Alan I Majerník

James P J Chong

A CONSERVED MECHANISM FOR REPLICATION ORIGIN RECOGNITION AND BINDING IN ARCHAEA

Keywords: DNA replication, origin of replication, archaea, methanogen, Cdc6

To date, methanogens are the only group within the Archaea where firing DNA replication origins have not been demonstrated in vivo. Here we show that a previously identified cluster of Origin Recognition Box (ORB) sequences do indeed function as an origin of replication in vivo in the archaeon Methanothermobacter thermautotrophicus. Although the consensus sequence of ORBs in M. thermautotrophicus is somewhat conserved when compared to ORB sequences in other archaea, the Cdc6-1 protein from M. thermautotrophicus (MthCdc6-1) displays sequence-specific binding that is selective for the MthORB sequence and does not recognize ORBs from other archaeal species. Stabilization of in vitro MthORB DNA binding by MthCdc6-1 requires additional conserved sequences 3' to those originally described for M. thermautotrophicus. By testing synthetic sequences bearing mutations in the MthORB consensus sequence, we show that Cdc6-ORB binding is critically dependent on the presence of an invariant guanine found in all archaeal ORB sequences. Mutation of a universally conserved arginine in the recognition helix of the winged helix domain of archaeal Cdc6-1 shows that specific origin sequence recognition is dependant on the interaction of this arginine with the invariant guanine. Recognition of a mutated origin sequence can be achieved by mutation of the conserved arginine to a lysine or glutamine. Thus, despite a number of differences in protein and DNA sequences between species, the mechanism of origin recognition and binding appears to be conserved throughout the archaea.

Introduction

DNA replication in archaea employs enzymes that are more similar to those utilised by eukaryotes than bacteria in the equivalent process. In general, the archaeal proteins are simplified versions of their eukaryotic counterparts [START_REF] Bernander | The archaeal cell cycle: current issues[END_REF][START_REF] Edgell | Archaea and the origin(s) of DNA replication proteins[END_REF]. For example, in eukaryotes a six subunit Origin Recognition Complex (ORC) binds to origins of replication and acts as a "landing pad" for the assembly of pre-replication and pre-initiation proteins such as Cdc6 and MCM. The closest homologues of ORC proteins in archaea are the Orc/Cdc6-like proteins, which share homology with eukaryotic Cdc6 and the C-terminal portion of ORC1. Most archaea possess two Orc/Cdc6 genes, although there are a number of exceptions: Methanopyrus kandleri, Methanocaldococcus jannashii and Methanococcus maripaludis have only distantly related Cdc6 homologues [3, 4, 5,], species of the Sulfolobus genus possess three [START_REF] Robinson | Identification of two origins of replication in the single chromosome of the archaeon Sulfolobus solfataricus[END_REF], whereas Halobacterium sp NRC-1 contains 10 Cdc6-like genes [START_REF] Berquist | An archaeal chromosomal autonomously replicating sequence element from an extreme halophile, Halobacterium sp. strain NRC-1[END_REF].

Structures of Cdc6 proteins from Pyrobaculum aerophilum [START_REF] Liu | Structure and function of Cdc6/Cdc18: implications for origin recognition and checkpoint control[END_REF] and Aeropyrum pernix [START_REF] Singleton | Conformational changes induced by nucleotide binding in Cdc6/ORC from Aeropyrum pernix[END_REF] show that archaeal Cdc6 proteins consist of 3 domains. The C terminal domain forms a winged helix domain (WHD) and binds DNA in a sequence-specific manner via a helix-turn-helix motif (HTH) [START_REF] Robinson | Identification of two origins of replication in the single chromosome of the archaeon Sulfolobus solfataricus[END_REF][START_REF] Capaldi | Biochemical characterization of Cdc6/Orc1 binding to the replication origin of the euryarchaeon Methanothermobacter thermoautotrophicus[END_REF][START_REF] Kasiviswanathan | DNA binding by the Methanothermobacter thermautotrophicus Cdc6 protein is inhibited by the minichromosome maintenance helicase[END_REF]. Comparison of archaeal HTH domain sequences reveals that "Class I", but not "Class II" Orc/Cdc6 proteins, possess a conserved motif on the second helix consistent with a DNA recognition helix [START_REF] Singleton | Conformational changes induced by nucleotide binding in Cdc6/ORC from Aeropyrum pernix[END_REF]. Classical WHD proteins recognize DNA sequences by inserting the recognition helix into the major groove of DNA, while the -hairpin "wing" interacts with the phosphodiester backbone [START_REF] Gajiwala | Winged helix proteins[END_REF]. Compatible with this, mutation of Arg 334 and Arg 335 residues in the putative recognition helix of MthCdc6-1 strongly reduced DNA binding whereas mutations in positively charged residues of the MthCdc6-1 "wing" (Arg 358 , Lys 360 , Arg 362 ) affected DNA binding only modestly [START_REF] Capaldi | Biochemical characterization of Cdc6/Orc1 binding to the replication origin of the euryarchaeon Methanothermobacter thermoautotrophicus[END_REF]. Recent data has shown that multiple copies of the A. pernix Cdc6-1/ORC1 dimer recognize and bind four A. pernix ORB elements, causing a specific change in the topology of the DNA and partial unwinding of an AT-rich region, as judged by sensitivity to DNase I and P 1 endonuclease [13]. Class II (Orc2/Cdc6-2) proteins display only partial origin sequence specific DNA binding and do not possess conserved sequences within the recognition helix, but rather show conservation in the wing motif of the protein which is implicated in a different mechanism of DNA binding [START_REF] Singleton | Conformational changes induced by nucleotide binding in Cdc6/ORC from Aeropyrum pernix[END_REF]. GC skew and Z-curve analyses have been used to predict origins of replication for a number of archaeal species, including M. thermautotrophicus and Methanosarcina mazei [START_REF] Lopez | Identification of putative chromosomal origins of replication in Archaea[END_REF][START_REF] Zhang | Single replication origin of the archaeon Methanosarcina mazei revealed by the Z curve method[END_REF], although these techniques have not been useful in other archaea such as M. jannashii or Sulfolobus solfataricus [START_REF] Zhang | Single replication origin of the archaeon Methanosarcina mazei revealed by the Z curve method[END_REF][START_REF] Zhang | Multiple replication origins of the archaeon Halobacterium species NRC-1[END_REF]. Whether or not predictions exist, experimental data are still required to confirm that putative origins act as sites of replication initiation in vivo. Functional archaeal replication origins have been experimentally verified by a number of methods [START_REF] Robinson | Identification of two origins of replication in the single chromosome of the archaeon Sulfolobus solfataricus[END_REF][START_REF] Berquist | An archaeal chromosomal autonomously replicating sequence element from an extreme halophile, Halobacterium sp. strain NRC-1[END_REF][START_REF] Maisnier-Patin | Chromosome replication patterns in the hyperthermophilic euryarchaea Archaeoglobus fulgidus and Methanocaldococcus (Methanococcus) jannaschii[END_REF][START_REF] Lundgren | Three replication origins in Sulfolobus species: synchronous initiation of chromosome replication and asynchronous termination[END_REF][19][START_REF] Norais | Genetic and physical mapping of DNA replication origins in Haloferax volcanii[END_REF]. Unusually for prokaryotes with circular chromosomes, a number of archaea including both of the Sulfolobus species examined have been shown to possess multiple origins of replication [START_REF] Robinson | Identification of two origins of replication in the single chromosome of the archaeon Sulfolobus solfataricus[END_REF][START_REF] Lundgren | Three replication origins in Sulfolobus species: synchronous initiation of chromosome replication and asynchronous termination[END_REF][START_REF] Norais | Genetic and physical mapping of DNA replication origins in Haloferax volcanii[END_REF][START_REF] Robinson | Extrachromosomal element capture and the evolution of multiple replication origins in archaeal chromosomes[END_REF]. To date, methanogens are the only group within the Archaea where replication origins have not been experimentally demonstrated.

The presence of Origin Recognition Box (ORB) repeats is specific to archaeal replication origins. 36 bp ORBs were first described in S. solfataricus near an AT-rich region that might act as a site for initial duplex melting. Several ORB sequences are typically located upstream of Class I Orc/Cdc6 genes and ORBs were identified in most sequenced archaea apart from methanogens [START_REF] Robinson | Identification of two origins of replication in the single chromosome of the archaeon Sulfolobus solfataricus[END_REF]. In vitro Cdc6 proteins specifically recognized S. solfataricus ORB sequences. This interaction is believed to be essential for DNA duplex melting and origin firing. Mutation of three consecutive bases in either of two positions in the SsORB consensus abolished binding of SsCdc6-1 protein to the mutated sequence [START_REF] Robinson | Identification of two origins of replication in the single chromosome of the archaeon Sulfolobus solfataricus[END_REF].

A cluster of repeats was identified in the upstream region of the MthCdc6-1 gene [START_REF] Lopez | Identification of putative chromosomal origins of replication in Archaea[END_REF] and was shown to contain a "mini-ORB" sequence of 5'-TTACA(g/c)TTGAA(a/c)N-3' [START_REF] Capaldi | Biochemical characterization of Cdc6/Orc1 binding to the replication origin of the euryarchaeon Methanothermobacter thermoautotrophicus[END_REF].

This sequence is a minimized version of the ORB sequences found in other archaea. MthCdc6-1

shows 40-fold higher binding affinity for mini-ORB sequences than for a random DNA sequence of the same length and cooperative binding to a 181 bp duplex containing three mini-ORB sequences. The affinity of MthCdc6-1 for the triple repeat is ~40-fold higher than that for a single site and 10-fold higher than expected for DNA containing three independent ORB sites [START_REF] Capaldi | Biochemical characterization of Cdc6/Orc1 binding to the replication origin of the euryarchaeon Methanothermobacter thermoautotrophicus[END_REF].

Here we show that the region predicted to contain the origin of replication in M. thermautotrophicus does indeed function as an origin in vivo. By presenting mutated ORB sequences to MthCdc6-1 in a previously described DNA filter binding assay [START_REF] Capaldi | Biochemical characterization of Cdc6/Orc1 binding to the replication origin of the euryarchaeon Methanothermobacter thermoautotrophicus[END_REF] we show that a single guanine that is invariant among all archaeal origin sequences described to date is critical for Cdc6 binding. In addition, we show that a number of single point mutations within the mini-ORB consensus have little or no effect on MthCdc6-1 binding in vitro, but that the DNA sequence required for tight binding of MthCdc6-1 extends beyond the 13 bp mini-ORB Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.

© 2007 The Authors Journal compilation © 2007 Biochemical Society consensus previously reported. We have generated mutant Cdc6 proteins that confirm a R334A mutation results in loss of origin sequence binding. In addition, we have shown that a R334K Cdc6 variant shows promiscuous binding of origin sequences where the conserved guanine has been changed and that a R334Q variant displays altered origin sequence specificity. The R334Q protein no longer binds the wild-type origin sequence, but recognizes sequences where the guanine has been replaced by a cytosine or adenosine. Our data suggest that a conserved mechanism is employed by the Class I archaeal Cdc6/ORC proteins in specifically recognizing replication origin sequences.

Experimental

M. thermautotrophicus Growth -cultures of M. thermautotrophicus DSM1053 were grown in defined mineral-salt media [START_REF] Nölling | Isolation and characterisation of plasmids from different strains of Methanobacterium thermoformicicum[END_REF] buffered with 50 mM MOPS (pH 7.4 at 20°C) at 58°C under strictly anaerobic conditions in a 3 L vessel (Applikon, Ltd) sparged with 240 ml/min H 2 /CO 2 and mixed at 650 rpm. Approximately 10 7 cells (equivalent to ~ 100 l of a culture at OD 0.4) were aerobically fixed by the addition of 1 ml ice-cold 77% ethanol and analysed by flow cytometry as described previously [23]. Flow cytometry was performed with an A40 miniFCM (Apogee Flow Systems).

Replication Intermediate Analysis -a 4 kb HindIII fragment containing the putative origin of replication from M. thermautotrophicus was cloned into pUC118 to produce pJC064 ("b" in Fig. 1A). The adjacent 5.29 kb HindIII fragment (upstream of MthCDC6-1) was cloned into pUC118 to produce pJC070 ("a" in Fig. 1A). Two 2.8 kb fragments were PCR amplified from M. thermautotrophicus genomic DNA using the primer pairs: 5'-GATATCTCATCTATCCTTGGGAGTGG-3' and 5'-CACCCTAAACTTGACAGGCTAATGG-3'; 5'-GTTCCACGTTTATACAGGATGCAAGG-3' and 5'-CTTAAGGAACTCCTGGAAAATGGAGG-3'). The purified products of these PCR reactions were used as a template to generate a control probe to the 5.7 kb HindIII fragment downstream of MthCdc6-1 ("c" in Fig. 1A). M. thermautotrophicus cells were harvested aerobically into 133 mM EDTA / 33% glycerol, rapidly cooled in dry ice/ethanol and recovered by centrifugation (6,500 g, 10 min, 4°C). Cell pellets were lysed by three rounds of freezing in liquid nitrogen and grinding in a pestle and mortar. The lysate was diluted 4-fold (20 mM Tris-HCl, pH 8.0 / 5 mM EDTA / 10% sucrose) and incubated at 60°C for 30 min in 2% SDS.

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Genomic DNA was recovered by isopropanol precipitation, RNase digestion and two rounds of phenol:chloroform extraction before being purified by cesium chloride gradient ultracentrifugation. Genomic DNA was dialysed against HindIII restriction buffer plus EDTA before being digested with HindIII and added MgCl 2 . Replication intermediates were enriched, separated and visualised as previously described [START_REF] Liang | ORC and Cdc6p interact and determine the frequency of initiation of DNA replication in the genome[END_REF] using the probes described above.

Cloning, mutagenesis and isolation of MthCdc6-1 -MthCdc6-1 (ORF1412) was PCR amplified from genomic DNA and cloned into pQE30 (Qiagen) to produce pJC062. pJC062 was used as a template to produce R334A, R334K and R334Q MthCdc6-1 variants using QuikChange sitedirected mutagenesis (Stratagene). Cultures were grown overnight in 2x LB without IPTG induction and MthCdc6-1 protein was purified as previously described [START_REF] Capaldi | Biochemical characterization of Cdc6/Orc1 binding to the replication origin of the euryarchaeon Methanothermobacter thermoautotrophicus[END_REF] with the following modifications: MthCdc6-1 protein was immobilised on Talon Affinity Resin (BD Biosciences) pre-equilibrated with lysis buffer. The bound protein was washed with 10 column volumes of lysis buffer containing 75 mM imidazole before MthCdc6-1 was eluted (40 mM Tris-HCl, pH 8.0 / 400 mM potassium acetate / 20% glycerol / 125 mM imidazole / 1 mM 2-mercaptoethanol / 1 mM PMSF / 1 mM pepstatin) and desalted using a NAP-25 column (GE Biosciences). Aliquots of MthCdc6-1 were snap frozen in liquid nitrogen and stored at -80°C. Protein purity was checked by SDS-PAGE.

DNA binding assays -Protein-DNA binding was assessed using a modification of the method previously described [START_REF] Capaldi | Biochemical characterization of Cdc6/Orc1 binding to the replication origin of the euryarchaeon Methanothermobacter thermoautotrophicus[END_REF]. Oligonucleotides labelled with [ -32 P]ATP and T4 polynucleotide kinase were annealed to a complementary oligonucleotide. MthCdc6-1 was desalted into binding buffer (40 mM bis-Tris propane, pH 7.0 / 70 mM, potassium acetate / 20% glycerol / 3 mM DTT / 2 mM MgCl 2 / 0.1 mg/ ml BSA) using a NAP-10 column (GE Biosciences). 100 l reactions containing 0-1.2 mM MthCdc6-1 protein, 0.8 nM substrate and binding buffer were assembled in microtitre plates and assayed as described [START_REF] Capaldi | Biochemical characterization of Cdc6/Orc1 binding to the replication origin of the euryarchaeon Methanothermobacter thermoautotrophicus[END_REF]. Binding constants for the wild-type Cdc6 protein and Orb8 DNA sequence were calculated by curve fitting a simple independent binding sites model equation to experimental data in SigmaPlot [START_REF] Capaldi | Biochemical characterization of Cdc6/Orc1 binding to the replication origin of the euryarchaeon Methanothermobacter thermoautotrophicus[END_REF][START_REF] Clarke | Enzymology[END_REF]. The resulting B max value (65%) was then used to constrain fitting of the same equation to all other data. Where DNA binding was strongly disrupted we observed a poor fit of the equation and K d(app) was not determined (n.d.). Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.

Results

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GC skew methods have predicted a single origin of replication from the genomic sequence of M. thermautotrophicus [START_REF] Lopez | Identification of putative chromosomal origins of replication in Archaea[END_REF]. Consistent with this notion, a number of direct and inverted repeats have been identified in this region, although the number and exact sequence of the repeats varies between reports [START_REF] Capaldi | Biochemical characterization of Cdc6/Orc1 binding to the replication origin of the euryarchaeon Methanothermobacter thermoautotrophicus[END_REF][START_REF] Lopez | Identification of putative chromosomal origins of replication in Archaea[END_REF]. We observed 11 repeats of the previously reported consensus 5'-TTACA(c/g)TTGAA(a/c)N-3' [START_REF] Capaldi | Biochemical characterization of Cdc6/Orc1 binding to the replication origin of the euryarchaeon Methanothermobacter thermoautotrophicus[END_REF], where the repeats are regularly spaced some 49-53 nucleotides apart, possibly suggesting a phased binding of MthCdc6-1 (Supp. Fig. 1). In addition to the mini-ORB sequence, we noticed that all 11 sequences contained an additional conserved region 3' to the mini-ORB consensus of 5'-NN(a/t)(t/c)(c/g)NCNC-3' (Supp. Fig. 1).

Analysis of the putative origin-containing region was performed by probing genomic DNA isolated from exponentially growing M. thermautotrophicus cells and separated by neutral/neutral (N/N) two-dimensional agarose gel electrophoresis [26]. A functional origin of replication was detected on hybridisation of the putative origin sequence using a 4 kb probe (Fig. 1), as indicated by the bubble arc structure (arrow, Fig. 1C) and diminished Y-arc characteristic of a firing origin of replication. The discontinuity between the top of the bubble arc and the Yarc (the so-called "bubble-to-Y transition") is also highly characteristic of a firing origin of replication. In order to demonstrate that the bubble structure was specific to the fragment probed, the blot was stripped and hybridised to ~5 kb probes specific for adjacent fragments (Fig. 1B,D).

Both probes yielded a complete Y-arc, and the absence of any bubble arc structures, indicating that these regions of DNA contained only fork structures and suggesting that DNA replication in M. thermautotrophicus is bidirectional and proceeds via the movement of a pair of replication forks (Fig. 1). The 2D analysis consistently yielded the same results when using DNA isolated from a number (n=5) of different exponentially growing cultures (e.g. Fig. 2). We took additional samples from an actively growing culture of M. thermautotrophicus to examine the initiation of replication as cells entered stationary phase (Fig. 2). Growth stage was confirmed by flow cytometry (Fig. 2 A,C,E) before replication intermediates were analysed. We observed a decrease in the frequency of replication initiation from stationary phase samples (Fig. 2 F), but replication did not appear to completely cease. These results are typical of an asynchronous microbial culture, where a small population of cells can continue to proliferate even under limiting growth conditions. Our data are also consistent with the growth characteristics of methanogens, which are not known to produce a dormant stage or sporulate, as they normally exist in hydrogen-limited environments which would result in slow but continuous ('cryptic')

S t a g e 2 ( a ) P O S T -P R I N T

growth. Although GC skew analysis predicted only a single origin of replication in the genome of M. thermautotrophicus [START_REF] Lopez | Identification of putative chromosomal origins of replication in Archaea[END_REF], we also probed a 11.5 kb DNA locus around the ORF encoding the MthCdc6-2 gene (chromosomal position 1,460,708 -1,472,211 bp). Our results showed no bubble structures in this DNA region (not shown). These results indicate that there is no replication origin near the MthCdc6-2 gene.

The sequence requirements of MthCdc6-1 DNA binding were examined using a filter binding assay [START_REF] Capaldi | Biochemical characterization of Cdc6/Orc1 binding to the replication origin of the euryarchaeon Methanothermobacter thermoautotrophicus[END_REF]. 34 bp duplexes of ORBs 8, 9, 10 or 11 containing the previously described mini-ORB consensus sequence [START_REF] Capaldi | Biochemical characterization of Cdc6/Orc1 binding to the replication origin of the euryarchaeon Methanothermobacter thermoautotrophicus[END_REF] plus flanking regions were incubated with purified MthCdc6-1 (Fig. 3A). ORB8 DNA binding and apparent dissociation constants (K d(app) ) for MthCdc6-1 (Table 1) were comparable to those published previously, even though the maximal protein concentration used was lower than in those experiments [START_REF] Capaldi | Biochemical characterization of Cdc6/Orc1 binding to the replication origin of the euryarchaeon Methanothermobacter thermoautotrophicus[END_REF]. Interestingly, variations in the sequence of the regions flanking the ORB consensus had a small but measurable effect on DNA binding (Fig. 3A, Table 1). In contrast to DNase I footprinting experiments where SsCdc6-1 was able to recognize ORB sequences from both Pyrococcus and Halobacterium [START_REF] Robinson | Identification of two origins of replication in the single chromosome of the archaeon Sulfolobus solfataricus[END_REF], reactions containing MthCdc6-1 and ORB sequences from P. abyssi or S. solfataricus resulted in reduced binding (Fig. 3A). A 34 bp duplex of random sequence was not bound by MthCdc6-1, but the addition of increasing amounts of this and other competitor DNA into reactions containing the origin sequence resulted in more specific origin binding (Fig. 3B, Table 1). These results were obtained using two different DNA duplexes of random sequence, indicating that the observation is not a sequence-based artefact, although it does not exclude the possibility of an effect of the filter-binding assay.

In order to more exactly determine the sequence requirements of MthCdc6-1 for specific ORB binding, we challenged the wild-type Cdc6 protein with additional 34 bp duplexes containing point mutations in the consensus sequence. Interestingly, we found that most single mutations (SM) within the consensus sequence appeared to have little effect on DNA binding (Fig. 3C, Table 1). The exceptions to this were SM6 and SM7, where mutations at the same G (to C or A respectively) within the mini-ORB consensus sequence effectively resulted in a complete loss of binding of MthCdc6-1 (Fig. 3C, Table 1). Two double mutations (DM1 and DM3) within the consensus sequence also resulted in abrogation of binding (Fig. 3D sequence we observed (above), a similar partial inhibition of MthCdc6-1 binding was observed with duplexes where double mutations were made 3' to the consensus sequence (Fig. 3D, Table 1), implying that these 3' bases are also important for MthCdc6-1 / ORB interactions. This was confirmed by replacing 13 bp of sequence 3' to the original consensus sequence with a random sequence, which resulted in an almost complete loss of MthCdc6-1 binding (Fig. 3D, Table 1).

In order to test the hypothesis that the conserved guanine identified in our experiments interacts directly with the conserved Arg 334 previously characterized [START_REF] Capaldi | Biochemical characterization of Cdc6/Orc1 binding to the replication origin of the euryarchaeon Methanothermobacter thermoautotrophicus[END_REF], we generated and purified three MthCdc-1 variants: R334A, R334K, R334Q, with the selection of amino acids being based on well characterized bidentate protein-DNA interactions [START_REF] Luscombe | Amino acid-base interactions: a three-dimensional analysis of protein-DNA interactions at an atomic level[END_REF] (Fig. 4). These proteins, along with the wild-type protein were subjected to in vitro DNA binding assays in the presence of Orb8, SM6 (G to C), SM7 (G to A) and random sequence duplexes (Fig. 5). As previously reported [START_REF] Capaldi | Biochemical characterization of Cdc6/Orc1 binding to the replication origin of the euryarchaeon Methanothermobacter thermoautotrophicus[END_REF], the R334A mutation in MthCdc6-1 disrupted the interaction of Cdc6 with Orb8. R334A also showed poor binding of SM6, SM7 and the random sequence (Fig. 5A).

The R334K variant of Cdc6 showed a modest decrease in binding of Orb8 compared to wildtype, but was also able to bind the mutated origin sequences SM6 and SM7 (Fig. 5B). The R334Q variant was also able to bind SM6 and SM7, but in contrast to R334K or wild-type Cdc6, this protein did not bind the Orb8 sequence, thus showing an altered DNA sequence binding specificity compared to the wild-type MthCdc6-1 (Fig. 5C).

Discussion

DNA replication in the archaea is interesting both for understanding this fundamental process in the third domain of life and as a way of probing the mechanisms that underlie eukaryotic replication. The replication initiation process is still only partially understood in archaea, and well-characterised model systems are required for mechanistic studies. Within the archaea, the replication proteins of M. thermautotrophicus are among some of the best characterized to date. M. thermautotrophicus possesses a strikingly organized array of ORB sequences. Evenly spaced repeats flanking both sides of an AT-rich region upstream of the MthCDC6-1 gene (Fig 1A ) were first reported to contain twelve 13mers with significant homology to repeats found by GC skew analysis in Pyrococcus species [START_REF] Lopez | Identification of putative chromosomal origins of replication in Archaea[END_REF], although we find only eleven (Supp. Fig. 1). While the repeats of P. furiosus were identified as homologous to the functional ORBs identified in Sulfolobus those of M. thermautotrophicus were not [START_REF] Robinson | Identification of two origins of replication in the single chromosome of the archaeon Sulfolobus solfataricus[END_REF]. Limited homology between the mini-ORB sequences of M. thermautotrophicus and those of Sulfolobus and Pyrococcus has since been noted [START_REF] Capaldi | Biochemical characterization of Cdc6/Orc1 binding to the replication origin of the euryarchaeon Methanothermobacter thermoautotrophicus[END_REF]. Our study supports the notion that the active origin of replication in M. thermautotrophicus contains eleven full-length ORB sequences that are homologous to those previously identified in other archaea. Our data provide the first experimental confirmation that the putative site of DNA replication initiation in M. thermautotrophicus is in fact a functional replication origin.

As previously reported [START_REF] Singleton | Conformational changes induced by nucleotide binding in Cdc6/ORC from Aeropyrum pernix[END_REF][START_REF] Capaldi | Biochemical characterization of Cdc6/Orc1 binding to the replication origin of the euryarchaeon Methanothermobacter thermoautotrophicus[END_REF], we have shown that MthCdc6-1 can bind to Orb8 sequences in vitro. We have additionally shown that this interaction is improved in the presence of competitor DNA. One possible explanation for this observation is that MthCdc6-1 contains two separate DNA binding sites, and that binding of non-specific DNA to one site causes some change in the protein that stabilizes the binding of the specific DNA at the other site. We have determined that changes to the DNA sequences 3' to the mini-ORB consensus affect MthCdc6-1 binding. Replacement of the entire 3' region of the duplex used for DNA binding resulted in a complete loss of DNA binding, suggesting that the originally reported mini-ORB consensus is not sufficient for effective Cdc6/ORB interactions. Mutations located outside the mini-ORB sequence in the conserved 3' residues (DM4, DM5) did not completely disrupt binding, although they did have a significant effect, supporting the notion that specific contacts between MthCdc6-1 and the 3' sequence could be crucial for MthORB binding. The limited conservation in this region of the ORB suggests that the 3' sequence may play a role in enhancing Cdc6 / DNA binding, or stabilisation of the protein-DNA complex, rather than in sequence-specific recognition by MthCdc6 protein. It should be noted that the sequences 3' to the mini-ORB identified here were present in the original MthCdc6-1 DNA binding studies [START_REF] Capaldi | Biochemical characterization of Cdc6/Orc1 binding to the replication origin of the euryarchaeon Methanothermobacter thermoautotrophicus[END_REF], and can also be identified in a synthetic triple repeat of the mini-ORB that was shown to be bound by MthCdc6-1 [START_REF] Kasiviswanathan | DNA binding by the Methanothermobacter thermautotrophicus Cdc6 protein is inhibited by the minichromosome maintenance helicase[END_REF] (Supp. Fig. 2). Thus our novel findings are completely consistent with other studies in this species and we are confident that the 3' sequences identified by our experiments form part of the essential MthORB sequence required for MthCdc6-1 binding, although their contribution was not previously appreciated. ORB sequences from different archaeal species show weaker homology in this region and the MthORB diverges greatly from the consensus. We believe that the differences in this part of the ORB sequence are likely sufficient to explain the All the double mutations we tested affected the total amount of DNA bound by MthCdc6-1.

DM1 (CT to TG) showed a very strong reduction in binding that did not occur when either of these mutations was tested alone, suggesting that these bases may be involved in a cooperative interaction with Cdc6 protein residues. Two of the seven single point mutations that we tested in the consensus sequence had a significant effect on Cdc6-1 binding (SM6, G to C and SM7, G to A), and the location of this guanine residue corresponds directly to one of the "triple mutant" residues shown to disrupt Sulfolobus Cdc6-1 / ORB interactions [START_REF] Robinson | Identification of two origins of replication in the single chromosome of the archaeon Sulfolobus solfataricus[END_REF]. Interestingly, a guanine corresponding to the residue mutated in SM6 is completely conserved in the ORB sequences of all archaeal species where an origin has been described to date (Fig. 6A). Furthermore, guanine but not cytosine can form two hydrogen bonds with arginine via the major groove of a double helix without disrupting base pairing [START_REF] Luscombe | Amino acid-base interactions: a three-dimensional analysis of protein-DNA interactions at an atomic level[END_REF][START_REF] Bloomfield | Nucleic Acids: Structures, Properties, and Functions[END_REF]. Given that the R334A mutation in MthCdc6-1 has previously been shown to abolish ORB binding [START_REF] Capaldi | Biochemical characterization of Cdc6/Orc1 binding to the replication origin of the euryarchaeon Methanothermobacter thermoautotrophicus[END_REF], the most obvious mechanism by which the SM6 mutation might effect changes in MthCdc6-1 binding is if this base interacts with Arg 334 via hydrogen bonding. Such an interaction could be key either in the initial positioning of MthCdc6-1 on the DNA, or in stabilizing the protein-DNA interaction once the specific site has been recognized. Arg 334 is also completely conserved in the Class I archaeal Cdc6 proteins known to bind to origin sequences, but not in the Class II Cdc6 variants that do not (Fig. 6B). Our mutagenesis of MthCdc6-1 has confirmed that Arg 334 is essential for the Cdc6 interaction with guanine. Mutating this amino acid to lysine reduced the specificity of binding and enabled Cdc6 to bind to a wider range of origin sequences. Most interestingly, we were able to show that a MthCdc6-1 variant containing glutamine at position 334 displayed altered sequence specificity as it will no longer bind to the wild-type guanine-containing Orb8 sequence, but is now able to recognize cytosine or adenine in its place, albeit with a lower affinity than we observe for the wild-type interaction. Thus it appears that the potential to form hydrogen bonds through a major groove / recognition helix guanine-arginine interaction, as described here, is part of a generally conserved mechanism employed in replication origin binding by all archaeal ORC/Cdc6 proteins. Variation in the surrounding DNA sequences and changes to the amino acid sequence of the recognition helix in the WHD of Class I Cdc6 proteins could thus explain the sequence specificity observed in Cdc6-1 proteins and ORBs from different archaeal species. A, Confirmation that the introduction of the R334A mutation into MthCdc6-1 disrupts Orb8 as well as all other DNA binding. B, An MthCdc6-1 R334K variant shows slightly reduced affinity for Orb8, but promiscuous binding to the SM6 and SM7 substrates. C, The R334Q variant of MthCdc6-1 cannot bind Orb8, but is able to bind DNA duplexes containing the SM6 and SM7 mutations, demonstrating an altered specificity for DNA sequences for this protein.

0.35 ORB9 GCA••ATTTT•••••••••••••TC••CC•••AT 0.43 ORB10 ATCCAGAT•G•••••••••••••A••••••C•A• 0.62 ORB11 ATAA•AG•AT•••••••••••••CT•CCA•A••• 0.68 ORB8
In the presence of 10-fold excess random competitor 0.30

ORB8

In the presence of 100-fold excess random competitor 0.23

ORB8

In the presence of 1000-fold excess random competitor 0.24

ORB8

In the presence of 1000-fold excess random competitor 2 0.21

SM1 •••••••••••C•••••••••••••••••••••• 0.34 SM2 •••••••••••••T•••••••••••••••••••• 0.51 SM3 •••••••••••••••T•••••••••••••••••• 0.48 SM4 ••••••••••••••••C••••••••••••••••• 0.64 SM5 ••••••••••••••G••••••••••••••••••• 0.43 SM6 ••••••••••••••••••C••••••••••••••• n.d. SM7 ••••••••••••••••••A••••••••••••••• n.d. DM1 •••••••••••••TG••••••••••••••••••• n.d. DM2 •••••••••••••••••••GG••••••••••••• n.d. DM3 ••••••••••••••••••AG•••••••••••••• n.d. DM4 •••••••••••••••••••••••••••••T•T•• n.d. DM5 •••••••••••••••••••••••••G•A•••••• n.d. 3' end •••••••••••••••••••••TAATGCCATGGAA n.d. Pa ORB CAAC•C•G••C••G•G••••••A•ACC•TGGGGG 7.41 Ss ORB TTTACCTTAAGTTC•C••G•G••••C •AA GGGGT n.d.

  S t a g e 2 ( a ) P O S T -P R I N T Biochemical Journal Immediate Publication. Published on 23 Oct 2007 as manuscript BJ20070213 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20070213

  e 2 ( a ) P O S T -P R I N T Biochemical Journal Immediate Publication. Published on 23 Oct 2007 as manuscript BJ20070213 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20070213

  ), one of which, DM3 overlapped with SM6/7. A third double mutation in the consensus sequence, DM2, resulted in a significant decrease in total DNA binding by MthCdc6-1. Consistent with the extended MthORB S t a g e 2 ( a ) P O S T -P R I N T Biochemical Journal Immediate Publication. Published on 23 Oct 2007 as manuscript BJ20070213 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20070213 Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2007 The Authors Journal compilation © 2007 Biochemical Society

  consensus

  S t a g e 2 ( a ) P O S T -P R I N T Biochemical Journal Immediate Publication. Published on 23 Oct 2007 as manuscript BJ20070213 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20070213 Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2007 The Authors Journal compilation © 2007 Biochemical Society

  S t a g e 2 ( a ) P O S T -P R I N T Biochemical Journal Immediate Publication. Published on 23 Oct 2007 as manuscript BJ20070213 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20070213 Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2007 The Authors Journal compilation © 2007 Biochemical Society differences between the SsCdc6-1 and MthCdc6-1 proteins with regards to their abilities to bind ORBs from other species.

  Fig. 1. -The M. thermautotrophicus origin of replication is functional in vivo. A, Cartoon of the genomic region of M. thermautotrophicus showing the presence of the fragments used to probe the gels in B-D (marked as a-c, respectively), location of nearby genes and the relative position and orientation of ORB sequences. An A/T-rich region that could be the site of initial unwinding is indicated. B, 2D gel probed with genomic fragment upstream of putative replication origin showing only a Y-arc consistent with the presence of a fork. C, 2D gel probed with genomic fragment containing the putative origin of replication. Bubble arc is indicated with an arrow. D, 2D gel probed with genomic fragment downstream of putative replication origin showing only a Y-arc consistent with the presence of a fork.

Fig. 2 .

 2 Fig. 2. -Replication initiation decreases but does not cease as M. thermautotrophicus cells enter stationary phase. A, C, E, Flow cytometric analysis of cells taken at different stages of growth show a characteristic reduction in average genome content [23]. B, D, E, 2D gels of DNA samples taken from the corresponding growth stages show that initiation frequency decreases, but is not completely lost in stationary phase. Bubble arcs are indicated by arrows.

Fig. 3 .

 3 Fig. 3. -Specific DNA binding by MthCdc6-1. A, Variations in the Orb sequences found in M. thermautotrophicus have a small effect on the extent of MthCdc6-1 binding. Despite similarities in sequence, ORB sequences from other species are not bound by MthCdc6-1. Pa: Pyrococcus abyssi; Ss: Sulfolobus solfataricus. B, Addition of increasing quantities of competitor (random sequence) DNA results in more specific binding of MthCdc6-1 to Orb8 duplex. C, Single point mutations within the Orb8 sequence has little effect on Cdc6-Orb binding other than the mutation of a conserved guanine to cytosine in SM6, which abrogates Orb binding. D, Mutation of pairs of nucleotides within the mini-ORB consensus affects the ability of MthCdc6-1 to effectively bind DNA. Mutation of nucleotides 3' to the mini-ORB consensus also compromises the ability of MthCdc6-1 to effectively bind DNA. Substrate sequences and K d(app) values (where appropriate) are listed in Table 1. All points are the mean of 3 independent reactions. Error bars indicate ±1 SD. S t a g e 2 ( a ) P O S T -P R I N T

Fig. 4 .Fig. 5 .

 45 Fig.4. -Variants in MthCdc6-1 may affect Cdc6 / Orb interactions. A, Coomassie stained SDS-PAGE gel of purified WT and variant MthCdc6-1 proteins. Position and size of molecular weight markers are indicated. B, Likely non-disruptive interactionsbetween amino acid side chains and DNA via the major groove (based on[START_REF] Luscombe | Amino acid-base interactions: a three-dimensional analysis of protein-DNA interactions at an atomic level[END_REF]). From top to bottom the interactions illustrated are: arginine -guanine, lysine -guanine and glutamineadenosine.

Fig. 6 .

 6 Fig. 6. -A conserved mechanism employed in replication origin binding by all archaeal ORC/Cdc6 proteins? A, The SM6 guanine (highlighted) is conserved in all archaeal origins characterized to date, as shown by an alignment of the consensus sequences of mini-ORB regions from a number of species (based on [10]). B, ClustalW alignment of archaeal Cdc6 proteins in the region of Arg 334 (highlighted) showing that this amino acid is completely conserved in all Class I archaeal Cdc6 proteins, but is not present in Class II Cdc6 proteins. Mth -M. thermautotrophicus, Sso -S. solfataricus, Afu -A. fulgidus, Ape -A. pernix, Pfu -P. furiosus. S t a g e 2 ( a ) P O S T -P R I N T

1 S

 1 app) values for the binding of MthCdc6-1 to different Orb-sequence containing DNA duplexes. Values were derived by using a simple independent binding site model equation with B max fixed to the value obtained for Orb8 (65%). ORB8: UPPER CASE bases indicate consensus sequence S t a g e 2 ( a ) P O S T -P R I N T Biochemical Journal Immediate Publication. Published on 23 Oct 2007 as manuscript BJ20070213 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20070213 Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2007 The Authors Journal compilation © 2007 Biochemical Society t a g e 2 ( a ) P O S T -P R I N T Biochemical Journal Immediate Publication. Published on 23 Oct 2007 as manuscript BJ20070213 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20070213 Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2007 The Authors Journal compilation © 2007 Biochemical Society

  g e 2 ( a ) P O S T -P R I N T Biochemical Journal Immediate Publication. Published on 23 Oct 2007 as manuscript BJ20070213 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20070213 Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2007 The Authors Journal compilation © 2007 Biochemical Society

6 A

 6 g 2 ( a ) P O S T -P R I N T Biochemical Journal Immediate Publication. Published on 23 Oct 2007 as manuscript BJ20070213 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20070213 Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2007 The Authors Journal compilation © 2007 Biochemical Society g e 2 ( a ) P O S T -P R I N T Biochemical Journal Immediate Publication. Published on 23 Oct 2007 as manuscript BJ20070213 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20070213 Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2007 The Authors Journal compilation © 2007 Biochemical Society g e 2 ( a ) P O S T -P R I N T Biochemical Journal Immediate Publication. Published on 23 Oct 2007 as manuscript BJ20070213 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20070213 Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2007 The Authors Journal compilation © 2007 Biochemical Society Majerník and Chong, Figure g e 2 ( a ) P O S T -P R I N T Biochemical Journal Immediate Publication. Published on 23 Oct 2007 as manuscript BJ20070213 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20070213 Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2007 The Authors Journal compilation © 2007 Biochemical Society

Table 1 -

 1 kinetic data for the interaction between MthCdc6-1 and different ORB sequences

	Name	Sequence

THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20070213 Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2007 The Authors Journal compilation © 2007 Biochemical Society

© 2007 The Authors Journal compilation © 2007 Biochemical Society

Acknowledgements

We thank Alison Walters, Cyril Sanders and Marjan van der Woude for comments on the manuscript and Mrs Magdaléna Országhová for excellent technical assistance. This work was funded by a BBSRC David Phillips Research Fellowship (J.P.J.C.), grant APVT-51-024904 from the Science and Technology Assistance Agency (Slovak Republic) and a Royal Society International Short Visit / Fellowship (A.I.M.).

Additional Note

While this manuscript was under review, crystal structures of S. solfataricus cdc6-1 and cdc6-3 forming a heterodimer bound to ori2 DNA (Dueber EL, Corn JE, Bell SD, Berger JM., 2007, Science 317:1210-3); and that of A. pernix cdc6-1 bound to an origin sequence (Gaudier M, Schuwirth BS, Westcott SL, Wigley DB., Science 317:1213-6) have been published. Both papers provide structural data fully consistent with our conclusions. In particular, Gaudier et al clearly show the essential interaction through the major groove between a guanine (Gd10) in the ORB and an arginine (R345) in Cdc6-1 of A. pernix, which correspond to those identified in our system (and predicted from our results). In addition the structures indicate that the less-conserved 3' ORB sequence is important for DNA binding and suggest that there are two independent DNA binding sites that interact with each other -clearly supporting our data concerning nonsequence-specific competitor. The fact that our data are completely consistent with the findings of these crystal structures serves to reinforce the conserved nature of these interactions across all archaea. We have created models where substitution of lysine or glutamine for R345 in the A. pernix structure was visualised (see Supp. Fig. 3; thanks to Dale Wigley for providing the crystal co-ordinates before release and Fred Anston for modelling assistance). This has allowed us to visualize the conserved hydrogen bonds of the lysine substitution, and the potential of glutamine to disrupt this interaction and contribute to the binding phenotypes observed for our mutant


Franco Zoccarato

Lucia Cavallini

Silvia Bortolami

Adolfo Alexandre
email: adolfo.alexandre@unipd.it

SUCCINATE MODULATION OF H 2 O 2 RELEASE AT NADH:UBIQUINONE OXIDOREDUCTASE (Complex I) IN BRAIN MITOCHONDRIA

Keywords: Brain mitochondria, NADH:ubiquinone oxidoreductase (Complex I), H 2 O 2 production, Succinate mitochondrial membrane potential, ∆pH, mitochondrial pH gradient, ∆p, mitochondrial

Complex I is responsible for most of the mitochondrial H 2 O 2 release, both during the oxidation of NAD-linked substrates and during succinate oxidation. The much faster succinate-dependent H 2 O 2 production is ascribed to Complex I, being rotenone sensitive. We report high affinity succinate -supported H 2 O 2 generation in the absence as well as in the presence of glutamate/malate ( G/M) 1 or 2 mM each. In brain mitochondria, their only effect was to increase from 0.35 to 0.5 or to 0.65 mM the succinate concentration evoking the semimaximal H 2 O 2 release. G/M are still oxidized in the presence of succinate, as indicated by the oxygen consumption rates, which are intermediate between those of G/M and of succinate alone when all substrates are present together. This effect is removed by rotenone, showing that it is not due to inhibition of succinate influx. Moreover, α-ketoglutarate production from G/M, a measure of the activity of Complex I, is decreased but not stopped by succinate. It is concluded that succinate-induced H 2 O 2 production occurs in conditions of regular downward electron flow in Complex I. Succinate concentration appears to modulate the rate of H 2 O 2 release probably by controlling the QH 2 /Q ratio.

INTRODUCTION

Mitochondria are the major site of reactive oxygen species (ROS) production in mammalian cells and superoxide (O 2 -) appears to be the primary ROS produced as the result of single electron reduction of O 2 .

Mitochondrial Complex I is the most complicated and least understood electron carrier in the respiratory chain. Its primary function is to pump protons across the inner membrane. A second property of Complex I is a highly modulated O 2 -production. O 2 -from Complex I is generated on the matrix side of the inner membrane [START_REF] St-Pierre | Topology of superoxide production from different sites in the mitochondrial electron transport chain[END_REF] and is transformed into H 2 O 2 by mitochondrial Mn-SOD. The highly permeable H 2 O 2 is the measured species in the extramitochondrial space. The importance of ROS removal from the mitochondrial matrix is demonstrated by the poor survival of Mn-SOD null mice [START_REF] Melov | Mitochondrial disease in superoxide dismutase 2 mutant mice[END_REF] and by the increased life span of mice expressing mitochondrial catalase [START_REF] Schriner | Extension of murine life span by overexpression of catalase targeted to mitochondria[END_REF]. Complex III also is known to produce O 2 -(mainly on the cytosolic space). However this O 2 -release is largely dependent on the presence of the electron transfer inhibitor antimycin A [START_REF] Turrens | Ubisemiquinone is the electron donor for superoxide formation by Complex III of heart mitochondria[END_REF].

ROS have been considered important in various pathologies [START_REF] Halliwell | Free radicals in biology and medicine[END_REF] and aging [START_REF] Cadenas | Mitochondrial free radical generation, oxidative stress, and aging[END_REF]. More recently however it has been recognized that ROS have important physiological roles as signalling molecules, whose functions are just emerging [START_REF] Allen | Oxidative stress and gene regulation[END_REF]. Complex I is now considered the major site of O 2 -/H 2 O 2 production.

H 2 O 2 is generated during coupled respiration of NAD-linked substrates (the release is increased by the electron transfer inhibitor rotenone); it is also generated, at a much faster rate, during coupled succinate respiration (production is strongly inhibited by rotenone and by decreasing membrane potential). These properties are taken as evidence that succinatedependent peroxide generation occurs in Complex I uphill of the rotenone inhibition site ( i.e.

via energy-dependent reverse electron transfer from Complex II to Complex I [START_REF] Turrens | Generation of superoxide anion by the NADH dehydrogenase of bovine heart mitochondria[END_REF][START_REF] Hansford | Dependence of H 2 O 2 formation by rat heart mitochondria on substrate availability and donor age[END_REF][START_REF] Liu | Generation of reactive oxygen species by the mitochondrial electron transport chain[END_REF][START_REF] Korshunov | High protonic potential actuates a mechanism of production of reactive oxygen species in mitochondria[END_REF][START_REF] Votyakova | ∆Ψm-dependent and independent production of reactive oxygen species by brain mitochondria[END_REF][START_REF] Han | Effect of glutathione depletion on sites and topology of superoxide and hydrogen peroxide production in mitochondria[END_REF]). We have shown that the Parkinson disease toxin dopaminochrome (derived from dopamine) promotes a strong increase of H 2 O 2 production both with glutamate/malate (G/M) and in the further presence of succinate [START_REF] Zoccarato | Dopamine-derived dopaminochrome promotes H 2 O 2 release at mitochondrial Complex I. Stimulation by rotenone, control by Ca 2+ , and relevance to Parkinson Disease[END_REF], and that H 2 O 2 is removed by GSH peroxidase [START_REF] Zoccarato | Respiration-dependent removal of exogenous H 2 O 2 in brain mitochondria. Inhibition by Ca 2+[END_REF]. The succinate-dependent peroxide production is controlled by ∆pH across the inner membrane and is depressed when such gradient is abolished [START_REF] Lambert | Superoxide production by NADH:ubiquinone oxidoreductase (Comple I) depends on the pH gradient across the mitochondrial inner membrane[END_REF]. The physiological importance of succinate-sustained H 2 O 2 generation, being linked to a situation of reverse electron transfer, has been considered dubious [START_REF] Hansford | Dependence of H 2 O 2 formation by rat heart mitochondria on substrate availability and donor age[END_REF][START_REF] Liu | Generation of reactive oxygen species by the mitochondrial electron transport chain[END_REF][START_REF] Korshunov | High protonic potential actuates a mechanism of production of reactive oxygen species in mitochondria[END_REF][START_REF] Votyakova | ∆Ψm-dependent and independent production of reactive oxygen species by brain mitochondria[END_REF][START_REF] Han | Effect of glutathione depletion on sites and topology of superoxide and hydrogen peroxide production in mitochondria[END_REF][START_REF] Lambert | Superoxide production by NADH:ubiquinone oxidoreductase (Comple I) depends on the pH gradient across the mitochondrial inner membrane[END_REF][START_REF] Lambert | Inhibitors of the quinone-binding site allow rapid superoxide production from mitochondrial NADH:ubiquinone oxidoreductase (Complex I)[END_REF].

In this study we analysed the production of H 2 O 2 in brain mitochondria in the presence of both the NAD-linked substrates G/M (or pyruvate/malate, which gave the same results) and succinate, i.e. close to in vivo conditions. We show that succinate-dependent peroxide production initiates at low physiological concentrations and is only marginally modified by the presence of G/M. Furthermore, G/M oxidation is progressively decreased, but not abolished by succinate and the two substrates appear to compete for electron transfer.

We propose that the sharp dependence of the H 2 O 2 generation rate on succinate concentration is an important modulator in the overall cellular ROS production.

MATERIALS AND METHODS

Reagents

Amplex Red (10-acetyl-

Preparation of Rat Brain Mitochondria

Brain mitochondria were isolated from cerebral cortices of 6-7 weeks old rats as described [START_REF] Zoccarato | Respiration-dependent removal of exogenous H 2 O 2 in brain mitochondria. Inhibition by Ca 2+[END_REF].

Standard Incubation Method

Mitochondria (0.4 -0.6 mg/ml) were incubated at 30 °C in 125 mM KCl, 1.

Assay of α α α α-Ketoglutarate

The α-KG assay was performed as in [START_REF] Sanadi | α-Ketoglutarate dehydrogenase from pig heart[END_REF][START_REF] Mccormarck | The effects of calcium ions and adenine nucleotides on the activity of pig heart 2-oxoglutarate dehydrogenase complex[END_REF]. In brief mitochondria (4 mg/ml) were incubated in the standard incubation medium under agitation for 10 minutes with the indicated amount of substrates. During incubation substrate concentrations of glutamate, malate and succinate were maintened constant by addition of 0,3 mM glutamate and malate and 0,5 mM succinate at 5 minutes. Reactions were stopped by addition of cold PCA ( 12 Values are reported as n moles /min/mg protein. Similar results were obtained by spinning down mitochondria at the end of incubation and measuring α-KG in the PCA-treated supernatant. Also, including catalase (5 µg/ml) in the incubation medium was without significant effect on α-KG accumulation.

Other assays

Mitochondrial Membrane potential (∆ψ) was measured using fluorescence quenching of the cationic dye safranine (3 µM) at 495 nm (excitation) and 586 nm (emission) as in [START_REF] Zoccarato | Respiration-dependent removal of exogenous H 2 O 2 in brain mitochondria. Inhibition by Ca 2+[END_REF]. NAD(P) reduction was monitored fluorimetrically at 348 nm (excitation) and 464 nm (emission) as in [START_REF] Zoccarato | Respiration-dependent removal of exogenous H 2 O 2 in brain mitochondria. Inhibition by Ca 2+[END_REF]. Mitochondrial Respiration was monitored in the presence of HRP and Amplex Red with a Clark-type oxygen electrode in a 1.6 ml closed chamber with continuous stirring. production.

RESULTS AND DISCUSSION

H 2 O 2 release is controlled by ∆ ∆ ∆ ∆pH

The succinate-induced H 2 O 2 release is dependent on ∆p across the inner mitochondrial membrane. However, ∆p is the sum of two components, the difference in electrical potential ∆ψ and the difference in pH (∆pH). It has been shown recently that ∆pH is a major controller of succinate-dependent peroxide production [START_REF] Lambert | Superoxide production by NADH:ubiquinone oxidoreductase (Comple I) depends on the pH gradient across the mitochondrial inner membrane[END_REF]. In fact, the inclusion of the K + /H + ionophore nigericin (in a high K + medium) allows K + equilibration and hence H + equilibration (∆pH = zero): this results in a corresponding equivalent increase of ∆ψ to maintain ∆p constant (ref 16, and results not shown). In these conditions, the succinate promoted H 2 O 2 release is at its lowest. The succinate titration of peroxide release in the presence of nigericin and G/M (Fig. 4) shows that the overall production rate was depressed relative to Fig. 1B (dotted line) but that the pattern was unchanged. On the contrary, increasing ∆pH by omitting phosphate (whose influx via the P i /H + symporter decreases ∆pH), promoted a higher succinate-dependent H 2 O 2 release [START_REF] Lambert | Superoxide production by NADH:ubiquinone oxidoreductase (Comple I) depends on the pH gradient across the mitochondrial inner membrane[END_REF], without changing the general behaviour in the presence of G/M (Fig. 4).

Conclusions

The results presented show that the succinate stimulation of mitochondrial H 2 O 2 release occurs at low concentrations and that the presence of G/M modifies only marginally the succinate promotion of peroxide release. Furthermore, G/M oxidation is decreased but not prevented by succinate.

When both NADH and succinate are present (the in vivo situation) it appears that they compete for Complex I (or a part of it), the first pushing electrons down and the second pushing electrons up into the complex. The two processes appear to occur at the same time, possibly due to competition between regular quinone reduction from NADH and re-binding of the reduced species due to its higher concentration during the operation of Complex II. [succinate] (mM) Oxygen (natoms / min x mg)

  %) and samples left in ice 20 minutes before centrifugation. Enzymatic fluorimetric titration of NADH formed by α-KG oxidation was carried out in 100 µl aliquots of the supernatant neutralized with KOH in 200 µl (final volume) of medium containing 100 mM Na-phosphate (pH 7.3), CaCl 2 5 mM, MgCl 2 1 mM, EGTA 5 mM, NAD + 1 mM , and 0.2 mg/ml (160 mU) of α-KG dehydrogenase in a plate reader Fluoroskan at 355/460 nm ex/em. The reaction was started by addition of 100 µM CoASH. α-KG concentrations were calculated from the titration curve of the rates obtained with known comparable amounts of standard α-KG.

Fig. 3 .

 3 Fig. 3. α α α α-KG is produced during coupled oxidation of G/M and succinate. Mitochondria (4 mg/ml) were incubated 10 min with 1 mM G/M and succinate as indicated. Extra G/M (0.3 mM each) and succinate (0.5 mM) were added at 5 min. The values are means ± SD from duplicate incubations from five preparations.

Fig. 4 .

 4 Fig. 4. The H 2 O 2 release rates are controlled by ∆ ∆ ∆ ∆pH. Conditions as in Fig. 1 with 1 mM G/M. The titration was performed in the presence of 100 nM nigericin (□) or in the absence of phosphate ( ). The dotted line represent values from Fig. 1, B (trace in the presence of G/M 1 mM). Data are means ± SD from at least four independent experiments.

Complex I dependent H 2 O 2 release with succinate and G/M

  peroxide release by succinate was increased to 0.6 -0.65 mM. The rotenone-inhibited H 2 O 2 production rates with succinate are also reported in Fig.1B. It is likely that this residual H 2 O 2 release also originated from Complex I, since succinate promoted a large NAD(P) reduction in the presence of rotenone, likely via malate dehydrogenase. Similar results were obtained with piericidin (not shown). Anyway, in the hypothesis that the peroxide generation with succinate and rotenone was not originating at Complex I, these rates were subtracted from the succinate and succinate-G/M values of Fig.1B. The results are reported in Fig.1C. They represent certainly Complex I-dependent H 2 O 2 releases with succinate and G/M. The generally accepted view is that succinate promotes H 2 O 2 release by pushing electrons back into Complex I, in a reverse mode of electron transfer. With NAD-linked substrates and in the absence of succinate, H 2 O 2 release is slow and can be increased in a non-physiological situation, i.e. in the presence of rotenone-type electron transfer inhibitors and high ∆pH [17].

	Succinate does not prevent electron flow from G/M in Complex I
	Fig.1A shows the fast H 2 O 2 production rate in brain mitochondria oxidizing succinate (trace were contributing to O 2 consumption when they were present together. In the presence of
	a) and the much slower peroxide generation with G/M as the substrates (trace b). The rotenone, the O 2 consumption rate with succinate was not significantly affected by G/M,
	production of H 2 O 2 as a function of succinate concentration, in the absence and presence of showing that there was no limitation to succinate availability. The overall titration of O 2
	G/M, is reported in Fig.1B. The maximal H 2 O 2 release (with 3 mM succinate) was unaffected consumption with increasing succinate, in the absence and presence of constant (1 mM) G/M
	by G/M. The half maximal peroxide production with succinate alone was at approximately is reported in Fig. 2. Above 0.2 mM the succinate oxidation rate was faster than that of G/M 0.35 mM. With G/M (1 mM each), a concentration which produced maximal ∆p, the H 2 O 2 and the addition of G/M induced a decrease of O 2 consumption to intermediate values. Only

release at zero succinate was about 0.15 nmol/mg/min, and the further addition of succinate promoted a sharp increase of peroxide production, with a half maximal stimulation at about 0.5 mM. A similar behaviour was obtained at higher (2 mM) G/M. In this situation the half maximal stimulation of Alternatively peroxide release was reported to be increased in cytochrome c depleted mitochondria

[START_REF] Kushnareva | Complex I-mediated reactive oxygen species generation: modulation by cytochrome c and NAD(P) + oxidation-reduction state[END_REF]

. The findings reported in the present study show that low succinate promotes H 2 O 2 release also in the presence of high NAD-linked substrates. It can be questioned if succinate in these conditions operates a "reverse" mode of electron flux, i.e. if the oxidation of G/M is prevented. To solve this problem first of all we analysed the rates of O 2 consumption with G/M (1 mM each), succinate and the mixture. Typical results are reported in Table

1

showing the experiment with 0.5 mM succinate (a concentration that promotes a large H 2 O 2 release). The oxidation rate was higher with succinate than with G/M, likely reflecting the lower number of protons pumped by succinate, which does not feed electrons to Complex I. However, the O 2 consumption rate with the mixture was intermediate between that of the two constituents alone, a possible indication that both succinate and G/M above about 1.5 mM succinate, G/M became incapable to decrease the succinate oxidation rate. These results confirm that a fraction of the overall oxidation rate was indeed dependent on G/M when both G/M and succinate were supplied to mitochondria.

Biochemical Journal Immediate Publication.

Published on 3 May 2007 as manuscript BJ20070215 Although

  the results just presented are a strong evidence in favour of the hypothesis that electrons actually flow downward in Complex I also during the succinate-promoted H 2 O 2 release in the presence of G/M, they are not a direct demonstration that this really occurs. As a second approach to prove this point we measured the generation of α-KG with G/M, in the absence and presence of succinate. The accumulation of α-KG is directly connected to the oxidation of G/M, via glutamate dehydrogenase, or via glutamate-oxaloacetete (OAA) transaminase, where OAA originates from malate oxidation. As shown in Fig.3, α-KG was accumulated during the oxidation of G/M, but not during succinate oxidation. With G/M, the further presence of succinate progressively decreased, but did not abolish, α-KG production.At 1 mM succinate, α-KG production was still 30% of the control without succinate. These results are in agreement with the data on respiration presented above.These experiments demonstrate conclusively that electrons keep flowing down fromComplex I in the presence of succinate and during the process of succinate-supported H 2 O 2

Table 1 O 2 consumption rate measured in mitochondria oxidizing different respiratory substrates.

 1 Electron backflow with succinate could mean just binding of QH 2 originating from Complex II to the quinone binding site on Complex I with O 2 -generation occurring right there (as speculated in ref.17) instead of electrons going uphill to a different site of O 2 production. In this view, the fact that G/M are still oxidized in the presence of succinate does not contradict any previously drawn conclusions. The high mitochondrial QH 2 /Q ratio induced by succinate appears the major determinant of H 2 O 2 release. Similarly, G/M alone induce high H 2 O 2 release rates in cytochrome c depleted mitochondria [20]. In the latter situation the removal of cytochrome c decreases the rate of electron flow and consequently increases QH 2 /Q. A similar effect could occur following NO • inhibition of cytochrome oxidase [21,22]. Amplex Red was added at the arrow. B) H 2 O 2 production was measured in the absence (Ο) and presence of G/M, 1 mM ( ) or 2 mM each ( ). Where present, rotenone was 2.5 µM ( ) without G/M. C) Net H 2 O 2 releases calculated by subtracting the rotenone rates from the no rotenone rates in B. Traces in A are representative of duplicate traces from at least seven independent experiments. Data in B are means ± SD from at least five independent experiments.

	Succinate concentration in vivo is controlled by the activity of the other dehydrogenases,
	which are NAD-dependent. It is significant that in vivo succinate concentration oscillates
	between 0.3 and 1 mM, as determined by Sato et al. [23] in perfused heart.
	Preliminary results appear to indicate a similar organization of H 2 O 2 release in heart
	mitochondria.

Fig.

2

. Succinate does not prevent G/M oxidation. Mitochondria (0.9 mg/1.6 ml) were incubated as in Table

1

with variable succinate and constant G/M (1mM each). Succinate alone ( ) and in the presence of G/M (□). Data are means ± SD from at least five independent experiments.

' 2007 The Authors Journal compilation ' 2007 Biochemical Society

Biochemical Journal Immediate Publication. Published on 3 May 2007 as manuscript BJ20070215 ' 2007 The Authors Journal compilation ' 2007 Biochemical Society

Acknowledgements : This work was supported by Ministero dell'Istruzione, dell'Università e della Ricerca. Progetti di Ricerca di rilevante Interesse Nazionale 2004 "Apoptosis and Mitochondria: New Targets in Neoplastic, Degenerative, and Immunologic Diseases".

Piericidin was a gift of Prof. Giorgio Lenaz.