The Plant Cell, Vol. 22: 2336–2352, July 2010, www.plantcell.org ã 2010 American Society of Plant Biologists

DNA Replication Factor C1 Mediates Genomic Stability and Transcriptional Gene Silencing in Arabidopsis W OA

Qian Liu,a,1 Junguo Wang,a,1 Daisuke Miki,b,c Ran Xia,a Wenxiang Yu,a Junna He,a Zhimin Zheng,b,c Jian-Kang Zhu,b,c,d and Zhizhong Gonga,d,e,2 a State Key Laboratory of Plant Physiology and Biochemistry, College of Biological Sciences, China Agricultural University, Beijing 100193, China b Institute for Integrative Genome Biology and Department of Botany and Plant Sciences, University of California, Riverside, California 92521 c Center for Plant Stress Genomics and Technology, 4700 King Abdullah University of Science and Technology, Thuwal 23955- 6900, Kingdom of Saudi Arabia d China Agricultural University–University of California, Riverside Center for Biological Sciences and Biotechnology, Beijing 100193, China e National Center for Plant Gene Research, Beijing 100193, China

Genetic screening identified a suppressor of ros1-1, a mutant of REPRESSOR OF SILENCING1 (ROS1; encoding a DNA demethylation protein). The suppressor is a mutation in the gene encoding the largest subunit of replication factor C (RFC1). This mutation of RFC1 reactivates the unlinked 35S-NPTII transgene, which is silenced in ros1 and also increases expression of the pericentromeric Athila retrotransposons named transcriptional silent information in a DNA methylation- independent manner. rfc1 is more sensitive than the wild type to the DNA-damaging agent methylmethane sulphonate and to the DNA inter- and intra- cross-linking agent cisplatin. The rfc1 mutant constitutively expresses the G2/M-specific cyclin CycB1;1 and other DNA repair-related genes. Treatment with DNA-damaging agents mimics the rfc1 mutation in releasing the silenced 35S-NPTII, suggesting that spontaneously induced genomic instability caused by the rfc1 mutation might partially contribute to the released transcriptional gene silencing (TGS). The frequency of somatic homologous recombi- nation is significantly increased in the rfc1 mutant. Interestingly, ros1 mutants show increased telomere length, but rfc1 mutants show decreased telomere length and reduced expression of telomerase. Our results suggest that RFC1 helps mediate genomic stability and TGS in Arabidopsis thaliana.

INTRODUCTION The formation of the DNA replication fork can be stalled or arrested during DNA replication if the DNA structure is chemically During DNA replication, DNA polymerase a interacts with DNA or physically altered by double-strand breaks (DSBs). DSBs primase to form a complex for initiating synthesis of a 15-20 can be repaired by homologous recombination (HR), which will mer DNA primer using an RNA primer. Replication factor C (RFC), reestablish a formal replication fork. DNA damage can activate a clamp-loader complex consisting of five different subunits, the S-phase replication checkpoint pathway that helps stabilize binds DNA at the template–primer junctions and displaces replication forks and prevents the breakdown of replication forks polymerase a to terminate DNA primer synthesis. The binding (Branzei and Foiani, 2009). Chromosome ends called telomeres of RFC to DNA creates a loading site for recruiting the DNA are natural DNA breaks. However, telomeres have specific DNA sliding clamp proliferating cell nuclear antigen (PCNA), a ring- structures that are protected by various proteins with negative or shaped homotrimer. RFC is an AAA+-type ATPase that requires positive effects on telomere length (Smolikov et al., 2004). When ATP hydrolysis for opening and closing PCNA around DNA the DNA replication machinery meets telomeres, it competes during DNA replication, repair, and recombination (Majka and with telomerase (Shore and Bianchi, 2009). Telomeres can be Burgers, 2004). elongated by some mutations, such as in Rfc1 and DNA poly- merase a, suggesting that the replication machinery regulates telomere length (Adams and Holm, 1996). Abnormal regulation of telomere maintenance may evoke a DNA damage response 1 These authors contributed equally to this work. 2 Address correspondence to [email protected]. leading to the repair of telomeres by HR. The author responsible for distribution of materials integral to the DNA replication is a highly regulated process that accurately findings presented in this article in accordance with the policy described replicates both the primary DNA sequence and chromatin struc- in the Instructions for Authors (www.plantcell.org) is: Zhizhong Gong ture from the parent strands. The transmission of heterochro- ([email protected]). matin structure and DNA methylation is an essential process W Online version contains Web-only data. OA Open Access articles can be viewed online without a subscription. after the DNA has been replicated and is tightly regulated by www.plantcell.org/cgi/doi/10.1105/tpc.110.076349 various proteins (Shultz et al., 2007; Kloc and Martienssen, 2008; Replication Factor C and Epigenetic Instability 2337

Martienssen et al., 2008; Probst et al., 2009). Furthermore, DNA polymerase « was isolated in a root-sensitive-to-abscisic epigenetic markers can be changed after the replication ma- acid (ABA) screen and is also involved in TGS in an RdDM- chinery has passed the DNA replication fork during cell differen- independent manner (Yin et al., 2009). In this study, we charac- tiation and development. In fission yeast, DNA polymerase terized another ros1 suppressor, RFC1, the largest subunit of the a interacts with the heterochromatin protein Swi6, a homolog RFC complex in Arabidopsis. Mutations in RFC1 lead to devel- of HETEROCHROMATIN PROTEIN1 (HP1), an important deter- opmental defects and earlier flowering. Our results indicate that minant of the epigenetic imprint in vivo, and influences the RFC1 is an important mediator of TGS, DNA replication, DNA localization of Swi6 (Nakayama et al., 2001). In Arabidopsis repair, HR, and telomere length regulation. thaliana, DNA polymerase a physically and genetically interacts with LIKE HETEROCHROMATIN PROTEIN1 (LHP1) (Barrero et al., 2007), suggesting that the DNA replication machinery RESULTS involved in the epigenetic imprint is conserved among different organisms. Early studies in yeast that included screens for The rfc1 Mutation Releases the TGS of 35S-NPTII in the transcriptional silencing genes identified several genes that ros1 Mutant encode DNA replication-related proteins, including Rfc1 and some chromatin structure modulators (Ehrenhofer-Murray et al., The accession used in this study, C24 RD29A-LUC, carries a T-DNA 1999; Smith et al., 1999). Homotrimeric PCNA is considered locus that contains active (i.e., unsilenced) RD29A-LUC and 35S- an essential component in recruiting the general mediators NPTII genes. This accession is herein referred to as the C24 wild for chromatin imprinting of epigenetic markers (Probst et al., type. ros1 mutations lead to the silencing of both RD29A-LUC 2009). Genetic studies in Arabidopsis have also identified some and 35S-NPTII,andtheros1-1 mutant is thus sensitive to other DNA replication- and repair-related proteins involved in the kanamycin (Gong et al., 2002a). A kanamycin-resistant mutant, regulation of transcriptional gene silencing (TGS); these include named rfc1-1, was isolated from ethyl methanesulfonate– BRU1, Chromatin assembly factor (CAF-1), REPLICATION mutagenized ros1-1 plants, for which the M2 generation was PROTEIN A2 (RPA2), DNA polymerase a, and DNA polymerase « grown on Murashige and Skoog (MS) medium supplemented (Takeda et al., 2004; Elmayan et al., 2005; Kapoor et al., 2005b; with 50 mg/L kanamycin. Genetic analysis using F2 seedlings Ono et al., 2006; Schonrock et al., 2006; Xia et al., 2006; Yin et al., from rfc1-1 ros1-1 backcrossed with ros1-1 on 50 mg/L kana- 2009; Liu et al., 2010). mycin indicated that the rfc1-1 mutation was caused by a single The well-studied phenomenon of TGS in plants is mediated by recessive nuclear gene (Figure 1A). We tested the kanamycin- small interfering RNA (siRNA)-directed DNA methylation (RdDM), sensitive phenotypes of rfc1-1 ros1-1 on different concentrations a dynamic process in which the DNA methylation patterns of kanamycin. As shown in Figure 1B, the rfc1-1 ros1-1 mutant depend on the production of siRNAs at different developmental was more tolerant to kanamycin than ros1-1 but less tolerant stages or under different conditions (Henderson and Jacobsen, than the C24 wild type or rfc1-1. Immunoblot analysis indicated 2007; Matzke et al., 2007, 2009; Law and Jacobsen, 2009; that rfc1-1 ros1-1 expressed more NPTII protein than ros1-1 but Teixeira et al., 2009). RdDM usually occurs in transposons and less than the C24 wild type (Figure 1C). We crossed rfc1-1 ros1-1 DNA repeat regions accompanied by the formation of hetero- with the C24 wild type and isolated the single rfc1-1 mutant and chromatin and accounts for ;30% of the total DNA methyla- tested its kanamycin sensitivity. rfc1-1 mutants had a slightly tion in Arabidopsis (Matzke et al., 2009). DNA methylation can increased kanamycin sensitivity and a slightly reduced quantity be reversibly removed by a ROS1 (for REPRESSOR OF of NTPII protein relative to the C24 wild type (see 350 mg/L Kan in SILENCING1) family of DNA glycosylase/lyases, including ROS1, Figures 1B and 1C), suggesting that the rfc1-1 mutation did not DEMETER, and two DEMETER-like proteins, DML2 and DML3 simply act to increase NPTII expression in the C24 wild type. (Gong et al., 2002a; Kapoor et al., 2005a; Morales-Ruiz et al., To determine whether the rfc1-1 mutation influences the ex- 2006). ROS1 was the first-characterized DNA demethylation pression of RD29A-LUC and the endogenous RD29A gene, we enzyme and was originally isolated during a screen for genes compared their expression after treatment with 50 mMABAfor4h whose mutations silence the expression of the foreign transgene (for RD29A expression) or 200 mM NaCl treatment for 4 h (for RD29A-LUC in a T-DNA region (Gong et al., 2002a). This T-DNA LUC image). As shown in Figure 1D, ros1-1 or rfc1-1 ros1-1 emitted region contains an RD29A-LUC gene and a 35S-NPTII gene. little or no luminescence, but the C24 wild type and rfc1-1 emitted ROS1 mutations lead to TGS of both RD29A-LUC and 35S-NPTII strong luminescence. Real-time RT-PCR analysis showed that by different regulation mechanisms (i.e., the former depends on endogenous RD29A was highly expressed in the C24 wild type RdDM, but the latter does not). When silenced RD29A-LUC was and rfc1-1 but was silenced in both ros1-1 and rfc1-1 ros1-1 (Figure used as a screening marker to identify ros1 suppressors, most 1E). We further used real-time RT-PCR to check the expression genes isolated were in the RdDM pathway (He et al., 2009a, of the pericentromeric Athila retrotransposons named transcrip- 2009b); when silenced 35S-NPTII was used as a screening tional silent information (TSI). As shown in Figure 1F, the transcripts marker for ros1 suppressors, most isolated genes are indepen- of TSI were more expressed in rfc1-1 and rfc1-1 ros1-1 than in dent of RdDM (Kapoor et al., 2005b; Xia et al., 2006; Wang et al., the C24 wild type or ros1-1.WealsocheckedtheexpressionofTSI 2007; Shen et al., 2009). The latter group of genes include ROR1/ in an rfc1-2 mutant of Columbia accession (a T-DNA insertion RPA2A (Xia et al., 2006), TOUSLED protein kinase (Wang et al., mutant; see below) and found that TSIs were expressed at a higher 2007), DNA polymerase a (Liu et al., 2010), and chlorophyll a/b level than in the wild type. These results indicate that the mutations binding protein gene underexpressed1 (Shen et al., 2009). ABO4/ in RFC1 release the TGS in some genomic loci. 2338 The Plant Cell

Figure 1. The rfc1-1 Mutation Releases the Transcriptional Gene Silencing of 35S-NPTII in ros1-1 and TSI in the Wild Type and ros1-1.

(A) Kanamycin-resistant phenotype of rfc1-1 ros1-1, ros1-1, C24 wild type (WT), and the F2 progeny of rfc1-1 ros1-1 crossed with ros1-1. Seeds were directly sown on MS medium containing 50 mg/L kanamycin (Kan) and were grown for 7 d before they were photographed. (B) Kanamycin resistance of rfc1-1 ros1-1, ros1-1, rfc1-1, and C24 wild type on MS medium containing different concentrations of kanamycin. (C) NPTII expression as determined by immunoblot analysis in rfc1-1 ros1-1, ros1-1, rfc1-1, and C24 wild type. NPTII antibody was used for immunoblot. Total proteins with Coomassie blue staining were used as protein loading control. (D) Luciferase luminescence images of rfc1-1 ros1-1, ros1-1, rfc1-1, and C24 wild-type seedlings treated with 200 mM NaCl for 4 h. (E) Endogenous RD29A gene expression as determined by real-time RT-PCR in rfc1-1 ros1-1, ros1-1, rfc1-1, and C24 wild-type seedlings treated with 50 mM ABA for 4 h. Total RNAs extracted from the C24 wild type without ABA treatment were used for normalizing each sample. The representative data were from one of three biologically independent experiments that showed similar results, each experiment with triple technical repetitions. The level of ACTIN was used as an internal control. Error bars indicate 6SD, n =3. (F) TSI expression as determined by real-time RT-PCR in rfc1-1 ros1-1, ros1-1, rfc1-1, and C24 wild type, or Columbia wild type and rfc1-2 mutant (a T-DNA insertion mutant; see below). RNAs from C24 wild type were used as a standard control for normalizing each sample. ACTIN was used as an internal control. Three biologically independent experiments were done with similar results. The representative one is from one experiment with three technical repetitions. Error bars indicate 6SD, n =3.

The rfc1-1 Mutation Does Not Change DNA Methylation at the C24 wild type. For the 35S promoter, no methylation differ- the RD29A Promoter or at Other DNA Repeat Regions ence was found among the different genomic DNAs when they were digested with DdeI and XmiI. We further compared the To test whether the rfc1 mutation affects DNA methylation lev- DNA methylation levels in the centromere, rDNA, and the Ta3 els, we analyzed the DNA methylation patterns in the RD29A transposon using two methylation-sensitive enzymes, HpaII promoter, the 35S promoter, the 5S rDNA region, the 180-bp (mCmCGG methylation sensitive) and MspI (mCCGG methylation centromere DNA repeat region, and the pericentromeric retro- sensitive), and we did not find any apparent differences among transposon Ta3 using methylation-sensitive restrictive enzymes. the C24 wild type, ros1-1, and rfc1-1 ros1-1 (Figure 2A). These RD29A and 5S rDNA are targeted by RdDM, and the 35S results suggest that RFC1 mutation does not influence the DNA promoter, the 180-bp centromere, as well as the Ta3 retrotrans- methylation at these sites. poson are not controlled by RdDM (Chan et al., 2006). As rfc1-1 reported previously, MluI(AmCGCGT) and BstUI (CGmCG) en- The Mutation Decreases the H3K9 Dimethylation at 35S ros1-1 zymes did not completely cleave the RD29A promoter in ros1-1 the Promoter in because of hypermethylation at these two sites (Figure 2A). The Because previous studies indicate that the expression of rfc1-1 mutation did not change the DNA digestion pattern 35S-NPTII is related to histone modification (Xia et al., 2006; produced by MluI and BstUI in ros1-1. By contrast, MluI and Wang et al., 2007; Yin et al., 2009), we performed chromatin BstUI enzymes could efficiently digest the RD29A promoter in immunoprecipitation (ChIP) to check the histone modifications Replication Factor C and Epigenetic Instability 2339

Figure 2. The rfc1-1 Mutation Did Not Influence DNA Methylation but Changed Levels of H3K9m2. (A) DNA methylation in the RD29A promoter, 35S promoter, 180-bp centromere, Ta3 transposon, and 5S rDNA regions digested by different DNA methylation-sensitive enzymes and detected by DNA gel blot analysis, hybridized with the indicated probe sequences. WT, wild type. (B) Levels of H3K9me2 in the 35S promoter, in the total RD29A (both transgenic and endogenous RD29A promoter), and in the endogenous RD29A promoter of rfc1-1 ros1-1, ros1-1, rfc1-1, and C24 wild type. C24 wild type was used as a standard control. Three biologically independent experiments were done with similar results. The data are from one experiment with triple technical repeats. Error bars indicate 6SD, n =3. in the 35S promoter region using antidimethylated histone H3 ent stages exhibit different sensitivities to MMS, another test was Lys 9 (H3K9me2), which is usually an epigenetic marker for het- done in which 5-d-old seedlings grown without MMS were erochromatin. Here, we used Ta3 as a control for H3K9me2 as transferred to MS medium containing 0 or 150 ppm MMS for 2 described previously (Liu et al., 2010). H3K9me2 was detected weeks. As shown in Figures 3A and 3B, rfc1-1 ros1-1 and rfc1-1 less in the 35S promoter of rfc1-1 ros1-1 than of ros1-1 but was were more sensitive to MMS than the C24 wild type or ros1-1 in detected at similar levels either in the total RD29A (including both both postgermination growth (the first test) and in seedling the transgenic and the endogenous RD29A promoter) or in the growth (the second test). We also compared the sensitivity of endogenous RD29A promoter in both rfc1-1 ros1-1 and ros1-1. rfc1-1 and the wild type to cisplatin. Cisplatin (cis-diamminedi- No clear difference in H3K9me2 level, however, was found chloroplatinum or cis-DDP), which is used as an anticancer between rfc1-1 and the C24 wild type in either the 35S or chemical, coordinates to DNA mainly through the nitrogen atoms RD29A promoter (Figure 2B). (specifically, the N7 atoms of purines) to produce intra- and interstrand DNA cross-links. During DNA replication, both types rfc1-1 Mutants Are Sensitive to DNA-Damaging Agents and of DNA cross-links induce DSBs, which are repaired predomi- Constitutively Express High Levels of DSB-Induced Genes nantly by HR during the S-phase (Frankenberg-Schwager et al., 2005; Osakabe et al., 2006). Seeds were sown on MS medium Although the DNA repair function of RFC1 has been studied in containing different concentrations of cisplatin, and seedling yeast and Aspergillus (McAlear et al., 1996; Kafer and Chae, growth was observed after 2 weeks. As shown in Figure 3C, both 2008), there is no direct evidence of RFC1 involvement in DNA rfc1-1 ros1-1 and rfc1-1 mutants were much more sensitive than repair in humans (Wood et al., 2001; Kafer and Chae, 2008). We the wild type or ros1-1 to 20 and 30 mM cisplatin. Figure 3D compared the sensitivity of rfc1-1 with the wild type to the indicates the number of true leaves of seedlings as affected by radiomimetic agent methylmethane sulphonate (MMS), which cisplatin concentration and genotype. These results suggest a causes DNA damage (Lundin et al., 2005). Seeds of the C24 wild critical role of RFC1 in repairing DNA damage in plant cells. type, ros1-1, rfc1-1, and rfc1-1 ros1-1 were sown on MS agar In a previous study on DNA polymerase «/ABO4, we found medium supplemented with 125 ppm MMS (or without MMS as a that several marker genes functioning in DSBs are constitu- control) and were kept in a growth chamber for 3 weeks to tively expressed in the abo4 mutant (Yin et al., 2009). We compare postgermination growth. Because seedlings at differ- determined the expression of the following DNA repair and/or 2340 The Plant Cell

Figure 3. The rfc1-1 Mutants Were Sensitive to DNA-Damaging Agents and Constitutively Expressed a Higher Level of Some DNA Repair-Related Genes.

(A) Growth of rfc1-1, rfc1-1 ros1-1, ros1-1, and C24 wild type after seeds were directly germinated and grown for 3 weeks on MS medium or on MS medium supplemented with 125 ppm MMS. (B) Growth of rfc1-1, rfc1-1 ros1-1, ros1-1, and C24 wild type (WT). Seedlings that had grown on MS medium for 5 d were transferred onto MS medium with or without 150 ppm MMS and were photographed 2 weeks later. (C) Growth of rfc1-1, rfc1-1 ros1-1, ros1-1, and C24 wild type as affected by different concentrations of cisplatin. Seeds were directly germinated and grown for 3 weeks on MS medium with or without different concentrations of cisplatin. (D) Average numbers of true leaves of rfc1-1, rfc1-1 ros1-1, ros1-1, and C24 wild type grown as described in Figure 3C. Sixty plants were counted for each treatment. Error bars indicate 6SD, n =60. (E) Relative expression levels of RD51A, Ku70, GR, BRCA,andMRE in rfc1-1 and the wild type as determined by real-time RT-PCR. Total RNA extracted from 1-week-old seedlings was reverse transcribed and used for qRT-PCR. The expression of each gene in C24 wild type was used as a standard normalization control. Three biologically independent experiments were done. Data are means of triple technical repetitions from one experiment. Error bars indicate 6SD, n =3. (F) and (G) Relative expression levels of RAD51 (F) and BRCA (G) in rfc1-1 and wild type as affected by treatment with 125 ppm MMS for different times. The relative expression of RAD51 or BRCA in C24 wild type without treatment was used as a standard normalization control. Three biologically independent experiments were done. Data are means of triple technical repetitions for one experiment. Error bars indicate 6SD, n =3. cell cycle checkpoint genes: KU70, encoding a repairing pro- 3G).TherelativeincreaseinexpressioninresponsetoMMSdid tein in the nonhomologous end joining (NHEJ) pathway; RAD51, not clearly differ between rfc1-1 and the wild type. These encoding a recombinase in HR repair; BREAST CANCER results indicate that DNA repair-related genes were spontane- SUSCEPTIBLITY1 (BRCA1); and GAMMA RESPONSE1 (GR1) ously induced by rfc1-1 mutation. in HR. Under normal growth conditions, rfc1-1 mutants express higher levels of GR1, RAD51, KU70,andBRCA1 than the C24 Treatment with DNA-Damaging Agents Mimics the rfc1 wild type (Figure 3E). Expression of MRE11 did not clearly differ Mutation in Releasing TGS of 35S-NPTII between rfc1-1 and the C24 wild type (Figure 3E). MMS treat- ment greatly induced the expression of RAD51 and BRCA1 in To determine whether the releasing of TGS is directly related to both the C24 wild type and the rfc1-1 mutant (Figures 3F and genomic instability caused by DNA damage, we compared the Replication Factor C and Epigenetic Instability 2341

shown in Figure 4A, ros1-1 seedlings showed greater kanamycin resistance on the medium with 50 ppm MMS or 10 mM cisplatin than on the medium without MMS or cisplatin. ros1-1 seedlings grew better on the medium with 50 ppm MMS than on medium with 10 mM cisplatin. We further used quantitative RT-PCR (qRT- PCR) to compare the transcripts of NPTII under MMS or cisplatin treatment. Seedlings (7 d old) were transferred to MS medium or MS medium containing 50 ppm MMS or 10 mM cisplatin for five additional days. As shown in Figure 4B, both MMS and cisplatin treatment increased the transcripts of NPTII, but the transcripts were still lower in ros1-1 than in rfc1-1 ros1-1, rfc1-1, or the wild type. These results suggest that treatment with DNA-damaging agents is able to directly release the TGS of 35S-NPTII in ros1-1.

rfc1-1 Mutants Exhibit Highly Spontaneous Somatic Homologous Recombination

DSBs, which are the most dangerous threat to the stability of the genome, can be repaired by NHEJ and somatic homologous recombination. NHEJ, a process of religation of broken DNA ends without any DNA template, usually occurs in the G1 phase of the cell cycle because sister chromatids are not available to provide a template for HR in this phase. HR can use an unbroken sister chromatid strand as a template to accurately repair an- other broken sister chromatid strand. Abiotic and biotic stresses can trigger DNA damage and efficiently stimulate recombination in plants (Pecinka et al., 2009; Yin et al., 2009). Because rfc1-1 mutants were sensitive to DNA-damaging agents, we used the Arabidopsis recombination reporter line 651 (in C24 background) to analyze the somatic homologous recombination (SHR) in the rfc1-1 mutant. Line 651 contains two incomplete but overlapping parts of a b-glucuronidase (GUS) gene in an inverted orientation, and these parts can reconstitute a functional GUS protein when recombination events occur (Lucht et al., 2002). The reporter gene was introduced into the rfc1-1 mutant, and SHR events Figure 4. Treatment with DNA-Damaging Agents Released the TGS of 35S-NPTII. were compared between the wild type and the rfc1-1 mutant. We compared the number of GUS spots in seedlings grown for (A) Seedling growth on MS medium or MS medium supplemented with different periods (Figure 5). Generally, only a few spots (most 50 mg/L kanamycin (Kan), 50 mg/L kanamycin and 50 ppm MMS or were in cotyledons) could be observed on each wild-type or 50 mg/L kanamycin and 10 mM cisplatin. Seeds were directly sown on ros1-1 mutant seedling (Figures 5A to 5C). By contrast, a high the different media and were photographed after 2 weeks. WT, wild type. (B) The relative expression levels of NPTII as determined by qRT-PCR frequency of SHR was observed in the rfc1-1 and the rfc1-1 after MMS and cisplatin treatment. Three biologically independent ros1-1 mutants, especially in the cotyledons of 7-, 15-, and 20-d- experiments were done. The data are from one experiment with triple old seedlings (Figures 5A and 5B) or in the first two true leaves of technical repetitions. Error bars indicate 6sd, n = 3 (*P < 0.05). Con, the 15-d-old seedlings (Figures 5A and 5C) or 20-d-old seedlings control. (Figure 5A; not quantified); fewer spots were present in the younger true leaves (Figure 5A). The number of GUS spots greatly increased with time in both cotyledons and true leaves. growth of ros1 seedlings on MS medium containing 50 mg/L Because RFC1 is expressed at higher levels in older leaves (see kanamycin and supplemented or not supplemented with MMS Figure 8), this suggests that RFC1 might substantially contribute or cisplatin. If treatment with DNA-damaging agents released to the restriction of SHR in these older tissues. We also tested the the TGS of 35S-NPTII, the treated ros1-1 seedlings should SHR rate in seedlings treated with 10 mM cisplatin or 50 ppm be resistant to kanamycin. We sowed the seeds directly on MS MMS for 8 d. These treatments apparently increased the SHR in medium (as a seed germination and growth control) or on MS that more GUS spots occurred with cisplatin treatment or MMS medium containing 50 mg/L kanamycin, 50 mg/L kanamycin treatment than with the control in true leaves (it is difficult to plus 50 ppm MMS, or 50 mg/L kanamycin plus 10 mM cisplatin. compare numbers of GUS spots in cotyledons) in the wild type Here, we used 50 ppm MMS and 10 mM cisplatin because higher (Figures 5D and 5E), ros1-1, rfc1-1,orrfc1-1 ros1-1 (Figure 5F for concentrations of these two agents will greatly inhibit seedling cisplatin treatment, it is hard to count the GUS spots for MMS growth, making comparison of seedling growth difficult. As treatment). 2342 The Plant Cell

Figure 5. The rfc1-1 Mutation Increased SHR. (A) SHR, as indicated by GUS spots, in the wild type (WT), ros1-1, rfc1-1, and rfc1-1 ros1-1 seedlings grown for 7 d (left), 15 d (middle), and 20 d (right). (B) SHR events in 7-d-old seedlings as indicated by numbers of GUS spots on entire seedlings. About 50 seedlings were examined for the wild type and for each mutant. (C) SHR events in the first two true leaves of entire, 15-d-old seedlings as indicated by numbers of GUS spots. About 50 leaves were examined for the wild type and for each mutant. (D) SHR in seedlings of the wild type, ros1-1, rfc1-1,andrfc1-1 ros1-1 as affected by 25 mM cisplatin or 150 ppm MMS. Seven-day-old seedlings were exposed to cisplatin or MMS for eight additional days. (E) SHR events in entire seedlings of the wild type treated with 10 mM cisplatin or 50 ppm MMS. About 50 leaves were examined for each treatment. (F) SHR events in the first true leaf of seedlings treated with cisplatin. About 50 leaves were examined for the wild type and each mutant.

The rfc1-1 Mutation Reduces Cell Division, Delays the Cell DNA polymerase a mutants, the cell cycle was delayed and Cycle, and Causes a High Expression of Cyclin CYCB1;1 there was high expression of a G2-M marker gene cyclin CYCB1;1 (Yin et al., 2009; Liu et al., 2010). The CYCB1;1 rfc1-1 plants are smaller than the C24 wild type. We compared promoter-GUS reporter was introduced into the rfc1-1 mutant. the size of epidermal cells of mature leaves and stems in these two genotypes. As shown in Figures 6A and 6B, cell size did not As illustrated in Figure 6C, more GUS staining was detected in differ between the rfc1-1 mutant and the C24 wild type, indi- the root tips, shoot meristems, and young leaves of the rfc1-1 cating that the rfc1-1 plants are smaller because they have mutant than of the C24 wild type, indicating that the G2/M fewer cells rather than smaller cells. In DNA polymerase « and phases were longer in the rfc1-1 mutant. qRT-PCR confirmed Replication Factor C and Epigenetic Instability 2343

Figure 6. The rfc1-1 Mutation Reduced Cell Division, Delayed the Cell Cycle, and Expressed High Levels of Cyclin CYCB1;1. (A) and (B) Sizes of epidermal cells in leaves (A) and stems (B) of rfc1-1, rfc1-1 ros1-1, ros1-1, and C24 wild type (WT) at the same growth stage. Bars = 60 mm. (C) CYCB1;1-GUS expression in the wild type and rfc1-1 mutant. (D) Relative expression level of CYCB1;1 in rfc1-1, rfc1-1 ros1-1, ros1-1, and C24 wild type. The expression of CYCB1;1 in C24 wild type was used as a standard normalization control. Three biologically independent experiments were done. Data are means of triple technical repetitions from one experiment. Error bars indicate 6SD, n =3. (E) Induction of CYCB1;1 by 125 ppm MMS at different times in rfc1-1 and wild type. Three biologically independent experiments were done. Data are means of triple technical repetitions from one experiment. Error bars indicate 6SD, n =3. the higher expression of CYCB1;1 in rfc1-1 than in the C24 These results suggest that ROS1 has a unique role in negatively wild type (Figure 6D). MMS treatment induced a high level of regulating telomere length in different Arabidopsis accessions. CYCB1;1 expression in both rfc1-1 and the C24 wild type Increasing evidence suggests that many DNA repair-related (Figure 6E), suggesting that a delay in the G2/M phases is proteins play crucial roles in regulating telomere length (Gallego necessary for DNA repair. and White, 2005). Because rfc1 mutants are defective in DNA repair with increased SHR, we tested whether the RFC1 mutation ros1 Mutation Increases the Telomere Length and rfc1 influences telomere length. Because telomere length varies Mutation Decreases the Telomere Length and Reduces among different individuals, we collected 2-week-old seedlings Expression of Telomerase and used the total mixed DNAs from these plants. As shown in Figure 7C, the rfc1-1 mutation greatly decreased the telomere Previous studies suggest that the DNA replication machinery can length relative to the C24 wild type or ros1-1, while the telomere compete with telomerase and control telomere length (Adams length in rfc1-1 ros1-1 was comparable to that in the rfc1-1 and Holm, 1996). ROS1 physically interacts with replication mutant. We also checked three other DNA replication mutants, protein A2 in a yeast two-hybrid assay, suggesting that ROS1 pol a, ror1/rpa2a, and pol «. The telomeres in pol a mutants were might be involved in DNA replication (Kapoor et al., 2005b; Xia only a little shorter than in the wild type. The telomeres of the pol et al., 2006). We compared telomere length between ros1 and a ros1-1 mutant were a little longer than that of pol a but shorter the C24 wild type. As illustrated in Figure 7A, telomeres were a than that of ros1-1 and were similar to the wild type. The little longer in ros1 than in the C24 wild type. In the Columbia telomeres in the pol « mutant were much shorter than in the accession, telomere length was also longer in the ros1 mutant wild type. Telomeres of ror1 and ror1 ros1-1 mutants were similar than in the wild type (Figure 7B). ROS3 is an RNA binding protein in length and were a little shorter that those of the wild type or working in the same pathway as ROS1 for DNA demethylation ros1-1. Together, these results indicate that the four different (Zheng et al., 2008). The telomere length of ros3 was similar to replication mutants have different effects on telomere length. that of the wild type (Figure 7A). The telomere length of the ros1-1 Among them, rfc1-1 reduces telomere length the most and pol ros3 mutant was similar to that of ros3 and the wild type. a reduces telomere length the least. 2344 The Plant Cell

Figure 7. Telomere Length Was Increased by the ros1 Mutation but Decreased by the rfc1 Mutation.

(A) The ros1 mutation but not the ros3 mutation increased telomere length. Genomic DNAs (C24) were digested with BfuCI or Hinfl. DNA gel blot analysis was performed using 32P-labeled telomere repeat as the probe. Telomeres were longer in the ros1 mutant than in the wild type (WT), ros3,orros1 ros3 double mutant. Signals corresponding to telomeric sequences are indicated by an asterisk. (B) Telomere lengths of the wild type, ros1,andros3 in Columbia accession. Genomic DNAs were digested with Hinfl and used for DNA gel blot. Signals corresponding to telomeric sequences are indicated by an asterisk. (C) Telomere lengths of the wild type, ros1-1, rfc1-1, rfc1-1 ros1-1, pol a, pol a ros1-1, pol «, ror1/rpa2a, and ror1/rpa2a ros1-1 mutants. Genomic DNAs were digested with HinflorMboI and used for DNA gel blot. Signals corresponding to telomeric sequences are indicated by an asterisk. (D) Gene expression of TERT, POT1a,andBT2 in the wild type, ros1-1, rfc1-1,andrfc1-1 ros1-1. Two-week-old seedlings were used for total RNA extraction. qRT-PCR was used to determine the expression level of each gene. Three experiments were independently done, each with triple technical replicates. The data represent one experiment results. Error bars indicate 6SD, n =3.

Telomerase reverse transcriptase (TERT) is the catalytic sub- was expressed at lower levels in rfc1-1 ros1-1 and rcf1-1 than in unit of the telomerase complex responsible for the addition of ros1-1 or in the C24 wild type (Figure 8C). The flowers and telomere repeats for telomere extension (Riha et al., 2001). siliques of rfc1-1 ros1-1 and rfc1-1 were also smaller than Telomeres 1A (POT1A) (three POT1 homologs: POT1A, POT1B, those of the C24 wild type or ros1-1 (Figures 8D and 8E). rfc1-1 and POT1C in the Arabidopsis genome) could interact with produced about one-half the seeds as the C24 wild type or TERT for protection of the telomere (Rossignol et al., 2007). ros1-1 (Figures 8E and 8F). When rfc1-1 ros1-1 was crossed with TELOMERASE ACTIVATOR1 regulates the expression of BT2 (a ros1-1, the defective growth phenotypes cosegregated with BTB and TAZ domain protein), which controls telomerase activity kanamycin resistance, indicating that these phenotypes are in Arabidopsis (Ren et al., 2007). We compared the expression caused by the same rfc1-1 mutation. levels of TERT, POT1a, and BT2 among ros1-1, rfc1-1, rfc1-1 ros1-1, and the wild type (Figure 7D). We found that the expres- Map-Based Cloning of RFC1 sion of these three genes was a little higher in ros1-1 than in the wild type but was lower in rfc1-1 and rfc1-1 ros1-1 than in the wild To clone the RFC1 gene, we crossed rfc1-1 (C24 accession) with type. It seems that the elongated telomere in ros1-1 or shortened gl1 (without trichomes, in the Columbia accession), selected the telomere in rfc1-1 might be partially due to changed expression rfc1-1 seedlings based on their smaller and earlier flowering of TERT, POT1a, and BT2. phenotypes, and used them for mapping with simple sequence length polymorphism markers. RFC1 was narrowed down to rfc1-1 Mutation Causes Hypomorphic Growth Phenotypes BAC clone T6G21 (Figure 8G). We sequenced the candidate and Earlier Flowering gene AT5G22010 and found a single G-to-A mutation in position 5421 (counting from the first putative ATG in the genomic rfc1-1 ros1-1 or rcf1-1 mutants were smaller and flowered earlier sequence), which is suspected to change the splice acceptor than the C24 wild type or ros1-1 when growing in soil (Figures 8A site from AG to AA in the 19th intron. We compared the cDNAs and 8B). The flowering suppressor gene Flower Locus C (FLC) amplified by RT-PCR from the C24 wild type, ros1-1, and rfc1-1 Replication Factor C and Epigenetic Instability 2345

Figure 8. Phenotypic Analysis of the rfc1-1 Mutant and Map-Based Cloning of the RFC1 Gene.

(A) Growth of rfc1-1, rfc1-1 ros1-1, ros1-1, and C24 wild type in soil for 3 weeks. (B) Mature plants of the C24 wild type, ros1-1, rfc1-1, and rfc1-1 ros1-1 grown in soil for 4 weeks. (C) Relative expression of FLC in rfc1-1, rfc1-1 ros1-1, ros1-1, and C24 wild type. Total RNA extracted from 3-week-old seedlings was reverse transcribed. The expression in C24 wild type was used as a standard for normalization control. Data are means of triple technical repetitions from one of three biologically independent experiments. Error bars indicate 6SD, n =3. (D) Flower size of the C24 wild type, ros1-1, rfc1-1,andrfc1-1 ros1-1. (E) Silique size of the C24 wild type, ros1-1, rfc1-1, and rfc1-1 ros1-1. 2346 The Plant Cell

ros1-1. The cDNA from rfc1-1 has lost 54 bp compared with the the largest subunit of the replication factor C complex, which is C24 wild type or ros-1-1 line because the mutation in the strongly conserved in all organisms, including human, mouse, acceptor splice site of the 18th intron resulted in mis-splicing and yeast. The middle part of yeast RFC1 (human p140, and moved the acceptor splice site to the 19th exon. To deter- Arabidopsis RFC1/AT5G22010) is homologous with the four mine whether the rfc1-1 mutation led to specific phenotypes, we small subunits, including yeast Rfc2 (human p37, Arabidopsis constructed a vector containing the full-length genomic DNA of RFC2/AT1G63160), yeast Rfc3 (human p36, Arabidopsis AT5G22010, including the wild-type AT5G22010 gene contain- RFC3/AT5G27740), yeast Rfc4 (human p40, Arabidopsis RFC4/ ing 2065 bp upstream of the first putative ATG and 349 bp AT1G21690), and yeast Rfc5 (human p38, Arabidopsis RFC5/ downstream of the putative stop codon TAA and transformed AT1G77470) (Cullmann et al., 1995; Ellison and Stillman, 1998; this vector to rfc1-1 ros1-1 mutants. We obtained several trans- Venclovas et al., 2002; Majka and Burgers, 2004; Shultz et al., genic plants and randomly selected five homozygous lines of the 2007). RFC1 proteins from different organisms are also highly T4 generation, all with the complemented growth (one line in conserved at the N- and C-terminal regions. Deleting the Figure 8H) and kanamycin-sensitive phenotype (two lines in N-terminal region of RFC1 in both yeast and human RFC does Figure 8I), confirming that the RFC1 gene is AT5G22010. not decrease RFC clamp loader activity with PCNA (Uhlmann We obtained an additional rcf1 allele, named rfc1-2,asa et al., 1997; Gomes et al., 2000; Gomes and Burgers, 2001). T-DNA insertion line (SALK_140231) with the T-DNA inserted in The RFC1 amino acid sequence has several nuclear localiza- the 21st intron of RCF1 (Figure 8G). The homozygous plants with tion signals that were predicted with help of the psort program T-DNA insertions did not exhibit any apparent growth defects (http://psort.nibb.ac.jp/). We examined the subcellular localiza- during the vegetative growth stage and had a similar flowering tion of the RFC1-green fluorescent protein (GFP) fusion gene in time as the wild type (Figure 8J). We were unable to test the effect both transient assays and stable transgenic Arabidopsis plants. of the rfc2-1 mutation on silenced 35S-NPTII in the ros1-1 mutant RFC1 was fused in frame to the N terminus of GFP and was as rfc2-1 carries a T-DNA insertion with a functional NPTII gene. expressed under the control of a super promoter (Gong et al., Mature plants of rfc1-2, however, produced fewer seeds than the 2002b). With the aid of a confocal microscope, GFP fluorescence wild type and did not produce any seeds in some of the siliques was mainly detected in the nucleus in a transient assay of an (Figures 8F, 8K, and 8L). These results suggest an essential role epidermal leaf cell or in the root of T2 transgenic lines carrying for the RFC1 in seed reproduction in Arabidopsis. RFC1-GFP (Figure 8O). As a control, GFP itself was detected in We constructed an RFC1 promoter-GUS fusion gene to ana- the cytoplasm. lyze RFC1 expression in different tissues. GUS expression was analyzed in the T2 lines of transgenic Arabidopsis expressing the RFC1 promoter-GUS transgene. GUS expression was substan- DISCUSSION tial in young inflorescence tissues and older leaves. GUS ex- pression was also high at the edges of leaves (Figure 8M). We In yeast, there are four different RFC complexes that share the used real-time RT-PCR to measure the relative expression level four small subunits (Rfc2p, Rfc3p, Rfc4p, and Rfc5p) but differ in of RFC1. RFC1 expression was detected in roots, stems, and the specific large subunit and therefore have different or over- leaves but was greater in flowers and young siliques (Figure 8N), lapping roles in DNA metabolism (Banerjee et al., 2007). The which is consistent with the promoter-GUS expression pattern. Rfc1-5p complex is needed for loading PCNA during general The open reading frame of RFC1 was amplified by RT-PCR. DNA replication or repair. Rfc1p can be replaced in the core RFC1 is predicted to encode a peptide of 956 amino acids with complex Rfc2-5 by other alternative clamp loaders, such as an estimated molecular mass of 104.26 kD, pI = 9.69. RFC1 is Rad24-RFC (At1g04730 is its homolog in Arabidopsis) in yeast,

Figure 8. (continued). (F) The average number of seeds per silique among 50 mature siliques in the C24 wild type, ros1-1, rfc1-1,andrfc1-1 ros1-1, or Columbia wild type and rfc1-2. Error bars indicate 6SD, n =50. (G) Positional cloning of the RFC1 gene. The RFC1 gene was first mapped between BAC clone F7C8 and MDJ22. Fine mapping delimited the RFC1 gene to BAC clone F13M11 and T6G21. The sequencing of some candidate genes identified a G5421 to A5421 point mutation in the acceptor splice site of the 18th intron in AT5G22010 (counting from the first putative ATG). The mutation moved the acceptor splice site to the next 54 bp (creating a putative new splice site AG in the 19th exon). As a result, rfc1-1 cDNA has 54 fewer base pairs than the wild type (the light-gray uppercase letters indicate the lost base pairs in rfc1-1; the light-gray lowercase letter indicates the intron). (H) The growth phenotype of rfc1-1 ros1-1 complemented with RFC1 genomic DNA. Compare with (A). All plants were grown in soil for 3 weeks. (I) The kanamycin-resistant phenotype of rfc1-1 ros1-1 complemented with RFC1 genomic DNA. (J) The growth phenotypes of Columbia wild type and rfc1-2 in soil for 3 weeks. (K) Inflorescences of Columbia wild type and rfc1-2. (L) Siliques of Columbia wild type and rfc1-2. (M) RFC1 promoter-GUS analysis in different tissues. (N) Relative expression of RFC1 in different tissues as determined by qRT-PCR. (O) RFC1-GFP localization in a transient assay (top panels show the GFP control, and bottom panels show RFC1-GFP) or in a root of a transgenic plant expressing 35S-RFC1-GFP gene. Replication Factor C and Epigenetic Instability 2347

which loads the PCNA-like protein complex Rad17-Ddc1-Mec3 cation factor C is an important component that displaces Pol around DNA (Majka and Burgers, 2003) for DNA damage check- a-primase to terminate primer synthesis early in DNA replication point; Rad17-RFC in humans, which loads the Rad9-Rad1-Hus1 and loads PCNA, which recruits Pol d for continuing the lagging heterotrimer clamp onto DNA (Bermudez et al., 2003); and Elg1 strand synthesis or Pol « for leading strand synthesis (Toueille (Human ATAD5, Arabidopsis homolog At1g77620) (Sikdar et al., and Hubscher, 2004; Garg and Burgers, 2005). Similar to the 2009) or Ctf18 in yeast, both of which are necessary for mutations in ror1/rpa2a, pol «/abo4, and pol a (Xia et al., 2006; HR-mediated DSB repair and negatively regulate telomere Yin et al., 2009; Liu et al., 2010), the rfc1 mutation released the length and sister chromatid cohesion (Hanna et al., 2001; Mayer TGS of the originally silenced 35S-NPTII, reduced the levels of et al., 2001; Naiki et al., 2001; Bellaoui et al., 2003; Ben-Aroya heterochromatin marker H3K9me2 in ros1, and increased the et al., 2003; Kanellis et al., 2003; Banerjee and Myung, 2004; expression of TSI. Our studies suggest that the DNA replication Smolikov et al., 2004; Ogiwara et al., 2007a, 2007b; Maradeo and machinery plays a crucial role in coupling DNA replication with Skibbens, 2009; Parnas et al., 2009). In this study, we provide DNA repair to restore chromatin structure in Arabidopsis. evidence that RFC1 in Arabidopsis regulates many aspects of Like the ror1/rpa2a and abo4/pol « mutants, the rfc1-1 mutant DNA metabolism, including DNA replication, DNA repair, HR, had increased sensitivity to the DNA-damaging agent MMS and TGS, and the control of telomere length. also to the DNA cross-linking agent cisplatin, suggesting that Maintaining genomic stability and integrity of both genetic RFC1 is involved in DNA repair, while pol d and pol a mutants are and epigenetic information is an elementary requirement for not sensitive to DNA-damaging agents (Xia et al., 2006; Schuermann all organisms during DNA replication and repair. Our genetic et al., 2009; Yin et al., 2009; Liu et al., 2010). Consistent screen for ros1 suppressors identified three genes, including with their increased sensitivity to DNA-damaging agents, rfc1-1 Pola/INCURVATA2, ROR1/RPA2A (Xia et al., 2006; Liu et al., mutants expressed increased levels of DNA repair-related 2010), and RFC1 (in this study), that are directly involved in DNA genes, such as RAD51 and BRCA1, both of which are involved replication, repair, and HR in plants. Pol«/ABO4 was identified in in DNA repair and SHR (Li et al., 2004; Abe et al., 2005); by a screening for root growth sensitivity to ABA, but it can also contrast, the pol d mutation does not result in increased expres- suppress the kanamycin-sensitive phenotype of ros1 (Yin et al., sion of DNA repair-related genes relative to the wild type 2009). Recently, Pold has been cloned because its mutations (Schuermann et al., 2009). The DNA repair-related genes were lead to increased SHR (Schuermann et al., 2009). Although also greatly increased in other DNA replication mutants, such as PCNA and primase have not been characterized yet, several core ror1/rpa2a and abo4/pol « (Xia et al., 2006; Yin et al., 2009). These DNA replication components in plants have been identified, and results suggest that Pol d might have different roles in DNA they participate in virtually all repair pathways needed to syn- replication and repair than other core DNA replication proteins. thesize DNA. Except for Pold, whose effect on TGS has not been Interestingly, we found that treatment with DNA-damaging tested yet, all of the other four genes affect TGS in an siRNA- agents can mimic the rfc1 mutation in releasing TGS of both directed DNA methylation (RdDM)-independent pathway (Xia NPTII and TSIs in the ros1 mutant (Yin et al., 2009). During the et al., 2006; Yin et al., 2009; Liu et al., 2010; this study). The repair of damaged DNA in the promoter regions of some cancer T-DNA locus used in our study contains RD29A-LUC and cells, the key proteins involved in establishing and maintaining 35S-NPTII with different regulation mechanisms. In ros1 mu- TGS are recruited to the sites of DNA breaks, which leads to the tants, the RD29A promoter is targeted by siRNAs and hyper- silencing of the targeted genes (O’Hagan et al., 2008; Khobta methylated (Gong et al., 2002a). The 35S promoter is found to et al., 2010). Results in this study indicate that DNA damage is be hypermethylated or hypomethylated when using different also able to activate the original silenced genes. Together, these primers for bisulfite sequencing (Kapoor et al., 2005b; Liu et al., findings suggest that DNA damage could have different effects 2010). However, unlike RFC1 and other DNA replication-related on both the active genes and the silenced genes. proteins that could release the TGS of 35S-NPTII in ros1 when To our surprise, the rfc1-1 mutant exhibited an extremely high mutated (Xia et al., 2006; Yin et al., 2009; Liu et al., 2010; this SHR. Mutations in core replication machinery proteins, including study), mutations of genes in RdDM pathway do not release TGS Pol a, Pol « , and Pol d, and replication protein A2 (ROR1/RPA2A) of 35S-NPTII in ros1 mutant (He et al., 2009a), suggesting that are known to increase SHR, with a mutation in Pol a increasing the TGS mechanism in 35S promoter is unique. However, the SHR only to a moderate level and mutations in Pol «, Pol d, and mechanism that regulates TGS of 35S-NPTII requires further ROR1/RPA2A increasing SHR to a high level (Schuermann et al., study in the future. 2009; Yin et al., 2009; Liu et al., 2010). In the study of Pol d, During DNA replication in animals, DNA methylation and however, Schuermann et al. (2009) found that DNA damage histone modifications can be maintained through PCNA interac- caused by DNA-damaging agents MMS, cisplatin, or bleomycin tion with DNA methyltransferase and many histone modification does not, but blocking DNA replication by a hydroxyurea reagent proteins, including the CAF-1 nucleosome assembly complex greatly increases the SHR in the pol d mutant relative to the wild (Probst et al., 2009). In Arabidopsis, CAF-1 subunits FAS1 and type. These core DNA replication mutants lack the ability to FAS2 regulate TGS (Ono et al., 2006; Schonrock et al., 2006). A efficiently repair DSBs during DNA replication, which might conserved protein interaction between Pol a and HP1 in animal spontaneously activate the S-phase checkpoint pathway re- and yeast or between Pol a and LHP in plants has been identified, quired for replisome stabilization and resolution of stalled forks. suggesting that inheritance of the heterochromatin state coupled The observation that CYCB1;1 is highly expressed in these with DNA replication is conserved in both plants and other mutants also suggests that the delayed M/G2 phase could organisms (Nakayama et al., 2001; Barrero et al., 2007). Repli- provide more time for cells to repair the DNA damage in the 2348 The Plant Cell

S-phase in these mutants (Xia et al., 2006; Yin et al., 2009; Liu rfc1-1 causes the mis-splicing of RFC1 pre-mRNA. rfc1-1 plants et al., 2010). The rfc1-1 mutant had an extremely high SHR rate, are smaller, flower earlier, and produce fewer seeds than the wild suggesting that RFC1 is a critical protein for preventing HR type. Cell sizes, however, are similar for rfc1-1 and the wild type, during DNA replication/repair. Leading-strand synthesis and suggesting that the small size of rfc1-1 plants is due to reduced lagging-strand synthesis are coordinated during DNA replication cell division and, therefore, fewer cells. The result suggests that with the lagging-strand polymerase working at a higher speed once DNA replication has finished, the expansion of the cell is not than the leading-strand polymerase (Pandey et al., 2009). RFC1 greatly influenced by the rfc1 mutation. Interestingly, vegetative is the key mediator that loads PCNA, which recruits both lagging- growth was similar for rfc1-2 and the wild type, but rfc1-2 strand polymerase Pol d and leading-strand polymerase Pol «, produced much fewer seeds than the wild type or rfc1-1.In and mutations in either Pol d or Pol « greatly increase SHR. It is Arabidopsis, two checkpoint proteins, ATM (ataxia telangiectasia- conceivable that impairment of RFC1 would lead to more stalled mutated) and ATR (ataxia telangiectasia-mutated and Rad3- forks and DSBs in both leading and lagging strands and that such related), are essential regulators of the cell cycle; these proteins strands could be used as substrates for SHR during DNA sense DNA damage and activate downstream components of cell replication. Interestingly, more SHR occurred in the older tissues cycle progression and DNA repair. Mutations in both ATM and than in the younger tissues or meristems of Arabidopsis seed- ATR do not affect the vegetative growth but do affect reproductive lings. The meristem is the most active site for DNA replication processes (Garcia et al., 2003; Culligan et al., 2004). Similar and cell division, while older tissues support active DNA endo- phenotypes are also produced by the RAD51 mutant (Li et al., reduplication but not cell division (Schuermann et al., 2009). It 2004). The diverse phenotypes exhibited by rfc1-1 and rfc1-2 seems that the core DNA replication proteins play different roles suggest that the mutated proteins might have different effects on in the older tissues versus meristems for the regulation of SHR. vegetative and reproductive development or that these mutations These DNA replication proteins likely influence SHR greatly in the have different effects in C24 and Columbia accessions. In both older tissues but not so much in the meristems (Schuermann accessions, it seems that these mutations affect the reproductive et al., 2009). process more than vegetative growth. Telomere homeostasis is regulated by telomerase and a number of DNA repair and recombination proteins. Deletion of METHODS the telomerase gene TERT in Arabidopsis causes the mutant to lose 300 to 500 bp of telomere per generation (Fitzgerald et al., Plant Materials and Growth Conditions 1999). Human RFC1 (RFC p140) can bind to the telomeric repeat and inhibit telomerase activity (Uchiumi et al., 1999). Mutations in The C24 wild type, ros1 mutation, and other Arabidopsis thaliana mutants DNA replication-related proteins, such as Pol a, RFC1, CTF18, (C24 background) mentioned in this article all carry the RD29A-LUC and and ELG1 in yeast, and KU70, KU80, and replication protein A 35S-NPTII transgenes. The T-DNA line (SALK_140231; at5g22010) was 70a in Arabidopsis, increase telomere length (Gallego et al., obtained from the Arabidopsis Stock Center. Sterilized seeds were sown onto MS medium with 2% (w/v) sucrose and 0.8% (w/v) agar. After 2 d, 2003; Takashi et al., 2009). Here, we found that loss of function of during which the seeds germinated, the plates were transferred to a ROS1 increased telomere length, while the impairing of RFC1 phytotron at 228C under long-day (23 h light/1 h dark) conditions. and other replication proteins decreased telomere length in Generally, 7-d-old seedlings were transferred to soil and cultured in a Arabidopsis. Telomere length in the ros3 mutant, however, was growth room with 228C (light) or 188C (dark) under 16-h-light/8-h-dark not changed, although ROS3 is in the same DAN demethylation conditions. pathway as ROS1 (Zheng et al., 2008). Because ROS1 can physically interact with RPA2A/ROR1 (Kapoor et al., 2005b; Xia Firefly Luciferase Imaging Analysis et al., 2006), it might affect telomere length by interfering with RPA2A. These results suggest that the molecular mechanisms Seedlings that had grown on MS medium for 7 d were transferred onto regulating telomere length are different in Arabidopsis than in filter paper saturated with 200 mM NaCl. After 4-h treatment, these plants were sprayed with D-luciferin (NanoFuels) assay buffer and immediately yeast. The expression of TERT, POT1a, and BT2 was a little subjected to CCD imaging analysis in a dark environment. higher in ros1 but much lower in rfc1-1 or rfc1-1 ros1-1 than in the wild type. Although TERT activities are not increased in the KU70 and KU80 mutants, telomere lengthening is apparently mediated Mutation Screening, Map-Based Cloning, and Mutant Complementation by telomerase activity rather than by recombination (Gallego et al., 2003). The results suggest that the lengthening or short- The isolation of the rfc1-1 mutant was described before (Xia et al., 2006). ening of the telomere might be somehow related to TERT and The rfc1-1 ros1-1 mutant was backcrossed four times to ros1-1 before other telomere-related proteins that are likely regulated by genetic analysis. For the cloning of the RFC1 gene, rfc1-1 ros1-1 was ROS1, RFC1, and other related DNA replication-related proteins. crossed with wild-type gl1 (Columbia accession), and 1485 plants that Like other DNA replication-related genes, such as Pol a, Pol «, showed rfc1-1 developmental phenotypes were selected from the F2 progeny. Map-based cloning was performed using simple sequence and Pol d (Barrero et al., 2007; Schuermann et al., 2009; Yin et al., length polymorphism markers (see Supplemental Table 1 online). The 2009; Liu et al., 2010), RFC1 should be an essential gene in mutated gene was mapped to chromosome 5 between BAC T10F18 and Arabidopsis because another subunit in the same complex with MWD9. Sequencing analysis revealed a G-to-A mutation in gene RFC1, RFC3, is essential in Arabidopsis (Xia et al., 2009). The AT5G22010. mutations used in this study are probably weak alleles because A full-length genomic DNA fragment (;8.8 kb, from 22065 to +6755 bp the two mutations occur in the C-terminal region. Mutation in containing from the putative start codon ATG) was released from BAC Replication Factor C and Epigenetic Instability 2349

clone T6G21 and subcloned into the pCAMBIA1391 binary vector (http:// method, 5-d-old seedlings were transferred from standard MS medium www.cambia.org/daisy/cambialabs/materials.html) by restriction enzyme (without DNA-damaging agents) to MS medium containing MMS, and sites SalIandBstEII. The strain GV3101 carrying this construct was seedlings were assessed and photographed after 2 weeks. transformed to the rfc1-1 ros1-1 mutant (Clough and Bent, 1998). The transgenic plants with resistance to hygromycin were selected, and F4 CYCB1;1:GUS Activity Assay generation plants without any segregation were analyzed for kanamycin resistance and compared with the growth phenotypes of wild-type plants. The reporter line carrying the CYCB1;1:GUS transgene was crossed with rfc1-1. The homozygous lines for both GUS reporter and rfc1 were selected from F3 progeny and were used for GUS activity analysis. The RNA Isolation and Real-Time PCR homozygous lines carrying only the GUS reporter were used as controls. Total RNA was extracted from the seedlings under different conditions using Trizol reagent (Invitrogen). After the RNA was digested with RNase- Recombination Assays free DNase I (NEB) at 378C for 20 min, 4 mg of total RNA was reverse The mutant ros1-1 rfc1-1 (accession C24) was crossed with the recom- transcribed by M-MLV reverse transcriptase (Promega) in a 20-mL sample bination reporter line NllC4 No.651, which carries overlapping 566-bp volume. The cDNA products to be subjected to quantitative real-time sequences of the GUS gene in inverted orientation (C24 ecotype) (Puchta PCR were diluted 20-fold. Each 20-mL reaction contained 10 mL SYBR et al., 1995). The homozygous lines for both recombination reporter and Green Master mix (Takara), 0.2 mM (final concentration) of each gene- ros1 and/or rfc1 were selected from F3 progeny plants. To monitor the specific primer (see Supplemental Table 2 online), and 2 mL cDNA. Three- SHR frequency, we stained different-stage seedlings with GUS as de- step PCR (denaturation, 10 s at 958C; annealing, 10 s at 618C; extension, scribed above. About 50 individual seedlings were analyzed for each 15 s at 728C) was carried out on a PTC-200 DNA engine cycler (MJ sample. To analyze the effect of cisplatin or MMS on SHR, we transferred Research) with Chromo4 Detector. The Ct values presented are the seedlings to MS medium containing 10 mM cisplatin or 50 ppm MMS and means of three biological replicates (means +SD, n = 3). Results of qRT- grew them for another 7 d before GUS staining. PCR were verified by at least three independent experiments.

ChIP Gene Expression and DNA Gel Blot Analysis ChIP was performed as described previously (Xia et al., 2006) using 18-d- The transcript levels of endogenous RD29A and TSI were determined by old seedlings grown on MS medium under long-day conditions (23 h light/ real-time RT-PCR. ACTIN was used as the control. The NPTII level was 1 h dark). Immunoprecipitations were performed with anti-H3K9me2 determined by immunoblots. DNA methylation changes in the RD29A (Upstate; 17-648) and anti-H3Ac (Upstate; 06-599) antibodies. Real-time promoter, 35S promoter, ribosome DNA, centromere DNA regions, and PCR analysis was performed using specific primers listed in Supplemen- retrotransposon Ta3 were analyzed by DNA gel blots with methylation- tal Table 2 online. sensitive restriction enzymes as previously described (Gong et al., 2002a).

Assay for Detection of Telomere Length Histochemical GUS Analysis Genomic DNA extracted from 3-week-old seedlings was digested ; A genomic DNA fragment ( 900 bp) of the RFC1 promoter upstream of overnight with the restriction enzymes BfuCI or Hinfl. The samples of di- 9 ATG was amplified by specific primers 5 -GATGGGAAGCTTTGGATGG- gested DNA (30 mg) were subjected to DNA gel blot analysis as de- 9 9 9 GAAGTG-3 and 5 -TGCCCGTCTCGAGAATTCGACTAC-3 and then scribed previously (Gallego et al., 2003). The telomeric repeat probe cloned into the pCAMBIA1391 vector. The fused RFC1 promoter-GUS [59-(TTTAGGG)7] was directly synthesized, and the 59 end was labeled gene construct was transformed into Arabidopsis. The transgenic T2 by [g-32P]ATP using T4 polynucleotide kinase. lines, which were resistant to hygromycin, were subjected to a GUS activity assay as described before (Yin et al., 2009). Accession Numbers

Localization of the RFC1-GFP Fusion Protein Sequence data from this article can be found in the Arabidopsis Genome Initiative or GenBank/EMBL databases under the following accession The full-length cDNA of the RFC1 gene was amplified from total numbers: RAD51 (at5g20850), Ku70 (at1g16970), ATGR (at3g52115), cDNA from flowers (C24 accession) by the PCR primer pair: cDNA-SalI- BRCA (at4g21070), MRE11 (at5g54260), CYCB1;1 (at4g37490), TERT F(59-CGCGTCGACGTAGTCGAATTCTCGAGACGGGCAAATG-39)and (at5g16850), POT1a (at2g05210), BT2 (at3g48360), FLC (at5g10140), cDNA-SpeI-R (59-CGCACTAGTTCTCTTTCTCTTGGCACCAGAGCC-39). RFC1 (at5g22010), and ACTIN2 (atg3g18780). The PCR products were directly digested by SalIandSpeI and then cloned into the modified vector pCAMBIA1300. GFP was fused in frame Supplemental Data to RFC1 at its C terminus. The Agrobacterium tumefaciens strain GV3101 carrying RFC1-GFP constructs together with the P19 strain were injected The following materials are available in the online version of this article. into 4-week-old tobacco (Nicotiana benthamiana) leaves using a plastic Supplemental Table 1. Primer Pairs of the Markers Used for Map- syringe as described before (Walter et al., 2004). The transient GFP Based Cloning. fluorescence was imaged with a confocal laser scanning microscope (LSM510; Carl Zeiss). Supplemental Table 2. The Primers Used for Real-Time PCR and ChIP.

DNA Damage Assay

Two methods were used to measure the sensitivity to DNA-damaging ACKNOWLEDGMENTS agents. In one method, seeds were sown directly on solid MS medium containing the specified concentration of cisplantin or MMS, and seed- This work was supported by the National Nature Science Foundation of lings were assessed and photographed after 3 weeks. In the second China (30630004 and 30721062), the National Transgenic Research 2350 The Plant Cell

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