were lowered (Fig. 5B, lanes 10-13). Multiple cleavage sites due to the loss of specificity appeared (Fig. 5C, S9 substrate).

Taken together these data showed for the first time that an archaeal RNase HII requires complete hybridization of the RNA segment to the DNA template in order to confer specific cleavage of RNA-DNA/DNA duplexes.

PabRNase HII specifically cuts a single embedded ribonucleotide in a DNA duplex

We anticipated that PabRNase HII nuclease could act on a single ribonucleotide embedded in Overall, we showed that PabRNase HII is active on single embedded ribonucleotides in a DNA duplex and releases a major product consisting of a single ribonucleotide on the 5'-end of the downstream DNA segment.

DISCUSSION

Two types of RNase H, type 1 and type 2, have been identified in a multiplicity of archaeal genomes. While most archaeal microorganisms have only one type of RNase H, a few archaea such as Sulfolobus tokodaii and Haloferax volcanii possess both types of RNase H. Although the physiological significance of multiple rnh genes in single archaeal genomes is not well understood, RNases H are thought to be involved in important cellular processes [START_REF] Chai | Archaeoglobus fulgidus RNase HII in DNA replication: enzymological functions and activity regulation via metal cofactors[END_REF][START_REF] Haruki | Cleavage of a DNA-RNA-DNA/DNA chimeric substrate containing a single ribonucleotide at the DNA-RNA junction with prokaryotic RNases HII[END_REF][START_REF] Meslet-Cladiere | A novel proteomic approach identifies new interaction partners for proliferating cell nuclear antigen[END_REF][START_REF] Ohtani | Cleavage of doublestranded RNA by RNase HI from a thermoacidophilic archaeon, Sulfolobus tokodaii 7[END_REF][START_REF] Ohtani | Identification of the first archaeal Type 1 RNase H gene from Halobacterium sp. NRC-1: archaeal RNase HI can cleave an RNA-DNA junction[END_REF]. Interestingly, archaeal type 2 RNase H appears more universal because the encoding gene is distributed in almost all archaeal genomes. Sequence comparison within archaeal type 2 RNases H revealed a high degree of sequence similarity with conserved active site residues, suggesting that these enzymes may have common biochemical properties [START_REF] Chai | Archaeoglobus fulgidus RNase HII in DNA replication: enzymological functions and activity regulation via metal cofactors[END_REF][START_REF] Haruki | Gene cloning and characterization of recombinant RNase HII from a hyperthermophilic archaeon[END_REF]. In this report, we demonstrated that PabRNase HII, type 2 RNase H from P. abyssi, is as an alkaline enzyme. This property seems to be a hallmark of type 2 thermostable RNases H [START_REF] Chai | Archaeoglobus fulgidus RNase HII in DNA replication: enzymological functions and activity regulation via metal cofactors[END_REF][START_REF] Haruki | Gene cloning and characterization of recombinant RNase HII from a hyperthermophilic archaeon[END_REF][START_REF] Ohtani | Junction ribonuclease: a ribonuclease HII orthologue from Thermus thermophilus HB8 prefers the RNA-DNA junction to the RNA/DNA heteroduplex[END_REF]. In addition, PabRNase HII appeared to prefer the Mg 2+ ion for RNase activity rather than Mn 2+ or Co 2+ . Distinct metal dependencies have been described for Archaeoglobus fulgidus and Thermococcus kodakaraensis KOD1 RNase HII with Mn 2+ -or Co 2+ -preference, respectively [START_REF] Chai | Archaeoglobus fulgidus RNase HII in DNA replication: enzymological functions and activity regulation via metal cofactors[END_REF][START_REF] Haruki | Gene cloning and characterization of recombinant RNase HII from a hyperthermophilic archaeon[END_REF]. Metal ion usage by archaeal RNases HII may be a consequence of the environmental conditions they thrive. It may also dictate the substrate requirement for hydrolysis and confer a specialised function to the enzyme in the maintenance of genome integrity. Determination of kinetic parameters highlighted that the homologous archaeal enzymes, AfuRNase HII and PabRNase HII, showed distinct catalytic efficiencies for RNA-DNA/DNA substrates. These results mainly reflected differences in substrate binding affinity. In general, biochemical discrepancies observed between the three enzymes are possibly related to variations in secondary structure elements and physicochemical parameters (e.g., isoelectric point). Despite these subtle differences, archaeal RNase HII seem to possess conserved structural features required to specifically recognise a comparable region of the substrates, and to produce similar products. Like other type 2 archaeal RNase H, PabRNase HII behaved as an efficient endoribonuclease on RNA/DNA duplexes, stalling at particular sites [START_REF] Chai | Archaeoglobus fulgidus RNase HII in DNA replication: enzymological functions and activity regulation via metal cofactors[END_REF][START_REF] Haruki | Gene cloning and characterization of recombinant RNase HII from a hyperthermophilic archaeon[END_REF]. Moreover, most of the biochemical features of PabRNase HII overlapped those of the eukaryotic equivalent, type 2 RNase H, described as a key enzyme in Okazaki fragment processing [START_REF] Murante | Junction ribonuclease: an activity in Okazaki fragment processing[END_REF].

With diverse constructs representing replication-fork intermediates, PabRNase HII made structure-specific endonucleolytic cleavage in the RNA initiator, leaving a single ribonucleotide at the 5'-end of the RNA-DNA junction. Cleavage 5' to the junctional ribonucleotide required the presence of double-stranded substrates with the RNA segment fully annealed to the complementary strand. Gapped double-stranded substrates containing RNA-DNA junctions did not alter cleavage specificity. However, a single-stranded 5'-RNA flap was resistant to cleavage activity, indicating that PabRNase HII does not carry out this reaction at the replication fork. On the other hand, other results have demonstrated that the structure-specific nuclease, Flap endonuclease I (Fen I), can cleave substrates with RNA flap structures, bypassing the need for Rnase HII in Okazaki fragment processing. [START_REF] Murante | Calf RTH-1 nuclease can remove the initiator RNAs of Okazaki fragments by endonuclease activity[END_REF][START_REF] Sato | Cooperative regulation for Okazaki fragment processing by RNase HII and FEN-1 purified from a hyperthermophilic archaeon, Pyrococcus furiosus[END_REF]. Furthermore, we demonstrated that mismatches in the RNA portion, produced by erroneous priming and polymerising activities during initiation of DNA replication in eukaryotes [START_REF] Sheaff | Misincorporation of nucleotides by calf thymus DNA primase and elongation of primers containing multiple noncognate nucleotides by DNA polymerase alpha[END_REF][START_REF] Thomas | Fidelity of mammalian DNA replication and replicative DNA polymerases[END_REF], resulted in loss of specificity by PabRNase HII. These results demonstrate, for the first time, that the RNA residues in the vicinity of the RNA-DNA junction are key structural determinants for cleavage specificity of type 2 archaeal RNase H. Notably, archaeal type 2 RNase H seems to differ from eukaryotic type 2 [START_REF] Murante | Junction ribonuclease: an activity in Okazaki fragment processing[END_REF] in that it recognizes the RNA strand rather than the RNA-DNA junction. Possibly, the RNA portion of the RNA-DNA junction annealed to DNA template adopts an intermediate helical structure, which might target RNase HII recognition and induce specific cleavage. This hypothesis is sustained by the observation that RNA/DNA and DNA/DNA duplexes form A-type and B-type helices, respectively [START_REF] Chou | Solid-phase synthesis and high-resolution NMR studies of two synthetic double-helical RNA dodecamers: r(CGCGAAUUCGCG) and r(CGCGUAUACGCG)[END_REF][START_REF] Dickerson | The anatomy of A-, B-, and Z-DNA[END_REF].

We recently proposed a model of DNA replication in P. abyssi that involves the family B DNA polymerase, PabpolB, at the leading strand and the family D DNA polymerase, PabpolD, at the lagging strand [START_REF] Henneke | The hyperthermophilic euryarchaeota Pyrococcus abyssi likely requires the two DNA polymerases D and B for DNA replication[END_REF]. This model is reinforced by complementary studies demonstrating that PabpolB is likely the leading strand DNA polymerase [START_REF] Rouillon | DNA polymerase switching on homotrimeric PCNA at the replication fork of the euryarchaea Pyrococcus abyssi[END_REF] Moreover, we demonstrated that PabRNase HII is able to cleave at the 5'-end of single embedded ribonucleotides with similar efficiency as at cognate Okazaki fragments [START_REF] Matsunaga | Identification of short 'eukaryotic' Okazaki fragments synthesized from a prokaryotic replication origin[END_REF]. Since such structural substrates can appear in vivo during Okazaki fragment processing from intrinsic RNA ligation activity or erroneous nucleotide incorporation [START_REF] Rumbaugh | Creation and removal of embedded ribonucleotides in chromosomal DNA during mammalian Okazaki fragment processing[END_REF] and during exposure to external damaging agents [START_REF] Sonntag | Radiation-induced degradation of the sugar in model compounds and in DNA[END_REF], we suggest that PabRNase HII can participate in the removal of inappropriate ribonucleotides from the hyperthermophilic chromosome. These biochemical characteristics would imply that PabRNase HII promotes the initial step of the repair process as already observed in eukaryotes [START_REF] Rumbaugh | Creation and removal of embedded ribonucleotides in chromosomal DNA during mammalian Okazaki fragment processing[END_REF]. However, reconstitution of the were incubated with the S11 substrate (lanes 2-8) and a base-hydrolysed ladder (lane 1) was prepared as described (see Materials and methods section). 5'-end fluorescently labelled products were visualised with a Mode Imager Typhoon 9400 (GE Healthcare) and quantification was performed using Image Quant 5.2 software. (B) PabRNase HII was incubated with the 32-base single-stranded RNA oligonucleotide at the indicated amounts (lanes 1-4). An 8-nt RNA oligonucleotide was used as a ladder (lane 5). 5'-end fluorescently labelled products were visualised with a Mode Imager Typhoon 9400 (GE Healthcare). An 18-nt nucleotide was used as an appropriate ladder (lane 13). Fluorescent-labelled products were visualised with a Mode Imager Typhoon 9400 (GE Healthcare) and quantification was performed using Image Quant 5.2 software. Both substrates and the corresponding hydrolysed products were manually labelled. Lanes 1 and 6 are appropriate 11-nt and 12-nt ladders for hydrolysed S1 substrates. Lanes 7 and 12 are suitable 11-nt and 12-nt ladders for hydrolysed S10 substrates. Fluorescent-labelled products were visualised with a Mode Imager Typhoon 9400 (GE Healthcare) and quantification was performed using Image Quant 5.2 software.

  Kinetic parameters, Km and Vmax, were obtained from the fit and were used to calculate the catalytic efficiency (k cat /K m ) of PabRNase HII. The kinetics values are the average of at least triplicate determinations and are shown with standard deviations (SD). Any adjustments to the above are noted in the Figure Legends.

  could direct the cleavage activity of PabRNase HII. On the 40-gapped S4 intermediate composed of an upstream DNA primer and a downstream RNA-DNA fragment fully annealed to the complementary DNA template, PabRNase HII specifically cleaved the RNA segment, releasing one ribonucleotide attached to the DNA segment (Fig. 4B, lanes 1-4). During the elongation step, the size of the gap would decrease to reach the next RNA initiator. By reconstitution of model transient substrates, we demonstrated that both the 20-nt gapped S5 intermediate (Fig. 4B, lanes 5-8) and a nicked intermediate (data not shown) were specifically cleaved. Collectively, cleavage efficiencies of the gapped and nicked Okazaki fragment intermediates were comparable to those of double-stranded RNA-DNA fragments (Fig. 3B, lanes 2-5). However, on a 5'-RNA flap which can result from strand displacement activity by PabpolD of the next Okazaki fragment (11), PabRNase HII did not significantly release oligomers (Fig. 4B, lanes 9-12). It is of note that a faint intensifying band at 8-nt (Fig. 4B, lanes 9-12) did not correspond to a specific cleavage product. These results clearly indicated that PabRNase HII is not involved in the cleavage of single-stranded RNA initiator despite the presence of surrounding DNA duplexes. These data are consistent with our observations from Fig. 3B that PabRNase HII exclusively cuts double-stranded RNA-DNA/DNA

  DNA. A single ribonucleotide in a DNA duplex could arise via misincorporation of ribonucleotide during DNA synthesis or by ligation of the monoribonucleotide attached to the DNA after cleavage of Okazaki fragments by type 2 RNase H (26). To determine whether an embedded ribonucleotide in DNA (S10 substrate) is a hydrolysable substrate, endonuclease activity of PabRNase HII was carried out. Fig. 6B, lanes 8-11 demonstrated that PabRNase HII was able to recognise and to cleave endonucleolytically on the 5'-side of an embedded monoribonucleotide. Additional fragments, shorter than the released 11-nt, were faintly detectable. Basically, cleavage efficiencies of a single embedded ribonucleotide were similar to those of model Okazaki fragment S1 substrate (Fig. 6B, compare lanes 2-5 and lanes 8-11).

  Typically, PabpolD has the capacity to displace the downstream fragment including the RNA initiator, while PabpolB is not active on this substrate. In this situation, RNA-initiated DNA segments fully annealed to a DNA template would arise only at the leading strand. Because PabRNase HII cannot cleave 5'-RNA flap templates, PabRNase HII would recognize the annealed RNA primer at the leading strand and promotes its endonucleolytic cleavage. The resulting 5' phosphorylated junction ribonucleotide attached to the DNA would be subsequently displaced by PabpolB and cleaved by PabFen I, prior to ligation by PabDNA ligase I. Thus, the functional importance of RNase HII in the completion of leading strand DNA replication in P.abyssi awaits the in vitro reconstitution of this multi-step enzymatic process (manuscript in preparation). Despite common biochemical properties with the eukaryotic type 2 RNase H, single archaeal RNases HII could be cellular enzymes involved in the removal of RNA residues at the leading strand rather than at the lagging strand. Such biological assumptions would indicate that these microorganisms have evolved differently by targeting analogous enzymes to unrelated biological functions.

Fig. 1

 1 Fig. 1 Enzymatic properties of PabRNase HII. (A) Alignment of the amino acid sequences

Fig. 2

 2 Fig. 2 Ribonuclease activity by PabRNase HII. (A) Indicated amounts of PabRNase HII

Fig. 3

 3 Fig. 3 PabRNaseHII specifically cleaves RNA-initiated DNA segments fully annealed to

Fig. 4

 4 Fig. 4 PabRNase HII specifically cleaves the fully annealed RNA strand of Okazaki

Fig. 5

 5 Fig. 5 PabRNase HII specifically cuts the RNA-DNA/DNA when the RNA is completely

Fig. 6

 6 Fig. 6 PabRNase HII specifically cuts single embedded ribonucleotide in a DNA duplex.

TABLE 1 Oligonucleotide sequences used to create structural duplex substrates S1

 1 substrate comprises primers 2 and template 8; S2 substrate is primer 2; S3 substrate comprises primers 2 and template 8; S4 substrate consists of primers 2, 3 and template 7; S5 substrate contains primers 2, 4 and template 7; S6 substrate includes primers 2, 5 and template 9; S7 substrate consists of primer 2 and template 12; S8 substrate is composed of primer 2 and template 11; S9 substrate consists of primer 2 and template 10; S10 substrate comprises primer 6 and template 8; S11 substrate contains primer 1 and template 7. Deoxyribonucleotides and ribonucleotides are shown by uppercase and lowercase letters, respectively.

TABLE 2 Kinetic parameters of archaeal RNase HII.

 2 Hydrolyses of RNA-DNA/DNA substrates (S1) were carried out at 60°C in PabRNase HII reaction buffer as described in the Materials and methods section. The data were fit by nonlinear regression using the Marquardt-Levenberg algorithm (EnzFitter 2.0, BioSoft) to the Michaelis-Menten equation. Kinetic parameters, K m and V max , obtained from the fit were used to calculate the catalytic efficiency (k cat /K m ) of PabRNase HII. The kinetics values are the average of at least triplicate determinations and are shown with standard deviations (SD). Kinetic parameters of AfuRNase HII were extracted from previous studies (4).

	Primers	Length	Sequence
	1	32-nt	ugccaagcuugcaugccugcaggucgacucua
	2	30-nt	auucguaaucauGGTCATAGCTGTTTCCTG
	3	17-nt	TGCCAAGCTTGCATGCC
	4	37-nt	TGCCAAGCTTGCATGCCTGCAGGTCGACTCTAGAGGA
	5	12-nt	TGGGTGGGGTGG
	6	30-nt	ATTCGTAATCAuGGTCATAGCTGTTTCCTG
	Templates Length	Sequence
	7	87-nt	CAGGAAACAGCTATGACCATGATTACGAATTCGAGCTCGGTACCCGGGGATCCTCTAGAGTCGACCTGCAGGCATG CAAGCTTGGCA
	8	30-nt	CAGGAAACAGCTATGACCATGATTACGAAT
	9	30-nt	CAGGAAACAGCTATGACCCCACCCCACCCA
	10	30-nt	CAGGAAACAGCTATGACCAGGATTACGAAT
	11	30-nt	CAGGAAACAGCTATGACCGTGATTACGAAT
	12	30-nt	CAGGAAACAGCTATGACTATGATTACGAAT
	Table1

Table 2

 2

complete enzymatic process awaits further assessment.

ACKNOWLEDGEMENTS

We are grateful to Hannu Myllykallio for providing the expression clone encoding PabRNase HII and critical reading of the manuscript. This work was financially supported by the French institute of marine research and exploitation (Ifremer). Sébastien Le Laz thanks the University of Western Brittany for funding.


Marcus Barkowsky

Kun Wang

Romain Cousseau

Kjell Brunnström

Roger Olsson

Patrick Le Callet

Subjective quality assessment of error concealment strategies for 3DTV in the presence of asymmetric transmission errors

Keywords: subjective experiment, packet loss, video quality, visual discomfort 7 26 Short duration, 1%

à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

INTRODUCTION

Inspired by the rapidly increasing popularity of 3D movies, 3D services are becoming popular for the home environment as well. There are many alternative technologies for the representation of 3D video content, for example, multi-view, depth plus 2D, volumetric, and holographic but at the moment the stereoscopic presentation with glasses seems to be the most mature technology and it is available on traditional terrestrial cable, satellite, IPTV services, as well as hardcopy media i.e Blu-ray disk.

Likewise, a wide variety of transmission channels is currently available for broadcasting, ranging from satellite and terrestrial transmission to mobile channels. However, for the home environment, the channels currently used for IPTV seem most appropriate for 3D services in HDTV quality.

For encoding a stereoscopic video, several different possibilities exist, for example, Multiview Video Coding (MVC) [START_REF] Vetro | Status of MVC text, software, and conformance[END_REF]. Although MVC has a higher coding efficiency as it exploits the inherent redundancy of the left and the right view by inter-view prediction, in this paper, the simultaneous broadcast of the left and right view in H.264/AVC is favored for sake of simplicity and availability of well-known tools. These tools include packet loss tools and the state-of-the-art error concealment strategy implemented in the JM reference decoder [START_REF]AVC Software manual[END_REF]. It is also very likely that simulcast is the preferred broadcasting method initially, since it allows the broadcaster to use most of its 2D infrastructure even for 3D.

Several publications discuss the influence of transmission distortions on the quality of experience in the 2D case [START_REF] Mu | Visibility of individual packet loss on H.264 encoded video stream: a user study on the impact of packet loss on perceived video quality[END_REF][START_REF] Meenowa | Subjective assessment of HDTV content: comparison of quality across HDTV formats[END_REF][START_REF] Pinson | HDTV subjective quality of H. 264 vs. MPEG-2, With and Without Packet Loss[END_REF]. In the 3D case, a transmission distortion in one view or in both views is perceived differently. A degradation in one view or a temporal misalignment between the left and the right view leads to binocular rivalry. This binocular rivalry strongly degrades the quality of experience as it exhibits visual discomfort which might lead to headache or nausea [START_REF] Ukai | Visual fatigue caused by viewing stereoscopic motion images: Background, theories, and observations[END_REF]. Therefore, in this paper, the preference of the observers to several different error concealment strategies is reinvestigated for the 3D case.

In order to compare the influence of the error concealment strategies to the impact of coding artifacts, several codingonly conditions are included in the subjective experiment. The subjective experiment uses a combination of the Double Stimulus Continuous Quality Scale (DSCQS) as specified in ITU-R BT.500 [START_REF] Itu | Methodology for the subjective assessment of the quality of television pictures[END_REF] and the Absolute Category Rating (ACR) as specified in ITU-T P.910 [START_REF]Subjective video quality assessment methods for multimedia applications[END_REF]. DSCQS is used for training the subjects to calibrate their expectations for displaying 3D video content while ACR is used in the subjective experiment itself as more conditions can be judged. In addition to answering on a general five point ACR scale, the subjects were asked to indicate visual discomfort with a binary choice. This paper is organized as follows. In Sec. 2, the tested coding and transmission scenarios are detailed. The subjective experiment is described in detail in Sec. 3 and in Sec. 4, the results are presented and discussed in detail in Sec. 5, before concluding the work in Sec. 6.

CODING AND TRANSMISSION SCENARIO

Source Material

In total, 10 source stereoscopic video sequences (SRC) were extracted out of 7 different longer video clips for the subjective experiment. Each SRC is about 10 seconds long and has a higher resolution than SDTV, covered from low motion and low detail through high motion and high detail content. The scenes are summarized in Table 1.

Encoding and transmission

Several different scenarios, called Hypothetical Reference Circuits (HRC) according to the terminology of the Video Quality Experts Group (VQEG) [START_REF] Cermak | Test Plan for Evaluation of Video Quality Models for Use with High Definition TV Content. Video Quality Experts Group (VQEG)[END_REF] were used in creating the Processed Video Sequences (PVS). The SRCs are transformed into PVS according to Figure 1.

The H.264/AVC video encoder in its reference implementation JM 17.0 was used to create the sequences. Realistic parameters for slightly error-prone channels were selected by having one slice extending one line of macroblocks and inserting an Immediate Decoder Refresh (IDR) picture each 25 frames. Both settings limit the error propagation while not severely reducing the coding efficiency.

AVC Encoding

PVS

SRC

Network simulation (optional packet losses) AVC Decoding 3D error concealment (optional) Transmission errors were introduced by using an improved version of the RTP packet loss simulator "rtp_loss" provided by the Joint Video Team (JVT) in their reference software package. Only one of the two views was distorted in each HRC.

Adaptation to display

In order to avoid obvious patterns for the choice of the degraded view, the PVS were split in two groups as indicated by the rightmost column of Table 2. For group 1, the left view was distorted for all odd SRC and the right view was distorted for all even SRC. The inverse applies to group 2.

The transmission errors are grouped as follows. A "short" duration means that the bitstream is only degraded from 39% to 58% while a "long" degradation indicates that the packets are lost in between 10% and 70% of the 10 seconds PVS. This placement ensured that the scene cut at the beginning and end of the scene would not visually mask transmission error artifacts. The parameters were chosen similar to a previous publication in order to allow comparisons to the 2D case [START_REF] Barkowsky | Analysis of Freely Available Dataset for HDTV including Coding and Transmission Distortions[END_REF]. The percentage values were adapted in order to keep the same visual impairment in the sequences of 10 seconds as was previously seen in the sequences of 14 seconds.

Most of the transmission error scenarios are based on encoding at the highest evaluated video quality with a QP equal to 26. This allows for a large footroom for evaluating the quality of the introduced transmission artifacts. However, in order to learn about the relationship between quantization artifacts and transmission errors, HRC17 and HRC18 were included with the smallest transmission error impairment but a QP of 32 and 38. All bitstreams were decoded using JM15.1 as the more recent versions of the JM decoder were found to be incapable of decoding the error impaired bitstreams.

Four different error concealment strategies were applied. Error concealment strategy "A" consists of playing back the decoded video. This should be considered the most computationally intensive algorithm as it involves the sophisticated error concealment implemented in the H.264 software which uses spatial and spatial-temporal interpolation depending on the frame type. However, in the 3D case, only a single view is distorted and thus binocular rivalry may occur as the error concealment artifacts are visible only in one view.

Error concealment strategy "B" implements a switching to a 2D presentation when an error occurs in one view. As the other view is undistorted in our setup, this undistorted view is displayed to both eyes thus leading to a 2D impression without disparity.

In error concealment strategy "C", the last frame that was correctly received for both views is displayed while the effects of the transmission errors are affecting one view. Thus, the observer watches a video which pauses for a certain time, showing a 3D still image of the last correctly received frame and then the scene suddenly skips to the next correctly received 3D frame and continues playing.

The fourth error concealment strategy "D" is very similar to "C" but instead of stopping the video completely, it is assumed that a buffer of video frames exists which contains half a second of decoded content, corresponding to 12 frames in our experiment. These 12 frames are slowly played back during the recovery time of the decoder. As the exact time of the recovery is not known, the frames are played back with an exponentially increasing delay such as the last frame is displayed after 37 frames. The observer would thus see that the playback slows down, skips and then continues at normal speed.

As no error concealment would be necessary in the decoder, the methods "B", "C", and "D" are less computationally expensive than method "A".

SUBJECTIVE EXPERIMENT

The PVS set was evaluated in a subjective experiment performed at the University of Nantes, France. The PVS set was presented to the observers on a 23" Alienware Optx LCD display (120Hz, resolution 1920x1080p). The display uses active shutter glasses from the Nvidia 3D vision system. The maximum crossed and uncrossed disparity of each SRC sequence was manually determined in order to assure displaying in the comfortable viewing zone [START_REF] Chen | New Requirements of Subjective Video Quality Assessment Methodologies for 3DTV[END_REF]. Those objects that clearly belonged to pop-out effects were ignored. It was decided to use a viewing distance of 3 times the display height which is the same value used in the VQEG HDTV testplan [START_REF] Pinson | HDTV subjective quality of H. 264 vs. MPEG-2, With and Without Packet Loss[END_REF]. The lab environments adhere to the lab setup defined in the recommendation ITU-R BT.500-11 [START_REF] Cermak | Test Plan for Evaluation of Video Quality Models for Use with High Definition TV Content. Video Quality Experts Group (VQEG)[END_REF]. The display was positioned far enough from the wall to avoid any conflicts of the displayed 3D content with the real world. The room illumination was adjusted in such a way that, through the activated shutter-glasses, the luminance reflected from the background is 15% of the display's peak luminance; this corresponded to 50cd/m 2 without glasses. No flickering of the background light was perceived. The video sequences were displayed in uncompressed format in order to make sure that all observers were given the same presentation of the same video sequence. In order to assure that no temporal distortion was introduced by the player, the videos were preloaded into the computer's Random Access Memory (RAM) and special care was taken that the playout of twice the Full-HD resolution was performed without temporal jitter.

The subjective experiments were preceded by a training session. The DSCQS method was used for the training session in order to make the observers become accustomed to the PVSs, their characteristics, and the range of distortion that could be expected in the experiment itself. In the DSCQS method, the sequences are presented in pairs. In our case, the observer saw a degraded sequence and its corresponding unimpaired reference sequence in random order. For the rating session, a single stimulus method is preferable. In our experiment, the "absolute category rating with hidden reference" (ACR-HR) method was used. The observers were instructed to vote on a quality of experience scale with the training session in mind. The PVSs are presented in random order and they are rated independently on a category scale corresponding to the ITU five-point quality scale (excellent, good, fair, poor, and bad1 , which are later mapped to the scores 5, 4, 3, 2, and 1 respectively). An interactive test setup was used, the next presentation followed immediately after an observer validated his vote.

As some of the PVS exhibit binocular rivalry, visual discomfort may be perceived by the subjects. As visual discomfort is difficult to quantify for an untrained observer, a simple checkbox was integrated in the graphical user interface in order to get a binary decision. The average time for visualization and rating of a single PVS and preloading the next sequence is about 16 seconds, thus about one third of the time needed for DSCQS.

The subjective experiment contained a total of 200 videos: 10 references and 190 impaired sequences which were presented in semi-random order. Prior to the subjective experiment, the observers were screened for visual acuity using a Snellen Chart, color blindness using Ishihara Plates and stereoscopic acuity using a Randot Stereo test. From the original 30 naïve observers, 2 were rejected in the stereoscopic acuity testing. Thus, 28 observers participated in the experiments which took place in two sessions of approximately 50 minutes each with pauses after about 15 minutes of viewing time. After the subjective experiments, the observer's votes were screened according to ITU-R BT.500 and the VQEG HDTV testplan and no observers were rejected.

RESULTS

The Mean Opinion Scores (MOS) were calculated, which is an average of the numerical values that were assigned to the attributes of the ACR scale as described previously. Figure 2 displays the MOS over all observers and SRCs partitioned by 20 HRCs. The 95% confidence interval is shown in the diagram. The distribution of votes on the different categories in the complete test is approximately equal so the subjective experiment appears to be balanced. Regarding the evaluation of error concealment strategies for the videos with transmission errors, we can see that the error concealment method B (switch to 2D mode when an error occurs) used in the HRC number 6, 10, and 14, is significantly preferred to all other methods in all 3 error categories (1% packet loss for short duration, 5.9% for short duration and 1% for long duration). The MOS value of HRC6 and HRC10 is still voted as "good". The standard error concealment method of the H.264 decoder (version A), used in HCR number 5, 9 and 13, is preferred compared to the versions C (HCR 7, 11 and 15) and D (HCR 8, 12 and 16), when the percentage of errors is low (1% in both short and long duration). However, for a high percentage of packet loss of 5.9%, it becomes the opposite, where concealment case A (HRC9) is rated lowest.

There is no statistical significant difference between the method C and D except for a slight preference (HRC 16 compare to HRC15) for the D in the case of long distributed errors, indicating that "freezing" is even more annoying than slow motion. As shown in [START_REF] Barkowsky | Analysis of Freely Available Dataset for HDTV including Coding and Transmission Distortions[END_REF] for 2D, widely spread transmission errors were voted as being more disturbing than transmission errors occurring in bursts. This is corroborated in the 3D case. Among the 3 different categories of transmission errors, the MOS of the 1% packet loss for a longer duration is worse than the other two scenarios. The difference is particularly visible for the concealment methods C and D which get a MOS about 2.5 for the short duration but only 1.5 for the longer duration. There is an exception as it should be noted that for the error concealment case "A" (simulcast decoding with JM), the worst case is the strong error condition of 5.9% at short duration.

The QP value plays an important role on video quality as usual, a sharp decline occurs from HRC1 (QP26) to HRC4 (QP44). The 3D coding with a QP38 has statistically the same MOS as the error concealment C and D for the short duration error and the version A for the longer duration of errors. This may help in finding a link between the coding artifacts and transmission errors as will be further discussed later.

A surprising result from Figure 2 occurs in HRC19, the undistorted 2D video that displays the left view only. This video contains no transmission errors and coding artifacts, and can thus be compared to the 3D reference sequence (HRC0). It can be seen that 2D is slightly preferred to 3D presentation although the absolute difference is small. This characteristic varies for different video source contents. The comparison between the 2D and the 3D case is shown in Figure 3 for all SRC. 7 out of 10 SRCs are voted for 2D preference, and it is particularly obvious for SRC 10 and SRC4 where the difference is statistically significant. An in depth analysis of the distribution of the observers was performed. Only one observer out of 28 preferred 2D to 3D in a statistical significant manner on a 95% confidence. For all other observers no significant difference was indicated. With 75% confidence, we get 8 observers that preferred 2D over 3D and 2 for the opposite case. Besides the MOS values, the observer was asked to indicate for each sequence whether he felt visual discomfort. The percentage of the observers that used this checkbox is detailed in Figure 4. The boxplot shows for each HRC the median as a red line, the 25% and 75% values as a box and the 95% confidence interval as a whisker. The minimum and maximum values are indicated by additional markers. The number of the SRC with the highest visual discomfort is displayed at the top. The highest visual discomfort was indicated for SRC10 at HRC14. This particular video sequence was selected by nearly 64% of all observers. It can also be seen that SRC10 is often rated topmost in terms of visual discomfort.

The discomfort indicated for HRC3, 4 and 18 is supposed to be associated mainly to coding artifacts as the high compression corresponding to QP38 value, which usually gives rise to poor 2D visual quality. In 3D presentation, the influence of coding artifacts is not only limited to a degradation of the perceived quality of the video. In addition, the coding artifacts degrade the left and the right view differently and thus, the fusion in the Human Visual System (HVS) is hindered. One particular effect of this is related to the block based approach used by most coding algorithms. The artifacts usually occur at fixed absolute positions in each image. However, the depth effect due to stereopsis is produced by disparity that is the spatial offset of the same object in the left and right view. As a consequence, there is a conflict between the position of the object in depth and the artifacts that appear at zero disparity, e.g. on the screen plane. Therefore the coding quality expressed as QP value in our experiment is an important factor for both, the quality of the sequences and the visual discomfort. HRC5, 9 and 13 represent the error concealment method used in traditional H.264 decoding (case A). In all three cases and for all SRC, at least one observer indicated visual discomfort. It should be noted that HRC9 (5.9% packet loss in a short duration) has the highest visual discomfort score. This verifies that the standard H.264 concealment is not suitable for 3DTV applications.

From Figure 4 it seems that visual discomfort may be a function of the SRC. This has been further detailed in Figure 5 which lists the percentage of indications of visual discomfort for all presentations of the same SRC in the experiment. SRC8 and SRC10 are topmost. Those sequences have a lower resolution and were upscaled for the presentation. They also had the highest crossed and uncrossed disparity. The choice of the comfortable viewing distance was based on those two sequences. Therefore, the objects that are most in the foreground or background of the scenes are rendered just at the limits of the comfortable viewing zone. The content is highly detailed and the video was shot in bright sunlight, thus the background of the scene appears very bright as well. A camera pan is used to track the persons that act in the scenes. On the opposite, SRC5 has the lowest discomfort vote and it should be noted that the disparity range is smallest, the background is dark and it is a rendered scene with no persons using a still camera. Visual discomfort (Obs %) SRC

Figure 5: Comparison of visual discomfort of each SRC

The observers used the visual discomfort checkbox differently. In Figure 6 a sorted list of the percentage of sequences that were marked by each observer can be found. 3 observers did not vote for visual discomfort at all, while the most critical observer marked 30.5% of the sequences. As the number of observers is limited, the data on the x-and y-axis is quantized. In order to show all results, a uniformly distributed random noise was added. The highest and second highest discomfort record, at 60% and 50% of observers' votes are represented by HRC14 which are SRC8 and SRC10 encoded at QP26 with 1% of transmission errors and concealment B. The shape resembles to a lower left triangle thus there are no high MOS values at high visual discomfort values. This indicates that a good MOS value is directly linked to the absence of visual disturbance, thus the visual discomfort has an influence on the quality perceived by the observer.

In order to further evaluate the influence of the visual discomfort on the voting, all sequences were selected where at least 5 observers indicated visual discomfort. For those sequences the MOS of those observers that felt visual discomfort was averaged and the result was 1.9. The observers that did not feel visual discomfort for those sequences was averaged as well and resulted in 2.9. A student t-test was performed and it was seen that this is statistically significant. Thus the presence of visual discomfort and its indication had a significant influence on the voting of an observer.

So far, the analysis focused on the error concealment analysis. A high visual quality of the video content was guaranteed by using a low QP value. Using HRC17 and HRC18 the impact of coding durations in combination with error concealment can be evaluated. The best error concealment method B (switching to 2D) was used. Table 3 compares the mean value of all SRCs in terms of the MOS differences between coding only artifacts and 1% burst transmission artifacts with concealment B case. As expected, the observers preferred the error free case when the 3D video was played back without switching to 2D. However, the difference is small indicating the high performance of the error concealment strategy across different coding qualities. On the contrary, people indicated significantly more visual discomfort for the cases in which a switch to 2D was necessary. Both results appear to be stable across different levels of coding quality indicating that the impact of coding artifacts is independent on the impact of switching from 2D to 3D. Figure 8 presents the MOS in dependence of bitrate from HRC1 to HRC4 (coding only artifacts) in a semi-logarithmic scaling. The full HD content (1920x1080p) is indicated using solid lines whereas lower resolution content uses dashed lines. For most full HD contents used in our test, the influence of the bitrate on the evaluated video quality changes significantly from the lowest quality rating level to the second highest level (for example the MOS of SRC1 reaches from 1.9 to 4.3 while the bitrate increases from 0.9Mbit/s to 4.8Mbit/s). The curve flattens when the MOS is above the second highest level (Changing the MOS from 4.2 to 4.4 needs twice the bandwidth, from 4.7Mbit/s to 9.7Mbit/s).

The Figure demonstrates why a fixed bitrate may not be suitable for subjective experiments. SRC1 achieves a MOS value of 4.3 at 4.7 Mbit/s while the same bitrates for SRC2 and SRC3 only reach a MOS of 2.8. The diagram also indicates that a bitrate of at least 10Mbps is necessary in order to achieve a mark of "fair" or higher. It can be estimated that at least 20Mbps may be necessary to reach "good" results.

DISCUSSION

The main purpose of the experiment was to test and compare several different error concealment strategies. The results show that error concealment method B, switching to 2D when errors occur, certainly retains the highest perceived video quality when transmission errors occur; the standard method of concealing the frames in one view is worse in 3D probably due to the effects of binocular rivalry. The sophisticated 2D error-concealment algorithms do not guarantee that the interpolated content matches well with the second, undistorted view. This mismatch will cause visual discomfort to viewers and hence it does not help the perceived quality. Staying in an undistorted 3D presentation mode but slowing or pausing the play-back as was tested in case C and D usually perform poorer than the A method, except for the large amount of burst error scenario. Further experiments are necessary in order to analyze whether there is a higher sensibility and annoyance of the viewers for videos stopping in 3D than in 2D.

It was seen that 3D does not significantly outperform the 2D presentation and indeed it seemed that when videos with error concealment strategy B were presented, the main cause of annoyance seems to be related rather to the switch between 2D and 3D presentation and not to the time that 2D was presented. Thus, if errors occur, a longer presentation in 2D should be preferred to switching forth and back between 2D and 3D presentation.

The choice of the QP plays not only an important role on video quality, but our test results show an additional effect: For 3D videos a direct connection exists with visual discomfort. Higher QP values will generate more binocular rivalry and let viewers feel uncomfortable; thereby the degradation of the video quality due to coding may be more annoying for the observer than it was for 2D videos.

It was seen in the results that the error concealment conditions can be compared to coding degradations in terms of MOS in our test. In particular, the QP38 was found statistically equal to some of the QP26 coded conditions with error concealment. This result may be exploited in the context of joint source channel coding. It can be noted already that if the video is played back with switching to 2D as error concealment strategy, the degradation of MOS is not significant. This indicates that a channel code that is supposed to be effective in this scenario is difficult to design. It has to correct more errors than were used in the test (e.g. 5.9% of lost packets in the given time frame) with only a small reduction of the available bitrate for the video transmission. It should be noted that in the simulcast case, the left and the right view transmission approaches a repetition code. We used this effect for the error concealment case "B".

The second goal was to work towards establishing a reliable subjective test method for 3DTV. Several important prerequisites were presented in the setup such as the viewing environment. The subjective test method that was used in our experiment was Absolute Category Rating with Hidden Reference using only one single voting session. This is opposed to doing several subjective experiments and asking the observers to judge one isolated aspect of 3D quality in each of them such as depth, naturalness etc.

The particular subjective experiment setup used in our experiment may be regarded as reliable as the votes of the observers sufficiently coincide so that none of the observers was rejected. The confidence intervals of the different sequences and their cumulative results per SRC or HRC are in the range that is known from similar 2D subjective experiments.

A counter-indication of the suitability of the ACR method for 3D testing seems to be the missing preference of 3D presentation over 2D presentation. One problem may be related to the single stimulus method, e.g. the missing reference when suddenly viewing 2D content in the context of 3D. As the DSCQS method was used in the pre-test and three of the 2D conditions (HRC19) were included a first comparison is possible: In all three cases (SRC2, 5 and 9), 3D was preferred in the DSCQS test in a statistically significant way. On the contrary, for the ACR test, no statistical significance is detected and only in one case the MOS for 3D is higher than for 2D. Please note that the value of this comparison is limited by the fact that the training session was often the first contact of the viewers with a 3D LCD screen.

The addition of the checkbox on visual discomfort provided insight into the influence of binocular rivalry on the MOS. It should be noted however that the number of votes per observer spans a large range from 0% to 30.5%. This may be improved by replacing the optional voting as a checkbox by a forced choice vote.

CONCLUSION

In this paper, stereoscopic 3D video sequences were transmitted over a lossless or simulated error-prone channel and their quality of experience was evaluated by observers. The influence of coding degradations and the error concealment strategy on the subjective quality was discussed. It was seen that the coding artifacts influence not only the QoE value but also the visual comfort. For errorprone channels, the H.264 standard method of concealing the frames is not suitable for 3D videos as this impacts only one view. The best method of the four tested error concealment methods is to switch to 2D presentation which also uses the inherent redundancy of the transmitted information. It was also learned that the QoE value and the visual discomfort depends strongly on the properties of the reference sequence, e.g. the 3D scene content, the camera capturing, and the resolution.

The setup, execution, and analysis of a subjective experiment for 3D content were presented. It was shown that our proposal of using the ACR-HR method with an additional checkbox for visual discomfort provides important insights into the perceived quality of 3D video sequences.

Figure 1 :

 1 Figure 1: Setup of encoding, transmission and processing steps in order to generate the PVS

Figure 2 :

 2 Figure 2: Mean value of the MOS of all SRCs per HRC

Figure 3 :

 3 Figure 3: Comparison of 3D and 2D video presentation for different contents

Figure 4 :

 4 Figure 4: Comparison of visual discomfort versus HRCs

Figure 6 :

 6 Figure 6: Sorted list of percentage of sequences marked by observersFigure 7 unfolds the relationship between visual discomfort and MOS. The vertical axis is the percentage of observers who vote for discomfort for a sequence, and the horizontal axis shows the MOS. The scatter plot shows the position of each of the 200 video sequences. As the number of observers is limited, the data on the x-and y-axis is quantized. In order to show all results, a uniformly distributed random noise was added. The highest and second highest discomfort record, at 60% and 50% of observers' votes are represented by HRC14 which are SRC8 and SRC10 encoded at QP26 with 1% of transmission errors and concealment B. The shape resembles to a lower left triangle thus there are no high MOS values at high visual discomfort values. This indicates that a good MOS value is directly linked to the absence of visual disturbance, thus the visual discomfort has an influence on the quality perceived by the observer.

Figure 7 :

 7 Figure 7: Visual fatigue versus MOS by HRC

Figure 8 :

 8 Figure 8: Bitrates versus MOS of each SRCs

Table 1 . Source video sequences

 1

	SRC Resolution	frame rate	Characterization
	1	1920x1080p	25 Hz	Macro-Recording, time-lapse, surprising motion
	2	1920x1080p	25 Hz	Car racing preparation, high detail, colorful
	3	1920x1080p	25 Hz	Car race, high motion, large depth range
	4	1920x1080p	25 Hz	Animation, human characters, rare colors
	5	1920x1080p	24 Hz	Mesh grid rendering, high detail, small depth range
	6	1920x1080p	24 Hz	Rendered transparent glass ball, circular motion
	7	1280x720p	25 Hz	Group of parachutists, unsteady camera, flapping clothes
	8	1440x1080p	25 Hz	Market place with groups of people, skin colors
	9	1024x576p	25 Hz	Night scene, fireworks, large depth effects, sudden motion
	10	1024x576p	25 Hz	Uphill hiking group, natural colors, highly detailed trees

Table 2

 2

	lists all HRC conditions. In order to cover the range
	of typical coding qualities, in HRC1-4 the quantization
	parameter (QP) was varied from 26 to 44 with a stepsize of
	six. Incrementing the QP by six, doubles the quantization
	stepsize of the linear quantizer for the Discrete Cosine
	Transform (DCT) coefficients in the H.264 encoder. This
	also approximately halves the bitrate. Further information
	can be found in [10]. Please note that the bitrate at the same
	QP depends on the properties of the SRC. This approach
	was preferred to choosing fixed bitrate as it helps to cover
	the full range of QoE for each SRC.

Table 2 : List of processing conditions (HRC)

 2

	HRC	H.264	Packet Loss	Error	HRC
	Nr.	QP	Type	concealment	Group
	0
	1	26	None	-
	2	32	None	-
	3	38	None	-
	4	44	None	-
	5	26	Short duration, 1%	A	1
	6	26	Short duration, 1%	B	1
	12	26	Short duration, 5.9%	D	2
	13	26	Long duration, 1%	A	1
	14	26	Long duration, 1%	B	1
	15	26	Long duration, 1%	C	1
	16	26	Long duration, 1%	D	1
	17	32	Short duration, 1%	B	2
	18	38	Short duration, 1%	B	2
	19		2D presentation

  The videos are shown to the observers sequentially with one repetition and divided by a short gray sequence, e.g. PVS, REF, PVS, REF or REF, PVS, REF, PVS. The observers score on a 0-100 scale with two sliders displayed on the screen. The DSCQS method is very time consuming, in our case about 50 seconds for each trial. On the other hand, it allows the observers to compare in detail the quality degradation, therefore rendering it suitable for the training session.

Table 3 : MOS and visual discomfort differences between packet loss free scenarios and 1% burst packet loss scenarios

 3

		HRC1-HRC6	HRC2-HRC17	HRC3-HRC18
		QP26	QP32	QP38
	Mean value of
	MOS difference	0.236	0.19	0.15
	Mean value of
	Visual discomfort	-7.8%	-7.5%	-7.5%
	difference

The scale was presented in the observers' native language (French)

ACKNOWLEDGEMENT

This work has been partly conducted within the scope of the JEDI (Just Explore DImensions) ITEA2 project which is supported by the French industry ministry through DGCIS. In Sweden, the work was partly financed by VINNOVA (The Swedish Governmental Agency for Innovation Systems). We would like to thank Mårten Sjöström at the Mid Sweden University for his advice.