Avichai Snir

Daniel Levy
email: daniel.levy@biu.ac.il

Allan ) Chen

End of 9-Endings, Price Recall, and Price Perceptions ✩

Keywords: JEL Classification: E31, L16, K20, D40, D83, L81, M21, M31 prices, psychological price points, price recall, price perception, sticky prices, rigid prices, price rigidity, price flexibility, price stickiness, level effect, left-digit effect, image effect, right-digit effect, integer constraint, price control, price regulation

Prices that end with 9, also known as psychological price points, are common, comprising about 70% of the retail prices. They are also more rigid than other prices. We take advantage of a natural experiment to document an emergence of a new price ending that has the same effects as 9-endings. In January 2014, the Israeli government passed a new regulation prohibiting the use of non 0-ending prices, bringing an end to 9ending prices. We find that seven months after 9-ending prices have disappeared, 90ending prices acquired the same status as 9-ending prices had before the new regulation was adopted. Thus, 90-ending prices became the new psychological price points, partially eliminating the regulation's intended effect.

1 1. Introduction 9-ending prices are common, comprising about 70% of the retail prices. They are also more rigid than other prices [START_REF] Kashyap | Sticky prices: new evidence from retail catalogues[END_REF][START_REF] Blinder | Asking about prices: A new approach to understanding price stickiness[END_REF][START_REF] Levy | Price points and price rigidity[END_REF][START_REF] Knotek | The roles of menu costs and nine endings in price rigidity[END_REF], Anderson et al 2015[START_REF] Mcshane | Decision stages and asymmetries in regular retail price pass-through[END_REF]. Their ubiquity and rigidity are explained by 9endings' positive effect on revenues [START_REF] Anderson | Effects of $9 price endings on retail sales: evidence from field experiments[END_REF], which is attributed to 9endings being psychological price points, implying that they affect shoppers' price perceptions. 1 Two leading explanations for this effect are level-effect and image-effect. 2According to the level-effect, consumers have bounded rationality and, therefore, they round prices down or process price information L-to-R, and sometimes ignore the rightmost digit [START_REF] Schindler | Patterns of right most digits used in advertised prices[END_REF][START_REF] Stiving | An empirical analysis of price-endings with scanner data[END_REF][START_REF] Thomas | Penny wise and pound foolish: the left-digit effect in price cognition[END_REF].

According to the image-effect, 9-endings signal low prices and so, consumers are drawn to goods with 9-ending prices even when other goods have lower prices (Anderson andSimester 2003, Stiving 2000).

In January 2014, the Israeli government adopted a new regulation prohibiting the use of non 0-ending prices. We take advantage of this natural experiment to study the effects of eliminating 9-ending prices on consumers' price recall and price perceptions. We combine instore posted price data from supermarkets with three consumer survey data, collected before, immediately after, and 7-months after the regulatory change. In the surveys, shoppers were asked to recall the prices they have paid, and indicate any price change they have noticed.

We find that before the regulation, (1) the most common price-ending was 9, and (2) shoppers made larger errors in recalling 9-ending prices in comparison to other prices. In addition, [START_REF] Levy | Not all price endings are created equal: price points and asymmetric price rigidity[END_REF] report that (3) the likelihood of noticing a price change was smaller for 9-ending prices than for other prices. Immediately after the regulation, shoppers' price recall accuracy improved relative to the period prior to the change. Further, although 90 became the most common price ending immediately after the change, they had no negative effect on price recall accuracy.

One year after the regulation, however, we find that (1) 90 is the most common price ending, (2) shoppers make larger errors in recalling 90-ending prices than other prices, and (3) the likelihood of noticing a price change is smaller for 90-ending prices in comparison to other prices. Thus, after the regulation, 90-ending prices seem to have influence similar to the influence 9-ending prices had before the change; 90-ending prices are the new psychological price points. We further find that the effect of 90-endings is consistent with the image-effect. [START_REF] Ater | Round prices and price rigidity: evidence from outlawing odd prices[END_REF] also use this particular episode of regulatory change in Israel.

They, however, study a theory of price rigidity, while we study shoppers' price recall and price perceptions. 3, 4 A second related study is [START_REF] Aalto-Setala | How do markets behave? The adjustment of price endings[END_REF], who studies another natural experiment, the Euro changeover (Ehrmann, 2012). He looks at the dynamic adjustment of retail price endings in Finland during and following the 2002 Euro transition.

There are important differences between the Israeli and Finish episodes, since the Euro changeover involved a currency conversion. Despite this, we find interesting similarities between the two episodes, as discussed below.

In section 2, we discuss the regulatory change. In section 3, we describe the data. In section 4, we discuss the estimation results. We conclude in section 5.

Abolition of non 0-ending prices: a natural experiment

The 0.01 New Israeli Shekel (1-agora) and the NIS 0.05 (5-agora) coins were eliminated in 1991 and 2008, respectively. They are not in use since, but until January 2014, the retailers were free to set any price ending. 5 In credit-card transactions, shoppers paid the exact amount.

In cash transactions, however, the total bill was rounded to the nearest 10-agora. For example, if the total bill was between NIS 7.45 and NIS 7.54, the actual amount paid was NIS 7.50.

The Israeli shoppers, however, felt that they were losing in cash transactions because the asymmetric rounding was tilted towards the retailers. More importantly, the public demanded to put an end to 9-ending prices because they perceived them as "unfair" and "manipulative."

In response to these public demands, the Minister of Economic Affairs announced in October 2013 that beginning January 1, 2014, all retail prices must end with 0. 6 Figure 1 illustrates the dynamic effect of the regulation by showing the distribution of 3 Ater and Gerlitz (2016) test Blinder et al.'s (1991Blinder et al.'s ( , 1998) ) theory, which argues that 9-endings form barriers to price changes, and therefore 9-ending prices will be more rigid than other prices. They compare the probability of prices to adjust before and after the regulation banning 9-endngs. Before the regulation, they find that 0-ending prices were more likely to change, in comparison to non 0-ending prices. After the regulation, these differences disappeared. These findings, they conclude, support the price point theory. 4 In addition, they use different data and different methodology. They use two large datasets: (1) Israeli Consumer Council's bimonthly price data, and (2) internet price data collected from supermarket websites. We use two small datasets: (1) handcollected, in-store, posted price data, and (2) three survey datasets which we collected by surveying supermarket shoppers, where they were asked to recall the prices they paid and indicate whether they noticed any price change (upward, downward, or no-change). 5 The coins were eliminated because of their increased cost of production. For example, by 2008 the cost of minting a 5-agora coin was 16-agorot. In addition, the public was reluctant to accept them as a change, and vending machines, parking meters, and other coin-operated devices stopped accepting them. As of January 2014, only 0-edning prices (i.e., prices that end with .10, .20,…, .90) are allowed. See: www.boi.org.il/en/NewsAndPublications/PressReleases/Pages/070716e.aspx, and www.boi.org.il/press/eng/100815/100815d.htm (both accessed February 19, 2017). 6 See: www.jpost.com/National-News/Farewell-to-the-agora-and-the-phenomenon-of-prices-that-cant-be-paid-336741 (accessed March 1, 2017). The asymmetry was caused by the rounding rule that was adopted by the Bank of Israel for the non 0-ending prices. If a total bill ended with 1, 2, 3, or 4 agorot, the price was rounded down, and if it ended with 5, 6, 7, 8, or 9 agorot, then it was rounded up. Thus, in four cases, the price was rounded down and in five cases, it was rounded up. In a survey conducted by the Israel Consumer Council as part of its project of constructing a fairness index for the retail industry, Israeli consumers ranked 9-ending prices as the most unfair practice of the food retailers, well above price mistakes, misleading promotional campaigns, lack of transparency, etc. Source: www.consumers.org.il/item/fairness_index (in Hebrew), and www.consumers.org.il/?catid=%7B36654ED8-AA18-4882-A471-BCB94C7F103B%7D (both accessed February 18, 2017).

price endings at three points in time: before the regulation was announced, immediately after it went into effect, and 7-months after it went into effect. Panel (a) shows the distribution of price endings in July 2013-August 2013, two months before the regulation was announced, and half a year before non 0-ending prices were abolished. At that time, about 60% of the prices ended in 99 and 5% in 49. In total, in that period 72% of the prices were 9-ending. The share of 90-ending prices was only about 2.3%.

Panel (b) depicts the price endings' distribution in January 2014-February 2014, immediately after the regulation went into effect. As can be seen, the share of 00-ending prices increased from 4% before the regulation, to over 23% after the regulation. The share of 90 ending prices increased from 2.3% to over 47%. Thus, it seems that following the abolition of non 0-ending prices, many 99-ending prices were converted to 90-ending prices or were rounded up to 00-ending prices. Panel (c) shows that by July 2014-August 2014, the share of 90-ending prices has reached about 55%, while the share of 00-ending prices went down to 18%.

The figures reported by [START_REF] Aalto-Setala | How do markets behave? The adjustment of price endings[END_REF] suggest that the Finish experience was similar but in the reverse direction. Before the Euro changeover, the price level in Finland was similar to the price level in Israel. Also, there were no 1-and 5-penniä coins in Finland, like in Israel.

At that time about 72% of the prices were 90-ending, and only 0.1% of the prices were 9ending, like it was in Israel after the regulatory change. Similarly, after the Euro changeover, the share of 9-ending prices has increased to about 34%, while the share of 90-ending prices has decreased from 72% to below 21%. In terms of the speed of adjustment, Finish retailers needed two years until 9-endings emerged as their favorite price point. In Israel, in contrast, it took less than 7-months, until 90-endings became the Israeli retailers' favorite price point. 7 3. Survey Data: "before," "immediately-after," and "one-year after"

We conducted three surveys: (1) in October 2013-December 2013, (2) in January 2014-March 2014, and (3) in January 2015-March 2015. 8 Shoppers exiting stores were shown a list of goods, and were asked to mark the goods they had purchased and indicate their prices. In 7 Although the conditions in both countries were similar, in Israel it was a regulatory change that made 90-endings the retailers' preferred ending, whereas in Finland, it seems, the retailers voluntarily adopted 90 as their preferred price ending. We speculate that prior to the Euro changeover, 9-endings were not that valuable because of the lower value of the Finnish currency relative to the Euro. The conversion to the Euro, then, could have made the finer level of currency divisibility more valuable, making 9-ending prices more popular among Finish retailers. The Israeli retailers, however, were able to use 9endings, to take advantage of the finer level of divisibility in their pricing, until the regulation banned it. A possible reason for the difference between Finland and Israel might be the difference in their average per capita income. It could be that in Finland, because of their higher per capita income, the relative value of penniä was not high enough for the retail sector to use it in transactions. 8 These survey dates do not coincide exactly with the three sub-periods covered by the dataset presented in Figure 1. The latter dataset, which pertains to July-August 2013, January-February 2014, and July-August 2014, was obtained from the Israel's Central Bureau of Statistics only lately, after we have completed the surveys. Because of our survey method, we do not control the survey shoppers' identity, and therefore the shoppers included in the three survey samples are different.

the third survey, in addition to the above questions, they were also asked to indicate whether in their opinion the prices have increased, decreased or remained unchanged, relative to the previous week.

The list included regularly consumed goods in 20 categories.9 Table 1 gives summary statistics for three periods: (a) before the regulation went into effect, (b) immediately after it went into effect, and (c) one year after it was in effect. Along with some socio-demographic data, Table 1 presents the shares of 9-and 90-ending prices shoppers reported, and the average absolute percentage error they made in recalling prices. The error started at 9% in October 2013-December 2013, declined to 5.7% immediately after the regulation went into effect, and rose back, reaching 10% in survey 3, one year later. The share of 9-ending prices in 2013 is only 18.6%, as retailers cut the use of 9-endings in preparation of the regulatory requirement. From January 1, 2014 and thereafter, the share of 9-ending prices is zero, as required by the regulation. Consistent with Figure 1, the share of 90-ending prices was only 12.5% before the regulation went into effect, rose to 24% immediately after it went into effect, and continued rising, reaching 50% in survey 3, one year later.

Econometric model and estimation results

Before the regulation went into effect: October 2013-December 2013

We use the data from survey 1 to assess the effect of 9-endings on shoppers' price recall. [START_REF] Thomas | Penny wise and pound foolish: the left-digit effect in price cognition[END_REF] show that shoppers process price information L-to-R and often they are inattentive to the rightmost digits. Consistent with the image-effect, [START_REF] Levy | Not all price endings are created equal: price points and asymmetric price rigidity[END_REF] find that when shoppers use price endings as a signal for low prices, they are inattentive to the price itself. We thus expect that shoppers will be less precise in recalling 9ending prices in comparison to other prices.

To assess the effect of 9-endings on shoppers' price recall, we first focus on survey 1 data, covering the period October 2013-December 2013, i.e., when non 0-ending prices were still allowed. We estimate a random effect regression, and report robust standard errors, which are clustered at the participants' level. The dependent variable is the percentage absolute error.

See columns (1)-(4) of Table 2. Column (1) reports the results when the only RHS variable is a 9-ending dummy (1 for 9-ending price, 0 otherwise). In columns (2)-(4) we add controls.

When interpreting the results, note that the dependent variable is the percentage absolute error, and therefore negative coefficients indicate better price recall.

For 9-ending prices, shoppers' price recall error is 2% higher than for other prices. The addition of control variables (column 2) does not affect the coefficient of 9-endings. 10 The 9-ending effect could be due to level-effect or due to image-effect. If it is the former, then consumers should have made larger errors also when prices ended with other non-zero endings in comparison to round endings. To test this hypothesis, we include a control for prices that end in cents other than 9 (column 3). The coefficient of 9-endings is unaffected by the inclusion of the cent-dummy. The cent-dummy is statistically insignificant. These results are consistent with the hypothesis that 9-endings influence the shoppers' price perceptions through the image-effect.

We also test for the effect of 90-endings because later we test whether 90-endings have a negative effect on shoppers' price recall after the elimination of 9-endings. It could be that such an effect was present even before 9-endings were eliminated. We therefore add to the regression both a 90-ending dummy (1 for 90-ending price, 0 otherwise) and a 0-ending dummy (1 for 0-ending price but not 90-ending, 0 otherwise). See column (4).

The coefficient of 0-ending is negative and significant, consistent with the argument that 0-ending numbers are cognitively more accessible [START_REF] Dehaene | The number sense: How the mind creates mathematics[END_REF], and thus they are recalled better than non 0-ending prices [START_REF] Schindler | Patterns of right most digits used in advertised prices[END_REF]. The coefficient of 90-endings is also negative, and its absolute value exceeds the absolute value of the coefficient of other 0endings 2 ( 2.77, 0.1) p χ = < . Thus, before 9-endings were abolished, 90-endings, if anything, had a positive effect on price recall accuracy when compared to other 0-ending prices.

Immediately after the regulation went into effect: January 2014-March 2014

In columns 5-6 of Table 2, we use the data collected in survey 1 (October 2013-December 2013) and survey 2 (January 2014-March 2014), to test whether the shoppers' price recall has changed after the regulation banning non 0-ending prices. In column 5, the only independent variable is a dummy for the period after the regulation (1 if it is after January 1, 2014, 0 otherwise). We find that after January 2014, the average error the shoppers made decreased by 3%. In column (6), we add the same controls as in column (2). The coefficient estimate of the after-january-2014 dummy is unchanged.

In column (7) we test whether 90 endings had an effect on consumers' price recall immediately after the regulatory change, given that by January 2014 the share of 90-ending prices has already increased to almost 50%. We therefore add to the regression a dummy for 10 The control variables include: the age, goods' prices, household size, number of cars owned, number of supermarkets the shoppers frequent, the average amount spent per shopping trip, and dummies for gender (1 if woman, 0 if man), marital status (1 if married, 0 otherwise), academic degree (1 if an academic degree, 0 otherwise), ultra-religious (1 if the shopper identifies himself as ultra-religious, 0 otherwise) and frequent buyer (1 if shops more than once a week, 0 otherwise). We control for ultra-religious households because they tend to have low incomes and large families. We also include dummies for the shop and for product categories. To save space, the coefficients of the latter are not reported.

90-endings and use only the data from survey 2, covering the period immediately after the regulatory change, January 2014-March 2014. The coefficient of the 90-endings dummy is negative but statistically insignificant, suggesting that shoppers made similar, or even smaller errors, when recalling 90-ending prices in comparison to other prices.

A year after the regulation went into effect: January 2015-March 2015

Next, we use the data collected in survey 3 to study what happened one year after the regulatory change. See Table 3. We estimate random effects regressions, and report robust standard errors, which are clustered at the shoppers' level. The dependent variable is the percentage absolute error. In column 1, the only independent variable is a 90-ending dummy

(1 for 90-ending price, 0 otherwise). We find that the effect of 90-endings is positive and significant. Thus, one year after 9-endings were eliminated, shoppers made larger errors in recalling 90-ending prices in comparison to other prices.

For robustness, we add the controls listed in footnote 10. See column (2). The coefficient of 90-endings remains significant, although its value drops from 4% to 3%. This point estimate is still larger than the one we find for 9-endings before the regulatory change, 2%.

Thus, our results suggest that the effect of 90-endings after the regulation was introduced, is at least as large as the effect that 9-endings had before the regulatory change.

As an additional robustness check, we use data on sales. For 5 of the 7 shops sampled, we have data on the goods that were on sale. We use these data to add a dummy variable for sales

(1 if the good was on sale, 0 otherwise). See column (3). The coefficient of the sales dummy is negative and significant, suggesting that shoppers made smaller errors in recalling the prices of goods on sale. We find, however, that the effect of 90-endings is larger with control for sales than without.

To assess whether the effect of 90-endings on price recall is because of the level-effect or the image-effect, we use data on shoppers' recall of price changes. If they make larger errors in recalling 90-ending prices because they pay attention to leftmost digits only, then 90endings should have little effect on the shoppers' recall of price changes. If they use 90endings as a signal for low prices, however, then 90-endings should reduce the likelihood of noticing price changes. [START_REF] Levy | Not all price endings are created equal: price points and asymmetric price rigidity[END_REF] show that before 9-endings were abolished, shoppers in Israel were indeed less likely to notice a price change when the new price was 9-ending than when the new price ended in a different digit. We test whether 90-endings have a similar effect after 9endings were eliminated by estimating a random effect probit regression of the probability that shoppers correctly recall changes of 9-ending prices.

We use data from the 5 shops where shoppers were asked to indicate whether goods' prices had increased, decreased or remained unchanged. The dependent variable is a dummy variable that equals 1 if the shopper responded correctly, and 0 otherwise. The shoppers recalled price changes correctly in 53% of the cases. See column (1) in Table 4.

We find that shoppers are less likely to notice a price change if the price ends with 90. If a price is not 90-ending, the probability of noticing a price change is 63%. If a price is 90ending, then the probability of noticing a price change is 41%.

We run three robustness checks. First, we add the controls listed in footnote 10 (column 2). The effect of 90-endings is still negative and significant, although its value slightly decreases. Second, we add a control for sales (column 3). The coefficient of 90-endings remains significant. Third, we add the absolute percentage price change (column 4). The size and the significance of the coefficient of 90-ending remain the same.

Conclusion

Until January 2014, Israeli retailers were free to use 9-ending prices. Since January 1, 2014, the use of 9-endings has been prohibited, bringing an end to 9-ending prices, and limiting the retailers' price-setting flexibility. We find that prior to this regulatory change, 9endings had a negative effect on shoppers' price recall. Further, we find that immediately after the regulatory change, shoppers' price recall has improved.

The new regulation has accomplished one of its goals, i.e., the elimination of the asymmetric rounding of cash transaction bills, which favored the retailers. The outlawing of 9-ending prices, however, failed to accomplish its second goal, which was to stop the manipulative pricing which the public attributed to the frequent use of 9-endings by the retailers. One year after the regulatory change, 90-endings have "turned into" the new 9endings, in the sense that they seem to have similar manipulative effects on the shoppers. We find that 90-endings have acquired the status of 9-endings through the image-effect, suggesting that 90-ending prices are popular in the new setting because 90-endings are used by shoppers as a signal for low prices. That is, 90-ending prices are the new psychological price points.

These findings confirm once again that the market reacts to regulatory changes. The Israeli retailers had a preferred price ending, 9, but it was made illegal by regulators. The retailers' response was to find a new price ending, 90, which plays the same role. The shoppers benefited from the use of 9-endings as a signal for low prices until the regulatory change. After the change, all prices end with 0, eliminating the value of 0-endings as a signal.

The retail prices have converged to 90-endings, to which shoppers responded by ignoring the 0-endings and treating the 90-ending prices as 9-ending. The end result is that the retailers' and the shoppers' reaction to the regulatory intervention has partially eliminated the regulation's intended effect.

Fig. 1 .

 1 Fig. 1. The distributions of price-endings: (a) July-August 2013 (n = 3,211), (b) January-February 2014 (n = 3,178), and (c) July-August 2014 (n = 1,548) Notes: 1. The figure uses monthly price data that are collected regularly by the Israel's Central Bureau of Statistics for compiling the CPI. 2. The data include the prices of goods in 103 product categories, collected at supermarkets and drugstores in Israel.

Table 1

 1 Summary statistics: survey 1 (before the regulatory change), survey 2 (immediately after the regulatory change) and survey 3 (One-year after the regulatory change)

		(a)	(b)	(c)
		Survey 1	Survey 2	Survey 3
		Oct. 2013-Dec. 2013	Jan. 2014-March 2014	Jan. 2015-March 2015
		"Before"	"Immediately after"	"One-year after"
	Age	39.16	37.036	44.26
		(17.034)	(15.058)	(14.112)
	Household size	2.73	2.88	3.79
		(1.638)	(1.668)	(1.707)
	% of Women	52%	49%	56%
	% of Academic	40%	37%	39%
	% of Married	58%	54%	71%
	% of Ultra-religious	2%	3%	4%
	No. of cars owned	1.20	1.21	1.41
		(0.827)	(0.763)	(0.793)
	No. of shops visited	1.53	1.43	1.92
		(0.873)	(0.765)	(1.020)
	Average amount spent	340.59	384.97	446.48
	per shopping trip	(223.404)	(216.605)	(222.681)
	% of shopping more	37%	42%	12%
	than once a week
	Average recall error	8.94%	5.69%	10.05%
	% of 9-ending prices	18.64%	0.01%	N/A
	% of 90-ending prices	12.47%	24.05%	47%
	Responses (no. of	6.41	5.77	5.22
	products purchased per
	shopper)
	No. of shops	13	13	7
	No. of shoppers	364	740	513

Table 2

 2 Regression of the price recall error before and immediately after the regulatory change

			Before the regulatory		Immediately after the regulatory
			change			change
		(October 2013-December 2013)	(January 2014-March 2014)
	Variable	(1)	(2)	(3)	(4)	(5)	(6)	(7)
	9-ending	0.02***	0.02***	0.02**			0.01
		(0.007)	(0.005)	(0.005)			(0.006)
	Good's price		-0.001***	-0.001***	-0.001***		-0.002***	-0.002***
			(0.0003)	(0.0003)	(0.0003)		(0.0004)	(0.0004)
	Age		0.0001	0.0001	0.0001		-0.0002	-0.0002
			(0.0003)	(0.0003)	(0.0003)		(0.0001)	(0.0001)
	Household		0.004	0.004	0.004		0.002	-0.0004
	size		(0.003)	(0.003)	(0.003)		(0.002)	(0.002)
	No. of cars		-0.005	-0.005	-0.004		-0.0001	0.006*
			(0.004)	(0.004)	(0.004)		(0.003)	(0.003)
	No. of		-0.002	-0.002	-0.002		-0.0008	-0.002
	supermarkets		(0.005)	(0.005)	(0.0005)		(0.003)	(0.003)
	visited
	Average		0.00003	0.00003	0.00003		-0.00006	-0.002
	amount spent		(0.00002)	(0.00002)	(0.00002)		(0.002)	(0.002)
	Woman		0.01	0.01	0.01		0.006	0.005
			(0.010)	(0.010)	(0.010)		(0.004)	(0.004)
	Married		-0.02*	-0.02*	-0.02*		-0.01**	-0.006
			(0.010)	(0.010)	(0.010)		(0.004)	(0.004)
	Academic		0.001	0.001	0.001		-0.004	-0.003
	degree		(0.008)	(0.007)	(0.007)		(0.003)	(0.003)
	Ultra-		-0.02**	-0.03**	-0.03**		-0.01	-0.0004
	religious		(0.011)	(0.012)	(0.012)		(0.011)	(0.013)
	Frequent-		-0.005	-0.005	-0.005		0.001	0.006
	shopper		(0.008)	(0.008)	(0.008)		(0.003)	(0.004)
	Cent ending			0.01
				(0.007)
	90-ending				-0.02***			-0.002
					(0.005)			(0.004)
	0-ending				-0.01*
					(0.006)
	After January					-0.03***	-0.03***
	2014					(0.005)	(0.004)
	Constant	0.08***	0.06***	0.005**	0.06***	0.09***	0.09***	0.08***
		(0.004)	(0.020)	(0.021)	(0.020)	(0.004)	(0.009)	(0.009)
	n	2,333	2,333	2,333	2,333	6,892	6,892	4,378
	χ 2	15.97	1645.06	1930.80 2580.87	38.7	3848.7	16,910.3
	Notes:
	1. The table reports the results of a random effects regression. The dependent variable is the percentage absolute error in price
	recall.

2. The standard errors are robust, and they are clustered at the participants' level. 3. ***, **, and * indicate significance at 1%, 5%, and 10%, respectively.

Table 3

 3 Regression of the price recall error one year after the regulatory change, In the table, we report the results of a random effects regression. The dependent variable is a % absolute error in price recall. 2. We report robust standard errors, which are clustered at the participants' level. 3. ***, **, and * indicate significance at 1%, 5%, and 10%, respectively

	January 2015-March 2015
	Variable	(1)	(2)	(3)
	90-ending	0.04***	0.03**	0.06***
		(0.010)	(0.02)	(0.023)
	Good's price		0.0008	0.0009
			(0.0008)	(0.001)
	Age		-0.0003	-0.0006
			(0.0003)	(0.0005)
	Household size		0.006	0.006
			(0.004)	(0.005)
	No. of cars		0.02**	0.02**
			(0.008)	(0.009)
	No. of supermarkets		0.0006	-0.00007
	visited		(0.007)	(0.008)
	Average amount spent		-0.00007*	-0.00007*
			(0.00004)	(0.00004)
	Woman		0.004	0.004
			(0.004)	(0.01)
	Married		-0.0009	-0.007
			(0.010)	(0.014)
	Academic degree		-0.002)	-0.01
			(0.009)	(0.013)
	Ultra-religious		0.009	0.02
			(0.027)	(0.03)
	Frequent shopper		-0.02	-0.01)
			(0.014)	(0.02)
	Sales			-0.03
				(0.012)
	Constant	0.08***	-0.08**	-0.07*
		(0.008)	(0.035)	(0.044)
	n	2,587	2,587	1,855
	χ 2	15.67	293.90	215.68
	Notes:
	1.

Table 4

 4 Regression of the probability that shoppers notice a price change one year after the regulatory change, January 2015-March 2015 In the table, we report the results of a random effects probit regression. The dependent variable is a dummy variable that equals 1 if the consumer responded correctly (and 0 otherwise) whether the price has increased, decreased, or remained unchanged. 2. We report robust standard errors, which are clustered at the participants' level. 3. ***, **, and * indicate significance at 1%, 5%, and 10%, respectively

	Variable	(1)	(2)	(3)	(4)
	90-ending	-0.56***	-0.25***	-0.32***	-0.37***
		(0.068)	(0.077)	(0.107)	(0.106)
	Good's price		-0.01**	-0.01	-0.01
			(0.006)	(0.007)	(0.007)
	Age		-0.005	-0.005	-0.002
			(0.003)	(0.004)	(0.004)
	Household size		0.03	0.04	0.05
			(0.029)	(0.036)	(0.031)
	No. of cars		0.08	-0.12	-0.08
			(0.062)	(0.078)	(0.068)
	No. of supermarkets		-0.11**	-0.15**	-0.11*
	visited		(0.055)	(0.068)	(0.067)
	Average amount spent		0.0002	0.0003	0.0002
			(0.0002)	(0.0003)	(0.00003)
	Woman		-0.09	-0.14	-0.11
			(0.079)	(0.101)	(0.088)
	Married		-0.11	-0.29**	-0.28**
			(0.094)	(0.125)	(0.108)
	Academic degree		-0.01	-0.002	0.08
			(0.085)	(0.111)	(0.095)
	Ultra-religious		-0.28	-0.29	-0.29
			(0.196)	(0.22)	(0.195)
	Frequent shopper		0.02	0.04	-0.06
			(0.0129)	(0.177)	(0.151)
	Sales			-1.16***	-1.10***
				(0.171)	(0.173)
	Size of price change				0.23
					(0.395)
	Constant	0.34***	0.91***	1.00***	1.04***
		(0.065)	(0.253)	(0.328)	(0.286)
	n	2,191	2,191	1,441	1,373
		67.62	386.64	272.34	227.58
	Notes:
	1.

According to[START_REF] Landsburg | The armchair economist[END_REF], dollar-store owners in the late 19 th century began using 99¢ pricing to make it harder for cashiers to steal, and thus inadvertently discovered the benefits of 9-ending prices as psychological price points.

In the literature, level-effect and image-effect are sometimes described as left-digit effect and right-digit effect, respectively.

The products include: cottage cheese, soft cheese, semi-hard cheese, fresh milk, chocolate drink, Coca-Cola, Diet Coke, mineral water, tomatoes, cucumbers, oranges, lemons, chocolate spread, dark chocolate, peanut flavor snack, pastrami, eggs, white sugar, margarine and sunflower oil.


Chames Kermi

Susana Prieto

Siem Van Der Laan

Nikolay Tsanov

Bénédicte Recolin

Emmanuelle Uro-Coste

Bernadette Delisle

Domenico Maiorano
email: domenico.maiorano@igh.cnrs.fr

Rad18 is a maternal limiting factor silencing the UV-dependent DNA damage checkpoint in Xenopus embryos

In early embryos the DNA damage checkpoint is silent until the midblastula transition (MBT) due to maternal-limiting factors of unknown identity. Here, we identify the Rad18 ubiquitin ligase as one such factor in Xenopus. We show, in vitro and in vivo, that inactivation of Rad18 function leads to DNA damagedependent checkpoint activation, monitored by Chk1 phosphorylation. Moreover, we show that the abundance of both Rad18 and PCNA monoubiquitylated (mUb) are developmentally-regulated. Increased DNA abundance limits availability of Rad18 close to MBT thereby reducing PCNA mUb and inducing checkpoint derepression. Further, we show that this embryonic-like regulation can be reactivated in somatic mammalian cells by ectopic Rad18 expression thus conferring resistance to DNA damage. Finally, we find high Rad18 expression in cancer stem cells highly resistant to DNA damage. Altogether these data propose Rad18 as a critical embryonic checkpoint-inhibiting factor and suggest that Rad18 deregulation may have an unexpected oncogenic potential.

Introduction

Early embryonic cleavages are rapid, consisting of alternating S-and M-phases, with virtually absent Gap-phases [START_REF] Graham | Changes in the cell cycle during early amphibian development[END_REF]. In this contracted cell cycle, the S-phase checkpoint delaying cell division upon DNA damage [START_REF] Anderson | Ionizing radiation induces apoptosis and elevates cyclin A1-Cdk2 activity before but not after the midblastula transition in Xenopus[END_REF][START_REF] Hensey | A developmental timer that regulates apoptosis at the onset of gastrulation[END_REF] or unreplicated DNA [START_REF] Dasso | Completion of DNA replication is monitored by a feedback system that controls the initiation of mitosis in vitro: studies in Xenopus[END_REF][START_REF] Kimelman | The events of the midblastula transition in Xenopus are regulated by changes in the cell cycle[END_REF] is inefficient and may represent an adaptation to ensure rapid proliferation. The molecular mechanisms responsible for checkpoint inhibition in early embryos are poorly understood. Previous studies in Xenopus [START_REF] Conn | The DNA damage checkpoint in embryonic cell cycles is dependent on the DNA-to-cytoplasmic ratio[END_REF][START_REF] Dasso | Completion of DNA replication is monitored by a feedback system that controls the initiation of mitosis in vitro: studies in Xenopus[END_REF][START_REF] Kappas | Dissection of the XChk1 signaling pathway in Xenopus laevis embryos[END_REF] have shown that checkpoint activation depends upon the nuclear-to-cytoplasmic (N/C) ratio, due to absence of cell growth, and not upon transcription nor translation, suggesting titration of maternal limiting factors of unknown identity. Genetic data in C. elegans [START_REF] Holway | Checkpoint silencing during the DNA damage response in Caenorhabditis elegans embryos[END_REF][START_REF] Ohkumo | Deficiency of the Caenorhabditis elegans DNA polymerase eta homologue increases sensitivity to UV radiation during germ-line development[END_REF] have implicated a translesion DNA polymerase (TLS Pol) specialized in replication of damaged DNA (Sale et al., 2012, for review). Pol is recruited to DNA damage upon binding to PCNA, monoubiquitylated by the Rad6 (E2)-Rad18(E3) ubiquitin ligase complex while the USP1 ubiquitin hydrolase catalyzes the opposite reaction (Ulrich and Takahashi, 2013, for review). In S-phase, checkpoint activation relies upon replication fork uncoupling generated by DNA damage such as UV-irradiation. Excess singlestranded (ss)DNA, generated in this process by the action of the helicase, is the primary substrate initiating ATR-dependent checkpoint signaling [START_REF] Byun | Functional uncoupling of MCM helicase and DNA polymerase activities activates the ATR-dependent checkpoint[END_REF]. Here we provide evidence that checkpoint repression in Xenopus eggs is a consequence of replication fork uncoupling inhibition mediated by Rad18, a critical factor for PCNA mUb .

We also show that this regulation is reversible and can be reactivated by increasing Rad18 abundance, resulting in resistance to DNA damaging agents that is relevant to cancer recurrence.

Results and Discussion

Constitutive TLS Pol binding to chromatin at low N/C ratio

To understand the molecular grounds of embryonic checkpoint silencing, we used cellfree extracts derived from activated Xenopus eggs. This in vitro system faithfully reproduces the developmentally-regulated activation of the DNA damage checkpoint observed in vivo [START_REF] Anderson | Ionizing radiation induces apoptosis and elevates cyclin A1-Cdk2 activity before but not after the midblastula transition in Xenopus[END_REF][START_REF] Conn | The DNA damage checkpoint in embryonic cell cycles is dependent on the DNA-to-cytoplasmic ratio[END_REF][START_REF] Kappas | Dissection of the XChk1 signaling pathway in Xenopus laevis embryos[END_REF][START_REF] Newport | On the coupling between DNA replication and mitosis[END_REF]. This is achieved by adding a sufficient amount of sperm nuclei into a fixed volume of egg cytoplasm thus reaching a critical N/C ratio that triggers checkpoint activation (400 nuclei/l, [START_REF] Dasso | Completion of DNA replication is monitored by a feedback system that controls the initiation of mitosis in vitro: studies in Xenopus[END_REF].

Figure S1A-C shows that UV-irradiated sperm nuclei, added at low N/C ratio into egg extracts naturally synchronized in very early S-phase, fail to delay both DNA synthesis and mitotic entry, compared to high N/C ratio. Consistent with previous observations in vivo [START_REF] Conn | The DNA damage checkpoint in embryonic cell cycles is dependent on the DNA-to-cytoplasmic ratio[END_REF][START_REF] Kappas | Dissection of the XChk1 signaling pathway in Xenopus laevis embryos[END_REF], we did not observe Chk1 phosphorylation at low N/C ratio (Figure 1A, upper panel, lane 2) while this occurred normally at high N/C ratio, as expected [START_REF] Kumagai | The Xenopus Chk1 protein kinase mediates a caffeine-sensitive pathway of checkpoint control in cell-free extracts[END_REF]. Inhibition of Chk1 phosphorylation was also observed using a fixed amount of damaged sperm nuclei while increasing the extract volume (Figure S1D), thus strengthening the conclusion that checkpoint activation is sensitive to the N/C ratio and not to the total amount of DNA damage.

Using as readout for replication fork uncoupling the ssDNA binding protein RPA [START_REF] Recolin | Role of replication protein A as sensor in activation of the S-phase checkpoint in Xenopus egg extracts[END_REF][START_REF] Walter | Initiation of eukaryotic DNA replication: origin unwinding and sequential chromatin association of Cdc45, RPA, and DNA polymerase alpha[END_REF] we observed that RPA greatly accumulated onto chromatin in S-phase at high N/C ratio upon UV-irradiation, as expected, (Figure 1A; lower panel, lane 4), while RPA accumulation was strongly reduced at low N/C ratio (lane 2), suggesting inefficient replication fork uncoupling. This is consistent with previous observations in C. elegans embryos [START_REF] Holway | Checkpoint silencing during the DNA damage response in Caenorhabditis elegans embryos[END_REF][START_REF] Ohkumo | Deficiency of the Caenorhabditis elegans DNA polymerase eta homologue increases sensitivity to UV radiation during germ-line development[END_REF] as well as with reduced production of ssDNA in human embryonic stem cells [START_REF] Desmarais | Human Embryonic Stem Cells Fail to Activate CHK1 and Commit to Apoptosis in Response to DNA Replication Stress[END_REF]. In addition, UV-dependent accumulation of the ATR-Interacting Protein (ATRIP), recruited by RPA and required for checkpoint signaling, was also strongly abolished, while it was recruited normally at high N/C ratio.

At low N/C ratio, ATR was bound to chromatin and showed modest accumulation upon UV-irradiation, similar to ATRIP. Efficient replication fork uncoupling is observed at low N/C ratio by blocking DNA synthesis with aphidicolin, an inhibitor of replicative DNA polymerases (Figure S1E), suggesting that the uncoupling defect is specific to UVdamage. Interestingly, at low N/C ratio we observed that Pol is chromatin-associated with or without DNA damage (Figure 1A, lower panel, lanes 1-2). In contrast, at high N/C ratio Pol was recruited only after UV-irradiation (lanes 3-4). We have also verified the presence of replicative polymerases on chromatin at low N/C ratio (Figure S1F) and observed that Pol abundance is similar to that of Pol (Figure S1G). Strikingly, at low N/C ratio, PCNA mUb was observed on chromatin irrespective of DNA damage (Figure 1A, lanes 1-2), while at high N/C ratio PCNA mUb was present mainly upon UV irradiation (lane 4, lower panel), as previously reported (Chang et al., 2006). Damage-independent Pol recruitment at low N/C ratio was reduced by addition of Geminin, an inhibitor of replication fork formation (Figure S1H), suggesting replication fork dependency for binding. Since TLS Pol replicates past UV lesions, constitutive Pol binding may avoid forks stalling by UV lesions, thus preventing replication fork uncoupling and ssDNA formation. In turn, this leads to failure to recruit checkpoint factors (ATRIP) and precludes checkpoint activation. Constitutive TLS in early embryos may be important to tolerate not only external damage but also endogenous replication stress induced by high concentration of nucleotides. This possibility is in line with evidence suggesting that rNTPs incorporation causes replication stress and induces PCNA mUb in yeast, and that TLS activity is required for replication resumption [START_REF] Lazzaro | RNase H and postreplication repair protect cells from ribonucleotides incorporated in DNA[END_REF].

Rad6-Rad18 and not Pol is titrated from egg cytosol at high N/C ratio

Maternally-supplied inhibitor(s), present in limited amount in egg cytoplasm and progressively titrated on chromatin during embryonic cleavages may be responsible for checkpoint silencing [START_REF] Conn | The DNA damage checkpoint in embryonic cell cycles is dependent on the DNA-to-cytoplasmic ratio[END_REF][START_REF] Dasso | Completion of DNA replication is monitored by a feedback system that controls the initiation of mitosis in vitro: studies in Xenopus[END_REF]Kappas et al., 2000, and Graphical Abstract). Data shown in Figure 1A, and previous data in C. elegans [START_REF] Holway | Checkpoint silencing during the DNA damage response in Caenorhabditis elegans embryos[END_REF][START_REF] Ohkumo | Deficiency of the Caenorhabditis elegans DNA polymerase eta homologue increases sensitivity to UV radiation during germ-line development[END_REF], implicate TLS components. We analyzed the abundance of several TLS factors remaining in the cytoplasm after incubation with sperm nuclei at low or high N/C ratio and observed that USP1, Pol, PCNA, RPA, Chk1, and ATR levels did not change (Figure 1B and Figure S1I), suggesting that they are in excess over the DNA. By raising specific antibodies (Figure S2A), we observed that Rad6 and Rad18 were depleted from the extract and less abundant on chromatin at high N/C ratio (Figure 1B). Reduced Rad18 and Rad6 chromatin binding at high N/C ratio correlates with both reduced PCNA mUb and Pol chromatin binding in the absence of UV-damage, while USP1 binding did not significantly change, suggesting that Rad18 may be limiting near MBT. To investigate whether this is due to titration or destabilization, we analyzed Rad18 binding to chromatin at increasing N/C ratios, with or without the proteasome inhibitor MG132 (Figure S2B, upper panel) and observed a gradual decline in both conditions suggesting titration, although MG132 increased Rad18 abundance at high N/C ratio. Moreover, kinetic analysis of chromatin binding in vitro shows that Rad18 is absent from chromatin at the end of S-phase (Figure S2B, lower panel), while in presence of MG132 its abundance is increased, suggesting destabilization after replication. Altogether these results suggest that both titration and destabilization limit the availability of Rad18 at high N/C ratio.

In mammalian cells, Rad18 is recruited to chromatin upon DNA damage by physical interactions with the Dbf4 subunit of the Cdc7 protein kinase [START_REF] Yamada | ATR-Chk1-APC/CCdh1-dependent stabilization of Cdc7-ASK (Dbf4) kinase is required for DNA lesion bypass under replication stress[END_REF]. Interestingly, immunoprecipitation experiments show complex formation between Rad18 and the Xenopus Dbf4-related protein Drf1 at low N/C ratio, in the absence of damage (Figure 1C). This complex was virtually undetectable at high N/C ratio, even when an excess of Rad18 immunoprecipitates, compared to low N/C ratio, were analyzed (Figure 1C, right panel). This observation suggests that at low N/C ratio high Rad18 abundance promotes DNA damage-independent complex formation with Drf1, to constitutively target Rad18 to replication forks.

Rad18 and PCNA mUb are developmentally-regulated

Analysis of Rad18 and PCNA mUb abundance in embryos at different stages of development shows that Rad18 decreases during embryogenesis, starting from stage 4, and drops to very low levels at stage 6.5 (pre-MBT, Figure 1D). Rad18 decline is paralleled by a correspondent decrease in PCNA mUb . Similar to what observed in vitro (Figure S2B), injection of MG132 into embryos, at a dose that does not interfere with the timing of MBT onset [START_REF] Brandt | Developmental downregulation of Xenopus cyclin E is phosphorylation and nuclear import dependent and is mediated by ubiquitination[END_REF], did not affect the decline of Rad18 levels prior to stage 6, although it significantly increased Rad18 abundance at stage 6.5 (Figure 1E). This result suggests that in vivo both titration and destabilization limit Rad18 abundance near MBT. In contrast, Drf1 did not show significant changes up to stage 7, similar to what previously reported [START_REF] Collart | Titration of four replication factors is essential for the Xenopus laevis midblastula transition[END_REF][START_REF] Takahashi | Cdc7-Drf1 is a developmentally regulated protein kinase required for the initiation of vertebrate DNA replication[END_REF], suggesting that Rad18 is more limiting than Drf1. This possibility is supported by the observation that Drf1 is not depleted from egg cytoplasm at high N/C ratio (Figure S1I) and is consistent with Drf1 titration at a higher N/C ratio (around 3000 nuclei/l, [START_REF] Collart | Titration of four replication factors is essential for the Xenopus laevis midblastula transition[END_REF], while onset of the DNA damage checkpoint occurs at 400 nuclei/l [START_REF] Conn | The DNA damage checkpoint in embryonic cell cycles is dependent on the DNA-to-cytoplasmic ratio[END_REF][START_REF] Dasso | Completion of DNA replication is monitored by a feedback system that controls the initiation of mitosis in vitro: studies in Xenopus[END_REF][START_REF] Kappas | Dissection of the XChk1 signaling pathway in Xenopus laevis embryos[END_REF]. Hence, reduced Rad18 abundance and not Drf1 is likely responsible for DNA damage-dependent checkpoint activation, although it cannot be excluded that titration of Rad18 stabilizing factor(s) may also contribute. Quantification of Rad18 shows that its concentration in Xenopus eggs is relatively low (~ 0.25 ng/embryo, 3.5 nM; Figure S2C), over one thousand times less than PCNA. Genetic evidence in C. elegans proposed Pol as a repressor of the checkpoint [START_REF] Holway | Checkpoint silencing during the DNA damage response in Caenorhabditis elegans embryos[END_REF][START_REF] Ohkumo | Deficiency of the Caenorhabditis elegans DNA polymerase eta homologue increases sensitivity to UV radiation during germ-line development[END_REF][START_REF] Roerink | A broad requirement for TLS polymerases eta and kappa, and interacting sumoylation and nuclear pore proteins, in lesion bypass during C. elegans embryogenesis[END_REF]. Although we find that in Xenopus Pol is implicated, it is not limiting since it is not quantitatively depleted at high N/C ratio. We speculate that at high N/C ratio, reduced abundance of Rad18 may be counteracted by USP1 resulting in reduced PCNA mUb .

Rad6-Rad18 inhibits the UV-dependent DNA damage checkpoint in vitro at low N/C ratio

We next removed Rad18 from egg extracts using specific antibodies. As expected [START_REF] Bailly | Specific complex formation between yeast RAD6 and RAD18 proteins: a potential mechanism for targeting RAD6 ubiquitin-conjugating activity to DNA damage sites[END_REF], Rad18 depletion also partially removed Rad6 (Figure 2A, lane 2; Figure S2D), but not Pol, RPA, or PCNA. Rad18 depletion drastically reduced PCNA mUb upon UV-irradiation at low N/C ratio, as well as Pol chromatin binding, and importantly, induced UV-damage-dependent Chk1 phosphorylation (Figure 2B, lane 3).

In contrast, Rad18 depletion at high N/C ratio did not induce checkpoint hyper activation compared to a mock-depletion (Figure S2E), indicating that this phenotype is specific to low N/C ratio, and likely it is not due to accumulation of unrepaired DNA as neither Rad6 nor Rad18 are required for Nucleotide Excision Repair [START_REF] Hishida | RAD6-RAD18-RAD5-pathway-dependent tolerance to chronic low-dose ultraviolet light[END_REF].

Moreover, pre-and post-MBT embryos appear to have similar DNA repair capacity [START_REF] Anderson | Ionizing radiation induces apoptosis and elevates cyclin A1-Cdk2 activity before but not after the midblastula transition in Xenopus[END_REF]. Further, the N/C ratio, and not the total amount of damaged DNA, is critical for checkpoint activation [START_REF] Conn | The DNA damage checkpoint in embryonic cell cycles is dependent on the DNA-to-cytoplasmic ratio[END_REF], ruling out differences in DNA repair rates.

Reconstitution of Rad18-depleted extracts at low N/C ratio (Figure S2F) with a recombinant 6His-Rad6-Rad18 complex (Figure 2C) inhibited UV-damage-dependent Chk1 phosphorylation (Figure 2D, lane 5), excluding the implication of co-depleted proteins. Further, this complex, and not recombinant Rad6 rescued defective PCNA mUb in Rad18-depleted extracts, demonstrating that it is functional (Figure S2G). Furthermore, Chk1 phosphorylation was induced by UV-damage when recombinant PCNA K164R mutant that cannot be monoubiquitylated, and not wild-type PCNA (WT), was added to extracts at low N/C ratio (Figure S2H). Of note, the recently discovered Primpol (Helleday, 2013, for review), was not bound to chromatin at low N/C ratio (Figure S2I), ruling out active UV lesions bypass, or replication fork restart by this polymerase. Finally, recombinant 6His-Rad6-Rad18 repressed both RPA accumulation and Chk1 phosphorylation normally observed at high N/C ratio upon UV-irradiation (Figure 2E, compare lanes 2 and 3), suggesting inhibition of replication fork uncoupling.

Rad18 silences the UV-dependent DNA damage checkpoint in Xenopus embryos

To obtain evidence for checkpoint inhibition by Rad18 in vivo, we overexpressed either Rad18 WT or catalytic inactive Rad18 (C28F mutant) by microinjection of the corresponding mRNA into embryos at the 2-cell stage. Embryos injected with either water (Mock) or Rad18 WT mRNA developed normally and reached stage 6.5 with or without UV-irradiation (Figure 2F). In contrast, UV-irradiated embryos injected with Rad18 C28F were delayed in the embryonic cleavages from one cell cycle after UVirradiation (Figure S2J). Consistent with this phenotype, UV-irradiated embryos expressing the Rad18 C28F mutant accumulated Chk1 phosphorylation while expression of Rad18 WT inhibited it (Figure 2G, compare lane 3 with lane 4). Importantly, no spontaneous Chk1 phosphorylation was observed in embryos injected with either Rad18 mRNAs (Figure S2K, -UV) showing that Rad18 is not implicated in DNA damage-independent developmental activation of Chk1, as reported for Drf1 [START_REF] Collart | Titration of four replication factors is essential for the Xenopus laevis midblastula transition[END_REF]. These data altogether show that Rad18 inhibition is sufficient to give to the embryo the competence to activate the DNA damage checkpoint. Since Rad18 is also implicated in double strand break repair (Huang et al., 2009;[START_REF] Szuts | Role for RAD18 in homologous recombination in DT40 cells[END_REF][START_REF] Watanabe | RAD18 promotes DNA double-strand break repair during G1 phase through chromatin retention of 53BP1[END_REF], it might also contribute to silencing the checkpoint upon -irradiation, although we have not tested this possibility.

Reactivation of embryonic-like checkpoint silencing in mammalian cells by Rad18 upregulation

Next we analyzed the consequences of increasing Rad18 abundance in somatic mammalian cells. Rad18 overexpression did not induce significant cell cycle changes (Figure S3A), and consistent with two previous reports [START_REF] Bi | Rad18 regulates DNA polymerase kappa and is required for recovery from S-phase checkpoint-mediated arrest[END_REF][START_REF] Davies | Activation of ubiquitindependent DNA damage bypass is mediated by replication protein a[END_REF] induced constitutive PCNA mUb (Figure 3A). Importantly, overexpression of either Rad6, or Rad18 and Rad6, was not sufficient to induce constitutive PCNA mUb to a level similar to that of Rad18 alone (Figure S3B). Of note, and unlike what observed in Xenopus at low N/C ratio (Figure 1A), the amount of PCNA mUb observed in asynchronous cells expressing Rad18, increased after UV irradiation. Moreover, eGFP-Pol nuclear foci formed even in the absence of DNA damage only upon Rad18 overexpression (Figure 3B-C). Most importantly, UV-dependent Chk1 phosphorylation was significantly reduced in asynchronous cells expressing Rad18 (Figure 3D; Figure S3D) suggesting that in mammalian cells high Rad18 abundance is sufficient to inhibit UV-dependent checkpoint activation, in line with a previous observation in yeast [START_REF] Daigaku | Ubiquitin-dependent DNA damage bypass is separable from genome replication[END_REF]. A very similar result was obtained upon expression of PCNA K164R fused to ubiquitin (Figure 3E-F) that mimics constitutive PCNA mUb (Kanao et al., 2015).

Moreover, expression of Rad18 lacking Cdc7 phosphorylation sites (Rad18 ∆401-445 ), also required for Pol binding [START_REF] Durando | A non-catalytic role of DNA polymerase eta in recruiting Rad18 and promoting PCNA monoubiquitination at stalled replication forks[END_REF], did not induce constitutive eGFP-Pol nuclear foci and acts as a dominant negative since it inhibits eGFP-Pol foci formation after UV-damage (Figure S3C), consistent with a previous report [START_REF] Day | Phosphorylated Rad18 directs DNA polymerase eta to sites of stalled replication[END_REF]. This mutant did not inhibit Chk1 phosphorylation compared to Rad18 WT (Figure S3D), suggesting that checkpoint silencing depends upon Rad18 phosphorylation by Cdc7.

Similarly, the TLS-deficient Rad18 C28F mutant did not induce constitutive eGFP-Pol nuclear foci (Figure S3E). Finally, and entirely consistent with results in Xenopus (Figure 1A), high Rad18 expression also strongly repressed RPA foci formed upon UV irradiation in mammalian cells (Figure 4A), suggesting inhibition of replication fork uncoupling. Altogether these observations show that Rad18 overexpression is sufficient to induce constitutive PCNA mUb independently of Rad6, although we could not formally prove it in Xenopus, since we failed to express active recombinant Rad18 without Rad6. This suggests that when overexpressed Rad18 may either bypass the Rad6 requirement, or Rad18 may use another abundant E2 to catalyze PCNA mUb . This latter possibility may explain why in C. elegans Rad6 mutations did not delay mitotic entry in early embryos upon DNA damage [START_REF] Holway | Checkpoint silencing during the DNA damage response in Caenorhabditis elegans embryos[END_REF].

We next determined whether cells expressing Rad18 display increased resistance to DNA damage resulting from impaired checkpoint activation. To this end, we generated stable cell lines expressing ectopic Rad18 at a similar level to endogenous Rad18. Expression of Rad18 WT significantly increased cell viability upon exposure to either UV-irradiation or to the chemotherapy-relevant drug cisplatin, while Rad18 C28F did not (Figure 4B-C). Taken together these results link Rad18 expression to checkpoint inactivation and resistance to DNA damaging agents.

Rad18 is overexpressed in cancer stem cells highly resistant to DNA damage

Resistance to DNA damaging agents is linked to cancer recurrence. We observed high Rad18 expression in a colon cancer-derived cell line resistant to oxaliplatin (HCT116) compared to the HCT8 sensitive cancer cell line (Figure S4A), as well as in the highly DNA damage-resistant brain cancer glioblastoma (Figure 4D). Importantly, we observed high Rad18 expression specifically in glioblastoma cancer stem cells isolated from tumor biopsies (CD133 + ), and not in their differentiated counterparts that express Rad18 to similar levels than HeLa cells (Figure 4E). In contrast expression of Rad6 and of other TLS-, checkpoint-and proliferation-relevant proteins was not increased in glioblastoma (Figure S4B). This result is consistent with GEO Profiles data showing high Rad18 mRNA expression in glioblastoma cancer stem cells. Moreover, very recent data implicate Rad18 in therapeutic resistance of colon cancer cells [START_REF] Liu | Tumor suppressor miR-145 reverses drug resistance by directly targeting DNA damage-related gene RAD18 in colorectal cancer[END_REF].

Further, Rad18 downregulation in the U87 glioblastoma cell line induced sensitivity to cisplatin (Figure 4F) while Rad18 re-expression induced a dramatic increased viability, suggesting acquired resistance. Since glioblastoma is resistant to cisplatin, this observation puts forward Rad18 as a target for sensitizing glioblastoma to cisplatin.

Altogether our findings suggest that increased Rad18 expression has a positive effect on proliferation upon DNA damage by shunting checkpoint activation thus conferring resistance to DNA damage (see Graphical abstract), and show high Rad18 expression specifically in cancer stem cells that are implicated in the resistance to therapy.

In conclusion, this work suggests that constitutive PCNA mUb , driven by Rad18, is responsible for silencing the UV-damage checkpoint in Xenopus embryos by inhibiting replication fork uncoupling, a critical determinant for checkpoint signaling. Genetic data in C. elegans [START_REF] Holway | Checkpoint silencing during the DNA damage response in Caenorhabditis elegans embryos[END_REF][START_REF] Ohkumo | Deficiency of the Caenorhabditis elegans DNA polymerase eta homologue increases sensitivity to UV radiation during germ-line development[END_REF], and the presence of constitutive PCNA mUb in early Drosophila embryos (our unpublished observation), makes likely that this regulation may be conserved in other organisms. Recent data suggest that the DNA damage checkpoint affects TLS through regulation of a Rad18-Cdc7-Dbf4 complex [START_REF] Yamada | ATR-Chk1-APC/CCdh1-dependent stabilization of Cdc7-ASK (Dbf4) kinase is required for DNA lesion bypass under replication stress[END_REF]. Our observations show that Rad18 deregulation affects the DNA damage checkpoint, suggesting a cross talk between these two pathways. This may constitute an additional mechanism, aside from the mutator activity, linking TLS deregulation to cancer [START_REF] Albertella | The overexpression of specialized DNA polymerases in cancer[END_REF]. In this perspective, Rad18 deregulation might have a previously unrecognized oncogenic potential relevant to the therapeutic resistance of certain cancer subtypes, such as those of embryonic origin or those generated by dedifferentiation of somatic cells.

Experimental Procedures

Xenopus egg extracts preparation and use

Experiments with Xenopus were performed in accordance with current institutional and national regulations approved by the Minister of Research under supervision of the

Departmental Direction of Population Protection (DDPP). Interphasic and cycling

Xenopus egg extracts were prepared and used as described [START_REF] Murray | Cell cycle extracts[END_REF][START_REF] Recolin | Role of replication protein A as sensor in activation of the S-phase checkpoint in Xenopus egg extracts[END_REF]. UV-irradiation of sperm chromatin and isolation of chromatin fractions was as described [START_REF] Recolin | Role of replication protein A as sensor in activation of the S-phase checkpoint in Xenopus egg extracts[END_REF].

Xenopus embryos and microinjection experiments

Embryos were prepared by in vitro fertilization using standard procedures [START_REF] Sivel | Early Development of Xenopus laevis. A laboratory manual[END_REF], UV-irradiated at the 2-cell stage and microinjected with the indicated mRNAs. Total protein extracts were obtained by collecting staged embryos according to Nieuwkoop and Faber normal tables.

mRNA synthesis

In vitro transcription was performed using mMESSAGE mMACHINE Kit® (Ambion). mRNA was ethanol-precipitated and dissolved in water ready for microinjection.

Cell culture

Cells were cultured and maintained under standard conditions. For transient expression, HEK293T were transfected with calcium phosphate. Twenty-four hours post transfection cells were mock-or UV-irradiated and collected at indicated time points. Whole cell extracts were clarified by centrifugation.

Immunofluorescence microscopy and foci formation assay

Cells were grown on coverslips prior to co-transfection. Four hours after UV-C irradiation, cells were fixed with 3.2 % paraformaldehyde for 15 min at room temperature and washed three times with PBS. After washing twice with PBS + 3% BSA, cells were mounted with ProlongGold DAPI (Invitrogen). eGFP-Pol foci were analyzed with Leica DM6000 epifluorescence microscope (RIO imaging facility). Images were acquired using a Coolsnap HQ CCD camera (Photometrics) and metamorph software (Molecular Devices). The percentage of eGFP-Pol-expressing cells displaying eGFP-Pol foci was determined by scoring at least 200 nuclei for each condition. Nuclei containing under 30 foci were scored as negatives.

Generation of stable NIH3T3 cells expressing Rad18

Cells were infected with viral particles generated by transfecting Platinum-E ecotropic packaging cell line (Cell Biolabs) with retroviral vectors (pLPC-puro) encoding Rad18 variants (WT, C28F) using Lipofectamine ® (Invitrogen). The viral supernatant was collected to infect cells. Forty-eight hours post infection, cells were selected in puromycin (2.5 g/ml, Sigma)-containing medium. Selected populations were expanded and promptly used.

Cell viability experiments

Cells were plated at 1.0 x 10 4 per well in twelve-well plates and UV-irradiated or exposed to the indicated amount of cisplatin. 48 hours post irradiation, cell viability was determined using the CellTiter-Glo® Luminescent Cell Viability assay (Promega). siRNA U87 cells were co-transfected either with siRNA using JETPrime reagent (Polyplus).

Twenty-four hours after transfection, cells were trypsinized and seeded in 12 wells plates at 10 4 cells/well density. Twenty-four hours later cells were treated with cisplatin (Sigma).

Statistical methods

Unless otherwise stated, data are presented as mean values ± SD of three independent experiments. For data shown in Figures 1E,3F, 4B, S2B, S3E, and S4B, unpaired, twotailed t test were performed. p values are represented (***p<0.0001, **p<0.001, *p<0.01). All statistical analyses were calculated using Graphpad® Prism 6 software.

Significance was assumed when p < 0.01. (B) Expression of DNA damage response proteins in glioblastoma. Western blot of total extracts obtained from glioblastoma biopsies (Glioblstoma, grade 4) or differentiated counterparts (progenitors, CD133-) or Hela cells.

Authors contribution

pFastBac1 vector as the XhoI-BamHI DNA fragment. The recombinant plasmid pFastBacXRad18 was sequenced on both strands. The HsRad18 ∆401-445 and Rad18 C28F mutants were previously described (Huang et al., 2009;[START_REF] Watanabe | Rad18 guides poleta to replication stalling sites through physical interaction and PCNA monoubiquitination[END_REF]. The plasmid expressing PCNA(K164R)-Ubiquitin fusion was previously described (Kanao et al., 2015).

Expression of recombinant proteins

6His-XlRad18 Cter was expressed in the E. coli strain BL21 Star (DE3). Cells were grown at 37 °C over night and diluted 100-fold into fresh LB until OD 600 reaches 0.6.

Then cells were left to shake at room temperature for 30 minutes and the expression of the recombinant protein was induced by addition of 0.5 mM IPTG. Cultures were left to shake for 3 hours at room temperature and harvested by centrifugation. The PCNA 6His-K164R mutant was expressed and purified as previously described (Chang et al., 2006). A recombinant 6His-Rad6-Rad18 complex was expressed in baculovirus-infected cells and purified to homogeneity on a Nickel column as previously described [START_REF] Watanabe | Rad18 guides poleta to replication stalling sites through physical interaction and PCNA monoubiquitination[END_REF], followed by gel filtration. Recombinant Geminin was expressed in bacteria and purified as previously described [START_REF] Tada | Repression of origin assembly in metaphase depends on inhibition of RLF-B/Cdt1 by geminin[END_REF].

Antibodies

XlRad18 antibodies were raised against 6His-XlRad18 Cter (amino acids 243-496) expressed and purified in bacteria by nickel affinity chromatography (Qiagen). Crude serum was also affinity-purified by affinity chromatography using the same antigen used to immunize rabbits coupled to Sepharose by standard procedures. RPA2

antibodies were previously described [START_REF] Cuvier | ORC is necessary at the interphase-tomitosis transition to recruit cdc2 kinase and disassemble RPA foci[END_REF]. XPolη antibodies were raised against full recombinant protein expressed in bacteria as previously described [START_REF] Yagi | DNA polymerases eta and kappa are responsible for error-free translesion DNA synthesis activity over a cis-syn thymine dimer in Xenopus laevis oocyte extracts[END_REF]. Rad6 antibodies were generated by injection of rabbit with recombinant, baculovirus-expressd Xenopus Rad6. The following antibodies were also used: human phospho-Chk1 (Ser 345 , Cell Signaling; 2341; recognizes S 344 XChk1); Chk1 (G-4, sc-8408, Santa Cruz biotechnology); H3 (ab1791, AbCam); PCNA (PC10, Sigma); MCM4 [START_REF] Coue | Evidence for different MCM subcomplexes with differential binding to chromatin in Xenopus[END_REF]; MCM2 (ab4461, AbCam), ß-tubulin (T3526, Sigma), USP1 (14346-1-AP, Proteintech); Drf1 [START_REF] Yanow | Xenopus Drf1, a regulator of Cdc7, displays checkpoint-dependent accumulation on chromatin during an S-phase arrest[END_REF], Primpol [START_REF] Wan | hPrimpol1/CCDC111 is a human DNA primase-polymerase required for the maintenance of genome integrity[END_REF]. ATRIP was a kind gift of K. Cimprich (Stanford University, USA).

ATR antibodies were raised as previously described [START_REF] Hekmat-Nejad | Xenopus ATR is a replication-dependent chromatin-binding protein required for the DNA replication checkpoint[END_REF].

ORC1 antibody was a gift of M. Méchali.

Immunodepletion procedures

Rad18 was removed from egg extracts by two rounds of depletion with affinitypurified Rad18 antibodies coupled to DynaBeads (Invitrogen). This procedure allows minimal dilution of the extracts during the depletion procedure avoiding spontaneous checkpoint activation likely due to dilution of the Rad6-Rad18 complex. Egg supernatants were thawed and supplemented with cycloheximide on ice and beads were added to the extract in a 50% ratio (V:V). For immunoprecipitations, extracts were diluted ten-fold in XB buffer supplemented with protease inhibitors and incubated with Rad18 antibody for 1 hour at 4°C. Immunocomplexes were collected with Protein A sepharose, washed in XB buffer and neutralized in Laemmli buffer.

Figure 1 .

 1 Figures legend

Figure 3 .

 3 Figure 3. Ectopic Rad18 expression induces spontaneous TLS Pol foci and

Figure 4 .

 4 Figure 4. High Rad18 expression is associated with resistance to DNA damage

Figure1

  Figure1Click here to downloadFigure: Figure1.tif

Figure

  Figure S1 is related to Figure 1 because shows controls regarding the effect of the N/C ratio upon cell cycle progression in the presence of DNA damage and its relationships with replicative and translesion DNA polymerases in Xenopus egg extracts.

Figure

  Figure S2 is related to Figure 2 and shows important controls about the specificity of Rad18 depletion on checkpoint activation at low N/C ratio as well as the contribution of PCNA mUb both in vitro and in vivo in Xenopus.

Figure

  Figure S3 is related to Figure 3 and shows important controls about the specificity of the phenotypes observed upon Rad18 overexpression in mammalian cells.

Figure

  Figure S4 is related to Figure 4 because shows the expression of rad18 in colorectal cancer cell lines sensitivie or resistant to oxaliplatin and the expression of divers DNA damage markers in glioblastoma.

Figure

  Figure S3, related to Figure 3

Figure

  Figure S4, related to Figure 4

Acknowledgements

We thank M. Méchali for encouragement, J. Moreau for help with microinjection and M.

Lamers for Rad-Rad18 purification, P. Coulombe for help with stable cell lines, A. Eloualid for technical assistance, K. Cimprich for XRad18 cDNA, P. Kannouche for eGFP-Pol, C. Vaziri for Rad18 ∆401-445 , C. Masutani for PCNA-Ubiquitin, M. Akiyama (XPol), A. Kumagai & W. Dunphy (XDrf1), J. Huang (Primpol) for antibodies, P-H. Nasheuer for the gift of recombinant Pol, J. Sale & J. Hutchins for critical reading of the manuscript. Grants supporting this work were from ARC (N° 3156), FRM (DEQ20071210543), Ligue contre le Cancer (to C.K.) and ANR (CHECKDEV). None of the authors of this manuscript have a financial interest related to this work.

quantification is expressed as Relative Optical Density (ROD). Means and standard deviations are represented (**p <0.001; See also Figure S1). (B) Cycling egg extracts were incubated at room temperature for the indicated times in the absence or presence (C) of cycloheximide and analysed by western blot to detect the phosphorylated forms of MCM4 (arrows), a CDK1 substrate [START_REF] Hendrickson | Phosphorylation of MCM4 by cdc2 protein kinase inhibits the activity of the minichromosome maintenance complex[END_REF]. UV irradiation did not induced mitotic delay, similar to the non-irradiated control (right panel). In contrast, extracts supplemented with sperm nuclei at high N/C ratio were strongly delayed in interphase (middle panel). No MCM4 phosphorylation was observed in the presence of cycloheximide at both low and high N/C ratio (C) as expected [START_REF] Maiorano | Cell cycle regulation of the licensing activity of Cdt1 in Xenopus laevis[END_REF], due to inhibition of cyclin B synthesis, indicating failure to enter mitosis.

(D) Checkpoint suppression by decreasing the N/C ratio. Equal amounts of sperm nuclei (1000 nuclei/µl) UV-irradiated (lanes 1-4) or not (lane 5) were incubated at room temperature for sixty minutes with increasing volumes of egg extracts (dilution factors X3, X6, X9 respectively for lanes 2-4 compared to lane 1). Nuclei were then isolated and Chk1 phosphorylation analyzed by western blot with a specific antibody.

Chk1 serves here as loading control. (5'-CTGAGCATTATACATTTGAACGAATTCC-3') containing a synthetic EcoRI restriction site (underlined), and used as primers in PCR reactions with 5' or 3' primers specific of a X. laevis ovary cDNA library made in lambda gt10 vector [START_REF] Rebagliati | Identification and cloning of localized maternal RNAs from Xenopus eggs[END_REF]. PCR products were blunt-end ligated to pRSET expression vector (Invitrogen) to generate XlRad18 Nter or XlRad18 Cter . Recombinant plasmids were sequenced on both strands. Full-length X. laevis Rad18 was kindly provided by K. Cimprich (Stanford University, USA). The sequence of the X. laevis Rad18 gene has been deposited to the EMBL genebank (accession number CCQ71719.2). The PCNA 6His-K164R mutant was generated as previously described (Chang et al., 2006). The Xenopus Rad6 gene was obtained from NIBB Xenopus cDNA Resource (NIB, Japan). Full length XRad6 cDNA was amplified by PCR using 5' (5'- Patients and tumour samples.

Tumour sample were obtained from patients diagnosed for type IV grade glioma (i.e. glioblastoma) and undergoing surgery at the neurosurgery department of the Rangueil Hospital (Toulouse, France). All subjects provided their informed written consent before their surgery and the protocol followed the declaration of Helsinki guidelines and was approved by local ethics committee.

Supplemental References

Balin-Gauthier, D., Delord, J.P., Pillaire, M.J., Rochaix, P., Hoffman, J.S., Bugat, R., Cazaux, C., Canal, P., and Allal, B.C. (2008). Cetuximab potentiates oxaliplatin cytotoxic effect through a defect in NER and DNA replication initiation. British journal of cancer 98, 120-128. Balin-Gauthier, D., Delord, J.P., Pillaire, M.J., Rochaix, P., Hoffman, J.S., Bugat, R., Cazaux, C., Canal, P., and Allal, B.C. (2008). Cetuximab potentiates oxaliplatin cytotoxic effect through a defect in NER and DNA replication initiation. British journal of cancer 98, 120-128.