DNA Replication Factor C1 Mediates Genomic Stability andTranscriptional 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, Chinab Institute for Integrative Genome Biology and Department of Botany and Plant Sciences, University of California, Riverside,

California 92521c Center for Plant Stress Genomics and Technology, 4700 King Abdullah University of Science and Technology, Thuwal 23955-

6900, Kingdom of Saudi Arabiad China Agricultural University–University of California, Riverside Center for Biological Sciences and Biotechnology, Beijing

100193, Chinae 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

During DNA replication, DNA polymerase a interacts with DNA

primase to form a complex for initiating synthesis of a 15-20

mer DNAprimer using anRNAprimer. Replication factor C (RFC),

a clamp-loader complex consisting of five different subunits,

binds DNA at the template–primer junctions and displaces

polymerase a to terminate DNA primer synthesis. The binding

of RFC to DNA creates a loading site for recruiting the DNA

sliding clamp proliferating cell nuclear antigen (PCNA), a ring-

shaped homotrimer. RFC is an AAA+-type ATPase that requires

ATP hydrolysis for opening and closing PCNA around DNA

during DNA replication, repair, and recombination (Majka and

Burgers, 2004).

The formation of the DNA replication fork can be stalled or

arrested duringDNA replication if theDNA structure is chemically

or physically altered by double-strand breaks (DSBs). DSBs

can be repaired by homologous recombination (HR), which will

reestablish a formal replication fork. DNA damage can activate

the S-phase replication checkpoint pathway that helps stabilize

replication forks and prevents the breakdown of replication forks

(Branzei and Foiani, 2009). Chromosome ends called telomeres

are natural DNA breaks. However, telomeres have specific DNA

structures that are protected by various proteins with negative or

positive effects on telomere length (Smolikov et al., 2004). When

the DNA replication machinery meets telomeres, it competes

with telomerase (Shore and Bianchi, 2009). Telomeres can be

elongated by some mutations, such as in Rfc1 and DNA poly-

merase a, suggesting that the replication machinery regulates

telomere length (Adams andHolm, 1996). Abnormal regulation of

telomere maintenance may evoke a DNA damage response

leading to the repair of telomeres by HR.

DNA replication is a highly regulated process that accurately

replicates both the primary DNA sequence and chromatin struc-

ture from the parent strands. The transmission of heterochro-

matin structure and DNA methylation is an essential process

after the DNA has been replicated and is tightly regulated by

various proteins (Shultz et al., 2007; Kloc andMartienssen, 2008;

1 These authors contributed equally to this work.2 Address correspondence to gongzz@cau.edu.cn.The author responsible for distribution of materials integral to thefindings presented in this article in accordance with the policy describedin the Instructions for Authors (www.plantcell.org) is: Zhizhong Gong(gongzz@cau.edu.cn).WOnline version contains Web-only data.OAOpen Access articles can be viewed online without a subscription.www.plantcell.org/cgi/doi/10.1105/tpc.110.076349

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

Martienssen et al., 2008; Probst et al., 2009). Furthermore,

epigenetic markers can be changed after the replication ma-

chinery has passed the DNA replication fork during cell differen-

tiation and development. In fission yeast, DNA polymerase

a interacts with the heterochromatin protein Swi6, a homolog

of HETEROCHROMATIN PROTEIN1 (HP1), an important deter-

minant of the epigenetic imprint in vivo, and influences the

localization of Swi6 (Nakayama et al., 2001). In Arabidopsis

thaliana, DNA polymerase a physically and genetically interacts

with LIKE HETEROCHROMATIN PROTEIN1 (LHP1) (Barrero

et al., 2007), suggesting that the DNA replication machinery

involved in the epigenetic imprint is conserved among different

organisms. Early studies in yeast that included screens for

transcriptional silencing genes identified several genes that

encode DNA replication-related proteins, including Rfc1 and

some chromatin structure modulators (Ehrenhofer-Murray et al.,

1999; Smith et al., 1999). Homotrimeric PCNA is considered

an essential component in recruiting the general mediators

for chromatin imprinting of epigenetic markers (Probst et al.,

2009). Genetic studies in Arabidopsis have also identified some

other DNA replication- and repair-related proteins involved in the

regulation of transcriptional gene silencing (TGS); these include

BRU1, Chromatin assembly factor (CAF-1), REPLICATION

PROTEIN A2 (RPA2), DNA polymerase a, and DNA polymerase «

(Takeda et al., 2004; Elmayan et al., 2005; Kapoor et al., 2005b;

Ono et al., 2006; Schonrock et al., 2006; Xia et al., 2006; Yin et al.,

2009; Liu et al., 2010).

The well-studied phenomenon of TGS in plants is mediated by

small interfering RNA (siRNA)-directed DNAmethylation (RdDM),

a dynamic process in which the DNA methylation patterns

depend on the production of siRNAs at different developmental

stages or under different conditions (Henderson and Jacobsen,

2007; Matzke et al., 2007, 2009; Law and Jacobsen, 2009;

Teixeira et al., 2009). RdDM usually occurs in transposons and

DNA repeat regions accompanied by the formation of hetero-

chromatin and accounts for ;30% of the total DNA methyla-

tion in Arabidopsis (Matzke et al., 2009). DNA methylation can

be reversibly removed by a ROS1 (for REPRESSOR OF

SILENCING1) family of DNA glycosylase/lyases, including ROS1,

DEMETER, and two DEMETER-like proteins, DML2 and DML3

(Gong et al., 2002a; Kapoor et al., 2005a; Morales-Ruiz et al.,

2006). ROS1 was the first-characterized DNA demethylation

enzyme and was originally isolated during a screen for genes

whose mutations silence the expression of the foreign transgene

RD29A-LUC in a T-DNA region (Gong et al., 2002a). This T-DNA

region contains an RD29A-LUC gene and a 35S-NPTII gene.

ROS1mutations lead to TGS of bothRD29A-LUC and 35S-NPTII

by different regulation mechanisms (i.e., the former depends on

RdDM, but the latter does not). When silenced RD29A-LUC was

used as a screening marker to identify ros1 suppressors, most

genes isolated were in the RdDM pathway (He et al., 2009a,

2009b); when silenced 35S-NPTII was used as a screening

marker for ros1 suppressors, most isolated genes are indepen-

dent of RdDM (Kapoor et al., 2005b; Xia et al., 2006; Wang et al.,

2007; Shen et al., 2009). The latter group of genes include ROR1/

RPA2A (Xia et al., 2006), TOUSLED protein kinase (Wang et al.,

2007), DNA polymerase a (Liu et al., 2010), and chlorophyll a/b

binding protein gene underexpressed1 (Shen et al., 2009).ABO4/

DNA polymerase « was isolated in a root-sensitive-to-abscisic

acid (ABA) screen and is also involved in TGS in an RdDM-

independent manner (Yin et al., 2009). In this study, we charac-

terized another ros1 suppressor, RFC1, the largest subunit of the

RFC complex in Arabidopsis. Mutations in RFC1 lead to devel-

opmental defects and earlier flowering. Our results indicate that

RFC1 is an important mediator of TGS, DNA replication, DNA

repair, HR, and telomere length regulation.

RESULTS

The rfc1Mutation Releases the TGS of 35S-NPTII in the

ros1Mutant

The accession used in this study, C24 RD29A-LUC, carries a T-DNA

locus that contains active (i.e., unsilenced)RD29A-LUC and 35S-

NPTII genes. This accession is herein referred to as the C24 wild

type. ros1 mutations lead to the silencing of both RD29A-LUC

and 35S-NPTII, and the ros1-1 mutant is thus sensitive to

kanamycin (Gong et al., 2002a). A kanamycin-resistant mutant,

named rfc1-1, was isolated from ethyl methanesulfonate–

mutagenized ros1-1 plants, for which the M2 generation was

grown on Murashige and Skoog (MS) medium supplemented

with 50 mg/L kanamycin. Genetic analysis using F2 seedlings

from rfc1-1 ros1-1 backcrossed with ros1-1 on 50 mg/L kana-

mycin indicated that the rfc1-1mutation was caused by a single

recessive nuclear gene (Figure 1A). We tested the kanamycin-

sensitive phenotypes of rfc1-1 ros1-1 on different concentrations

of kanamycin. As shown in Figure 1B, the rfc1-1 ros1-1 mutant

was more tolerant to kanamycin than ros1-1 but less tolerant

than the C24 wild type or rfc1-1. Immunoblot analysis indicated

that rfc1-1 ros1-1 expressed more NPTII protein than ros1-1 but

less than the C24wild type (Figure 1C).We crossed rfc1-1 ros1-1

with the C24 wild type and isolated the single rfc1-1 mutant and

tested its kanamycin sensitivity. rfc1-1 mutants had a slightly

increased kanamycin sensitivity and a slightly reduced quantity

of NTPII protein relative to theC24wild type (see 350mg/L Kan in

Figures 1B and 1C), suggesting that the rfc1-1 mutation did not

simply act to increase NPTII expression in the C24 wild type.

To determine whether the rfc1-1 mutation influences the ex-

pression of RD29A-LUC and the endogenous RD29A gene, we

compared their expression after treatment with 50 mMABA for 4 h

(for RD29A expression) or 200 mM NaCl treatment for 4 h (for

LUC image). As shown in Figure 1D, ros1-1or rfc1-1 ros1-1 emitted

little or no luminescence, but the C24 wild type and rfc1-1 emitted

strong luminescence. Real-time RT-PCR analysis showed that

endogenous RD29A was highly expressed in the C24 wild type

and rfc1-1butwas silenced inboth ros1-1 and rfc1-1 ros1-1 (Figure

1E). We further used real-time RT-PCR to check the expression

of the pericentromeric Athila retrotransposons named transcrip-

tional silent information (TSI). As shown in Figure 1F, the transcripts

of TSI were more expressed in rfc1-1 and rfc1-1 ros1-1 than in

theC24wild type or ros1-1. We also checked the expression of TSI

in an rfc1-2 mutant of Columbia accession (a T-DNA insertion

mutant; see below) and found that TSIswere expressed at a higher

level than in the wild type. These results indicate that themutations

in RFC1 release the TGS in some genomic loci.

Replication Factor C and Epigenetic Instability 2337

The rfc1-1Mutation Does Not Change DNAMethylation at

the RD29A Promoter or at Other DNA Repeat Regions

To test whether the rfc1 mutation affects DNA methylation lev-

els, we analyzed the DNA methylation patterns in the RD29A

promoter, the 35S promoter, the 5S rDNA region, the 180-bp

centromere DNA repeat region, and the pericentromeric retro-

transposon Ta3 using methylation-sensitive restrictive enzymes.

RD29A and 5S rDNA are targeted by RdDM, and the 35S

promoter, the 180-bp centromere, as well as the Ta3 retrotrans-

poson are not controlled by RdDM (Chan et al., 2006). As

reported previously, MluI (AmCGCGT) and BstUI (CGmCG) en-

zymes did not completely cleave the RD29A promoter in ros1-1

because of hypermethylation at these two sites (Figure 2A). The

rfc1-1 mutation did not change the DNA digestion pattern

produced by MluI and BstUI in ros1-1. By contrast, MluI and

BstUI enzymes could efficiently digest the RD29A promoter in

the C24 wild type. For the 35S promoter, no methylation differ-

ence was found among the different genomic DNAs when they

were digested with DdeI and XmiI. We further compared the

DNA methylation levels in the centromere, rDNA, and the Ta3

transposon using two methylation-sensitive enzymes, HpaII

(mCmCGG methylation sensitive) andMspI (mCCGG methylation

sensitive), and we did not find any apparent differences among

the C24 wild type, ros1-1, and rfc1-1 ros1-1 (Figure 2A). These

results suggest that RFC1 mutation does not influence the DNA

methylation at these sites.

The rfc1-1Mutation Decreases the H3K9 Dimethylation at

the 35S Promoter in ros1-1

Because previous studies indicate that the expression of

35S-NPTII is related to histone modification (Xia et al., 2006;

Wang et al., 2007; Yin et al., 2009), we performed chromatin

immunoprecipitation (ChIP) to check the histone modifications

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 mMABA 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.

2338 The Plant Cell

in the 35S promoter region using antidimethylated histone H3

Lys 9 (H3K9me2), which is usually an epigenetic marker for het-

erochromatin. Here, we used Ta3 as a control for H3K9me2 as

described previously (Liu et al., 2010). H3K9me2 was detected

less in the 35S promoter of rfc1-1 ros1-1 than of ros1-1 but was

detected at similar levels either in the totalRD29A (including both

the transgenic and the endogenous RD29A promoter) or in the

endogenous RD29A promoter in both rfc1-1 ros1-1 and ros1-1.

No clear difference in H3K9me2 level, however, was found

between rfc1-1 and the C24 wild type in either the 35S or

RD29A promoter (Figure 2B).

rfc1-1Mutants Are Sensitive to DNA-Damaging Agents and

Constitutively Express High Levels of DSB-Induced Genes

Although the DNA repair function of RFC1 has been studied in

yeast and Aspergillus (McAlear et al., 1996; Kafer and Chae,

2008), there is no direct evidence of RFC1 involvement in DNA

repair in humans (Wood et al., 2001; Kafer and Chae, 2008). We

compared the sensitivity of rfc1-1 with the wild type to the

radiomimetic agent methylmethane sulphonate (MMS), which

causes DNA damage (Lundin et al., 2005). Seeds of the C24 wild

type, ros1-1, rfc1-1, and rfc1-1 ros1-1 were sown on MS agar

medium supplementedwith 125 ppmMMS (or withoutMMS as a

control) and were kept in a growth chamber for 3 weeks to

compare postgermination growth. Because seedlings at differ-

ent stages exhibit different sensitivities toMMS, another test was

done in which 5-d-old seedlings grown without MMS were

transferred to MS medium containing 0 or 150 ppm MMS for 2

weeks. As shown in Figures 3A and 3B, rfc1-1 ros1-1 and rfc1-1

were more sensitive to MMS than the C24 wild type or ros1-1 in

both postgermination growth (the first test) and in seedling

growth (the second test). We also compared the sensitivity of

rfc1-1 and the wild type to cisplatin. Cisplatin (cis-diamminedi-

chloroplatinum or cis-DDP), which is used as an anticancer

chemical, coordinates to DNAmainly through the nitrogen atoms

(specifically, the N7 atoms of purines) to produce intra- and

interstrand DNA cross-links. During DNA replication, both types

of DNA cross-links induce DSBs, which are repaired predomi-

nantly by HR during the S-phase (Frankenberg-Schwager et al.,

2005; Osakabe et al., 2006). Seeds were sown on MS medium

containing different concentrations of cisplatin, and seedling

growth was observed after 2 weeks. As shown in Figure 3C, both

rfc1-1 ros1-1 and rfc1-1mutants were muchmore sensitive than

the wild type or ros1-1 to 20 and 30 mM cisplatin. Figure 3D

indicates the number of true leaves of seedlings as affected by

cisplatin concentration and genotype. These results suggest a

critical role of RFC1 in repairing DNA damage in plant cells.

In a previous study on DNA polymerase «/ABO4, we found

that several marker genes functioning in DSBs are constitu-

tively expressed in the abo4 mutant (Yin et al., 2009). We

determined the expression of the following DNA repair and/or

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.

Replication Factor C and Epigenetic Instability 2339

cell cycle checkpoint genes: KU70, encoding a repairing pro-

tein in the nonhomologous end joining (NHEJ) pathway;RAD51,

encoding a recombinase in HR repair; BREAST CANCER

SUSCEPTIBLITY1 (BRCA1); and GAMMA RESPONSE1 (GR1)

in HR. Under normal growth conditions, rfc1-1mutants express

higher levels of GR1, RAD51, KU70, and BRCA1 than the C24

wild type (Figure 3E). Expression ofMRE11 did not clearly differ

between rfc1-1 and the C24 wild type (Figure 3E). MMS treat-

ment greatly induced the expression of RAD51 and BRCA1 in

both the C24 wild type and the rfc1-1 mutant (Figures 3F and

3G). The relative increase in expression in response toMMS did

not clearly differ between rfc1-1 and the wild type. These

results indicate that DNA repair-related genes were spontane-

ously induced by rfc1-1 mutation.

Treatment with DNA-Damaging Agents Mimics the rfc1

Mutation in Releasing TGS of 35S-NPTII

To determine whether the releasing of TGS is directly related to

genomic instability caused by DNA damage, we compared the

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, and MRE 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 ppmMMS 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.

2340 The Plant Cell

growth of ros1 seedlings on MS medium containing 50 mg/L

kanamycin and supplemented or not supplemented with MMS

or cisplatin. If treatment with DNA-damaging agents released

the TGS of 35S-NPTII, the treated ros1-1 seedlings should

be resistant to kanamycin. We sowed the seeds directly on MS

medium (as a seed germination and growth control) or on MS

medium containing 50 mg/L kanamycin, 50 mg/L kanamycin

plus 50 ppm MMS, or 50 mg/L kanamycin plus 10 mM cisplatin.

Here, we used 50 ppmMMS and 10mMcisplatin because higher

concentrations of these two agents will greatly inhibit seedling

growth, making comparison of seedling growth difficult. As

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 mMcisplatin. We further used quantitative RT-PCR (qRT-

PCR) to compare the transcripts ofNPTII under MMS or cisplatin

treatment. Seedlings (7 d old) were transferred to MSmedium 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.

rfc1-1Mutants 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

rfc1-1mutant. 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

were compared between thewild type and the rfc1-1mutant.We

compared the number of GUS spots in seedlings grown for

different periods (Figure 5). Generally, only a few spots (most

were in cotyledons) could be observed on each wild-type or

ros1-1 mutant seedling (Figures 5A to 5C). By contrast, a high

frequency of SHR was observed in the rfc1-1 and the rfc1-1

ros1-1mutants, especially in the cotyledons of 7-, 15-, and 20-d-

old seedlings (Figures 5A and 5B) or in the first two true leaves of

the 15-d-old seedlings (Figures 5A and 5C) or 20-d-old seedlings

(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.

Because RFC1 is expressed at higher levels in older leaves (see

Figure 8), this suggests that RFC1 might substantially contribute

to the restriction of SHR in these older tissues.We also tested the

SHR rate in seedlings treated with 10 mM cisplatin or 50 ppm

MMS for 8 d. These treatments apparently increased the SHR in

that more GUS spots occurred with cisplatin treatment or MMS

treatment than with the control in true leaves (it is difficult to

compare numbers of GUS spots in cotyledons) in the wild type

(Figures 5D and 5E), ros1-1, rfc1-1, or rfc1-1 ros1-1 (Figure 5F for

cisplatin treatment, it is hard to count the GUS spots for MMS

treatment).

Figure 4. Treatment with DNA-Damaging Agents Released the TGS of

35S-NPTII.

(A) Seedling growth on MS medium or MS medium supplemented with

50 mg/L kanamycin (Kan), 50 mg/L kanamycin and 50 ppm MMS or

50 mg/L kanamycin and 10 mM cisplatin. Seeds were directly sown on

the different media and were photographed after 2 weeks. WT, wild type.

(B) The relative expression levels of NPTII as determined by qRT-PCR

after MMS and cisplatin treatment. Three biologically independent

experiments were done. The data are from one experiment with triple

technical repetitions. Error bars indicate 6sd, n = 3 (*P < 0.05). Con,

control.

Replication Factor C and Epigenetic Instability 2341

The rfc1-1Mutation Reduces Cell Division, Delays the Cell

Cycle, and Causes a High Expression of Cyclin CYCB1;1

rfc1-1 plants are smaller than the C24 wild type. We compared

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

differ between the rfc1-1 mutant and the C24 wild type, indi-

cating that the rfc1-1 plants are smaller because they have

fewer cells rather than smaller cells. In DNA polymerase « and

DNA polymerase a mutants, the cell cycle was delayed and

there was high expression of a G2-M marker gene cyclin

CYCB1;1 (Yin et al., 2009; Liu et al., 2010). The CYCB1;1

promoter-GUS reporter was introduced into the rfc1-1 mutant.

As illustrated in Figure 6C, more GUS staining was detected in

the root tips, shoot meristems, and young leaves of the rfc1-1

mutant than of the C24 wild type, indicating that the G2/M

phases were longer in the rfc1-1 mutant. qRT-PCR confirmed

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, and rfc1-1 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.

2342 The Plant Cell

the higher expression of CYCB1;1 in rfc1-1 than in the C24

wild type (Figure 6D). MMS treatment induced a high level of

CYCB1;1 expression in both rfc1-1 and the C24 wild type

(Figure 6E), suggesting that a delay in the G2/M phases is

necessary for DNA repair.

ros1Mutation Increases the Telomere Length and rfc1

Mutation Decreases the Telomere Length and Reduces

Expression of Telomerase

Previous studies suggest that the DNA replicationmachinery can

compete with telomerase and control telomere length (Adams

and Holm, 1996). ROS1 physically interacts with replication

protein A2 in a yeast two-hybrid assay, suggesting that ROS1

might be involved in DNA replication (Kapoor et al., 2005b; Xia

et al., 2006). We compared telomere length between ros1 and

the C24 wild type. As illustrated in Figure 7A, telomeres were a

little longer in ros1 than in the C24 wild type. In the Columbia

accession, telomere length was also longer in the ros1 mutant

than in the wild type (Figure 7B). ROS3 is an RNA binding protein

working in the same pathway as ROS1 for DNA demethylation

(Zheng et al., 2008). The telomere length of ros3 was similar to

that of thewild type (Figure 7A). The telomere length of the ros1-1

ros3 mutant was similar to that of ros3 and the wild type.

These results suggest that ROS1 has a unique role in negatively

regulating telomere length in different Arabidopsis accessions.

Increasing evidence suggests that many DNA repair-related

proteins play crucial roles in regulating telomere length (Gallego

and White, 2005). Because rfc1 mutants are defective in DNA

repair with increasedSHR,we testedwhether theRFC1mutation

influences telomere length. Because telomere length varies

among different individuals, we collected 2-week-old seedlings

and used the total mixed DNAs from these plants. As shown in

Figure 7C, the rfc1-1 mutation greatly decreased the telomere

length relative to the C24 wild type or ros1-1, while the telomere

length in rfc1-1 ros1-1 was comparable to that in the rfc1-1

mutant. We also checked three other DNA replication mutants,

pol a, ror1/rpa2a, and pol «. The telomeres in pol amutants were

only a little shorter than in the wild type. The telomeres of the pol

a ros1-1mutant were a little longer than that of pol a but shorter

than that of ros1-1 and were similar to the wild type. The

telomeres in the pol « mutant were much shorter than in the

wild type. Telomeres of ror1 and ror1 ros1-1mutants were similar

in length and were a little shorter that those of the wild type or

ros1-1. Together, these results indicate that the four different

replication mutants have different effects on telomere length.

Among them, rfc1-1 reduces telomere length the most and pol

a reduces telomere length the least.

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.

Replication Factor C and Epigenetic Instability 2343

Telomerase reverse transcriptase (TERT) is the catalytic sub-

unit of the telomerase complex responsible for the addition of

telomere repeats for telomere extension (Riha et al., 2001).

Telomeres 1A (POT1A) (three POT1 homologs: POT1A, POT1B,

and POT1C in the Arabidopsis genome) could interact with

TERT for protection of the telomere (Rossignol et al., 2007).

TELOMERASE ACTIVATOR1 regulates the expression of BT2 (a

BTB and TAZdomain protein), which controls telomerase activity

in Arabidopsis (Ren et al., 2007). We compared the expression

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-

sion of these three genes was a little higher in ros1-1 than in the

wild type butwas lower in rfc1-1 and rfc1-1 ros1-1 than in thewild

type. It seems that the elongated telomere in ros1-1 or shortened

telomere in rfc1-1 might be partially due to changed expression

of TERT, POT1a, and BT2.

rfc1-1Mutation Causes Hypomorphic Growth Phenotypes

and Earlier Flowering

rfc1-1 ros1-1 or rcf1-1mutants were smaller and flowered earlier

than the C24 wild type or ros1-1when growing in soil (Figures 8A

and 8B). The flowering suppressor gene Flower Locus C (FLC)

was expressed at lower levels in rfc1-1 ros1-1 and rcf1-1 than in

ros1-1 or in the C24 wild type (Figure 8C). The flowers and

siliques of rfc1-1 ros1-1 and rfc1-1 were also smaller than

those of the C24 wild type or ros1-1 (Figures 8D and 8E). rfc1-1

produced about one-half the seeds as the C24 wild type or

ros1-1 (Figures 8E and 8F). When rfc1-1 ros1-1was crossedwith

ros1-1, the defective growth phenotypes cosegregated with

kanamycin resistance, indicating that these phenotypes are

caused by the same rfc1-1 mutation.

Map-Based Cloning of RFC1

To clone theRFC1 gene, we crossed rfc1-1 (C24 accession) with

gl1 (without trichomes, in the Columbia accession), selected the

rfc1-1 seedlings based on their smaller and earlier flowering

phenotypes, and used them for mapping with simple sequence

length polymorphism markers. RFC1 was narrowed down to

BAC clone T6G21 (Figure 8G). We sequenced the candidate

gene AT5G22010 and found a single G-to-A mutation in position

5421 (counting from the first putative ATG in the genomic

sequence), which is suspected to change the splice acceptor

site from AG to AA in the 19th intron. We compared the cDNAs

amplified by RT-PCR from the C24 wild type, ros1-1, and rfc1-1

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

(A) The ros1mutation but not the ros3mutation increased telomere length. Genomic DNAs (C24) were digested withBfuCI orHinfl. DNA gel blot analysis

was performed using 32P-labeled telomere repeat as the probe. Telomeres were longer in the ros1mutant than in the wild type (WT), ros3, or ros1 ros3

double mutant. Signals corresponding to telomeric sequences are indicated by an asterisk.

(B) Telomere lengths of the wild type, ros1, and ros3 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 ros1-1mutants. Genomic DNAs

were digested with Hinfl or MboI and used for DNA gel blot. Signals corresponding to telomeric sequences are indicated by an asterisk.

(D) Gene expression of TERT, POT1a, and BT2 in the wild type, ros1-1, rfc1-1, and rfc1-1 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.

2344 The Plant Cell

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, and rfc1-1 ros1-1.

(E) Silique size of the C24 wild type, ros1-1, rfc1-1, and rfc1-1 ros1-1.

Replication Factor C and Epigenetic Instability 2345

ros1-1. The cDNA from rfc1-1 has lost 54 bp compared with the

C24 wild type or ros-1-1 line because the mutation in the

acceptor splice site of the 18th intron resulted in mis-splicing

and moved the acceptor splice site to the 19th exon. To deter-

mine whether the rfc1-1mutation led to specific phenotypes, we

constructed a vector containing the full-length genomic DNA of

AT5G22010, including the wild-type AT5G22010 gene contain-

ing 2065 bp upstream of the first putative ATG and 349 bp

downstream of the putative stop codon TAA and transformed

this vector to rfc1-1 ros1-1 mutants. We obtained several trans-

genic plants and randomly selected five homozygous lines of the

T4 generation, all with the complemented growth (one line in

Figure 8H) and kanamycin-sensitive phenotype (two lines in

Figure 8I), confirming that the RFC1 gene is AT5G22010.

We obtained an additional rcf1 allele, named rfc1-2, as a

T-DNA insertion line (SALK_140231) with the T-DNA inserted in

the 21st intron of RCF1 (Figure 8G). The homozygous plants with

T-DNA insertions did not exhibit any apparent growth defects

during the vegetative growth stage and had a similar flowering

time as thewild type (Figure 8J).Wewere unable to test the effect

of the rfc2-1mutation on silenced 35S-NPTII in the ros1-1mutant

as rfc2-1 carries a T-DNA insertion with a functional NPTII gene.

Mature plants of rfc1-2, however, produced fewer seeds than the

wild type and did not produce any seeds in some of the siliques

(Figures 8F, 8K, and 8L). These results suggest an essential role

for the RFC1 in seed reproduction in Arabidopsis.

We constructed an RFC1 promoter-GUS fusion gene to ana-

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-

tial in young inflorescence tissues and older leaves. GUS ex-

pression was also high at the edges of leaves (Figure 8M). We

used real-time RT-PCR to measure the relative expression level

of RFC1. RFC1 expression was detected in roots, stems, and

leaves but was greater in flowers and young siliques (Figure 8N),

which is consistent with the promoter-GUS expression pattern.

The open reading frame of RFC1 was amplified by RT-PCR.

RFC1 is predicted to encode a peptide of 956 amino acids with

an estimated molecular mass of 104.26 kD, pI = 9.69. RFC1 is

the largest subunit of the replication factor C complex, which is

strongly conserved in all organisms, including human, mouse,

and yeast. The middle part of yeast RFC1 (human p140,

Arabidopsis RFC1/AT5G22010) is homologous with the four

small subunits, including yeast Rfc2 (human p37, Arabidopsis

RFC2/AT1G63160), yeast Rfc3 (human p36, Arabidopsis

RFC3/AT5G27740), yeast Rfc4 (human p40, Arabidopsis RFC4/

AT1G21690), and yeast Rfc5 (human p38, Arabidopsis RFC5/

AT1G77470) (Cullmann et al., 1995; Ellison and Stillman, 1998;

Venclovas et al., 2002; Majka and Burgers, 2004; Shultz et al.,

2007). RFC1 proteins from different organisms are also highly

conserved at the N- and C-terminal regions. Deleting the

N-terminal region of RFC1 in both yeast and human RFC does

not decrease RFC clamp loader activity with PCNA (Uhlmann

et al., 1997; Gomes et al., 2000; Gomes and Burgers, 2001).

The RFC1 amino acid sequence has several nuclear localiza-

tion signals that were predicted with help of the psort program

(http://psort.nibb.ac.jp/). We examined the subcellular localiza-

tion of the RFC1-green fluorescent protein (GFP) fusion gene in

both transient assays and stable transgenic Arabidopsis plants.

RFC1 was fused in frame to the N terminus of GFP and was

expressed under the control of a super promoter (Gong et al.,

2002b).With the aid of a confocal microscope, GFP fluorescence

was mainly detected in the nucleus in a transient assay of an

epidermal leaf cell or in the root of T2 transgenic lines carrying

RFC1-GFP (Figure 8O). As a control, GFP itself was detected in

the cytoplasm.

DISCUSSION

In yeast, there are four different RFC complexes that share the

four small subunits (Rfc2p, Rfc3p, Rfc4p, and Rfc5p) but differ in

the specific large subunit and therefore have different or over-

lapping roles in DNA metabolism (Banerjee et al., 2007). The

Rfc1-5p complex is needed for loading PCNA during general

DNA replication or repair. Rfc1p can be replaced in the core

complex Rfc2-5 by other alternative clamp loaders, such as

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, and rfc1-1 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.

2346 The Plant Cell

which loads the PCNA-like protein complex Rad17-Ddc1-Mec3

around DNA (Majka and Burgers, 2003) for DNA damage check-

point; Rad17-RFC in humans, which loads the Rad9-Rad1-Hus1

heterotrimer clamp onto DNA (Bermudez et al., 2003); and Elg1

(Human ATAD5, Arabidopsis homolog At1g77620) (Sikdar et al.,

2009) or Ctf18 in yeast, both of which are necessary for

HR-mediated DSB repair and negatively regulate telomere

length and sister chromatid cohesion (Hanna et al., 2001; Mayer

et al., 2001; Naiki et al., 2001; Bellaoui et al., 2003; Ben-Aroya

et al., 2003; Kanellis et al., 2003; Banerjee and Myung, 2004;

Smolikov et al., 2004; Ogiwara et al., 2007a, 2007b;Maradeo and

Skibbens, 2009; Parnas et al., 2009). In this study, we provide

evidence that RFC1 in Arabidopsis regulates many aspects of

DNA metabolism, including DNA replication, DNA repair, HR,

TGS, and the control of telomere length.

Maintaining genomic stability and integrity of both genetic

and epigenetic information is an elementary requirement for

all organisms during DNA replication and repair. Our genetic

screen for ros1 suppressors identified three genes, including

Pola/INCURVATA2, ROR1/RPA2A (Xia et al., 2006; Liu et al.,

2010), and RFC1 (in this study), that are directly involved in DNA

replication, repair, and HR in plants. Pol«/ABO4was identified in

a screening for root growth sensitivity to ABA, but it can also

suppress the kanamycin-sensitive phenotype of ros1 (Yin et al.,

2009). Recently, Pold has been cloned because its mutations

lead to increased SHR (Schuermann et al., 2009). Although

PCNAandprimase have not been characterized yet, several core

DNA replication components in plants have been identified, and

they participate in virtually all repair pathways needed to syn-

thesize DNA. Except for Pold, whose effect on TGS has not been

tested yet, all of the other four genes affect TGS in an siRNA-

directed DNA methylation (RdDM)-independent pathway (Xia

et al., 2006; Yin et al., 2009; Liu et al., 2010; this study). The

T-DNA locus used in our study contains RD29A-LUC and

35S-NPTII with different regulation mechanisms. In ros1 mu-

tants, the RD29A promoter is targeted by siRNAs and hyper-

methylated (Gong et al., 2002a). The 35S promoter is found to

be hypermethylated or hypomethylated when using different

primers for bisulfite sequencing (Kapoor et al., 2005b; Liu et al.,

2010). However, unlike RFC1 and other DNA replication-related

proteins that could release the TGS of 35S-NPTII in ros1 when

mutated (Xia et al., 2006; Yin et al., 2009; Liu et al., 2010; this

study), mutations of genes in RdDM pathway do not release TGS

of 35S-NPTII in ros1 mutant (He et al., 2009a), suggesting that

the TGS mechanism in 35S promoter is unique. However, the

mechanism that regulates TGS of 35S-NPTII requires further

study in the future.

During DNA replication in animals, DNA methylation and

histone modifications can be maintained through PCNA interac-

tion with DNA methyltransferase and many histone modification

proteins, including the CAF-1 nucleosome assembly complex

(Probst et al., 2009). In Arabidopsis, CAF-1 subunits FAS1 and

FAS2 regulate TGS (Ono et al., 2006; Schonrock et al., 2006). A

conserved protein interaction between Pol a and HP1 in animal

and yeast or betweenPola and LHP in plants has been identified,

suggesting that inheritance of the heterochromatin state coupled

with DNA replication is conserved in both plants and other

organisms (Nakayama et al., 2001; Barrero et al., 2007). Repli-

cation factor C is an important component that displaces Pol

a-primase to terminate primer synthesis early in DNA replication

and loads PCNA, which recruits Pol d for continuing the lagging

strand synthesis or Pol « for leading strand synthesis (Toueille

and Hubscher, 2004; Garg and Burgers, 2005). Similar to the

mutations in ror1/rpa2a, pol «/abo4, and pol a (Xia et al., 2006;

Yin et al., 2009; Liu et al., 2010), the rfc1 mutation released the

TGS of the originally silenced 35S-NPTII, reduced the levels of

heterochromatin marker H3K9me2 in ros1, and increased the

expression of TSI. Our studies suggest that the DNA replication

machinery plays a crucial role in coupling DNA replication with

DNA repair to restore chromatin structure in Arabidopsis.

Like the ror1/rpa2a and abo4/pol «mutants, the rfc1-1mutant

had increased sensitivity to the DNA-damaging agent MMS and

also to the DNA cross-linking agent cisplatin, suggesting that

RFC1 is involved in DNA repair, while pol d and pol amutants are

not sensitive to DNA-damaging agents (Xia et al., 2006; Schuermann

et al., 2009; Yin et al., 2009; Liu et al., 2010). Consistent

with their increased sensitivity to DNA-damaging agents, rfc1-1

mutants expressed increased levels of DNA repair-related

genes, such as RAD51 and BRCA1, both of which are involved

in DNA repair and SHR (Li et al., 2004; Abe et al., 2005); by

contrast, the pol dmutation does not result in increased expres-

sion of DNA repair-related genes relative to the wild type

(Schuermann et al., 2009). The DNA repair-related genes were

also greatly increased in other DNA replication mutants, such as

ror1/rpa2a and abo4/pol « (Xia et al., 2006; Yin et al., 2009). These

results suggest that Pol d might have different roles in DNA

replication and repair than other core DNA replication proteins.

Interestingly, we found that treatment with DNA-damaging

agents can mimic the rfc1 mutation in releasing TGS of both

NPTII and TSIs in the ros1 mutant (Yin et al., 2009). During the

repair of damaged DNA in the promoter regions of some cancer

cells, the key proteins involved in establishing and maintaining

TGS are recruited to the sites of DNA breaks, which leads to the

silencing of the targeted genes (O’Hagan et al., 2008; Khobta

et al., 2010). Results in this study indicate that DNA damage is

also able to activate the original silenced genes. Together, these

findings suggest that DNA damage could have different effects

on both the active genes and the silenced genes.

To our surprise, the rfc1-1mutant exhibited an extremely high

SHR. Mutations in core replication machinery proteins, including

Pol a, Pol «, and Pol d, and replication protein A2 (ROR1/RPA2A)

are known to increase SHR, with a mutation in Pol a increasing

SHR only to a moderate level and mutations in Pol «, Pol d, and

ROR1/RPA2A increasing SHR to a high level (Schuermann et al.,

2009; Yin et al., 2009; Liu et al., 2010). In the study of Pol d,

however, Schuermann et al. (2009) found that DNA damage

caused by DNA-damaging agents MMS, cisplatin, or bleomycin

does not, but blocking DNA replication by a hydroxyurea reagent

greatly increases the SHR in the pol dmutant relative to the wild

type. These core DNA replication mutants lack the ability to

efficiently repair DSBs during DNA replication, which might

spontaneously activate the S-phase checkpoint pathway re-

quired for replisome stabilization and resolution of stalled forks.

The observation that CYCB1;1 is highly expressed in these

mutants also suggests that the delayed M/G2 phase could

provide more time for cells to repair the DNA damage in the

Replication Factor C and Epigenetic Instability 2347

S-phase in these mutants (Xia et al., 2006; Yin et al., 2009; Liu

et al., 2010). The rfc1-1 mutant had an extremely high SHR rate,

suggesting that RFC1 is a critical protein for preventing HR

during DNA replication/repair. Leading-strand synthesis and

lagging-strand synthesis are coordinated during DNA replication

with the lagging-strand polymerase working at a higher speed

than the leading-strand polymerase (Pandey et al., 2009). RFC1

is the keymediator that loads PCNA, which recruits both lagging-

strand polymerase Pol d and leading-strand polymerase Pol «,

and mutations in either Pol d or Pol « greatly increase SHR. It is

conceivable that impairment of RFC1 would lead to more stalled

forks andDSBs in both leading and lagging strands and that such

strands could be used as substrates for SHR during DNA

replication. Interestingly, more SHR occurred in the older tissues

than in the younger tissues or meristems of Arabidopsis seed-

lings. The meristem is the most active site for DNA replication

and cell division, while older tissues support active DNA endo-

reduplication but not cell division (Schuermann et al., 2009). It

seems that the core DNA replication proteins play different roles

in the older tissues versus meristems for the regulation of SHR.

These DNA replication proteins likely influence SHRgreatly in the

older tissues but not so much in the meristems (Schuermann

et al., 2009).

Telomere homeostasis is regulated by telomerase and a

number of DNA repair and recombination proteins. Deletion of

the telomerase gene TERT in Arabidopsis causes the mutant to

lose 300 to 500 bp of telomere per generation (Fitzgerald et al.,

1999). Human RFC1 (RFC p140) can bind to the telomeric repeat

and inhibit telomerase activity (Uchiumi et al., 1999). Mutations in

DNA replication-related proteins, such as Pol a, RFC1, CTF18,

and ELG1 in yeast, and KU70, KU80, and replication protein A

70a in Arabidopsis, increase telomere length (Gallego et al.,

2003; Takashi et al., 2009). Here, we found that loss of function of

ROS1 increased telomere length, while the impairing of RFC1

and other replication proteins decreased telomere length in

Arabidopsis. Telomere length in the ros3 mutant, however, was

not changed, although ROS3 is in the same DAN demethylation

pathway as ROS1 (Zheng et al., 2008). Because ROS1 can

physically interact with RPA2A/ROR1 (Kapoor et al., 2005b; Xia

et al., 2006), it might affect telomere length by interfering with

RPA2A. These results suggest that the molecular mechanisms

regulating telomere length are different in Arabidopsis than in

yeast. The expression of TERT, POT1a, and BT2 was a little

higher in ros1 butmuch lower in rfc1-1 or rfc1-1 ros1-1 than in the

wild type. Although TERT activities are not increased in the KU70

and KU80mutants, telomere lengthening is apparently mediated

by telomerase activity rather than by recombination (Gallego

et al., 2003). The results suggest that the lengthening or short-

ening of the telomere might be somehow related to TERT and

other telomere-related proteins that are likely regulated by

ROS1, RFC1, and other related DNA replication-related proteins.

Like other DNA replication-related genes, such as Pol a, Pol «,

and Pol d (Barrero et al., 2007; Schuermann et al., 2009; Yin et al.,

2009; Liu et al., 2010), RFC1 should be an essential gene in

Arabidopsis because another subunit in the same complex with

RFC1, RFC3, is essential in Arabidopsis (Xia et al., 2009). The

mutations used in this study are probably weak alleles because

the two mutations occur in the C-terminal region. Mutation in

rfc1-1 causes the mis-splicing of RFC1 pre-mRNA. rfc1-1 plants

are smaller, flower earlier, and produce fewer seeds than thewild

type. Cell sizes, however, are similar for rfc1-1 and the wild type,

suggesting that the small size of rfc1-1 plants is due to reduced

cell division and, therefore, fewer cells. The result suggests that

onceDNA replication has finished, the expansion of the cell is not

greatly influenced by the rfc1 mutation. Interestingly, vegetative

growth was similar for rfc1-2 and the wild type, but rfc1-2

produced much fewer seeds than the wild type or rfc1-1. In

Arabidopsis, two checkpoint proteins, ATM (ataxia telangiectasia-

mutated) and ATR (ataxia telangiectasia-mutated and Rad3-

related), are essential regulators of the cell cycle; these proteins

sense DNA damage and activate downstream components of cell

cycle progression and DNA repair. Mutations in both ATM and

ATRdo not affect the vegetative growth but do affect reproductive

processes (Garcia et al., 2003; Culligan et al., 2004). Similar

phenotypes are also produced by the RAD51 mutant (Li et al.,

2004). The diverse phenotypes exhibited by rfc1-1 and rfc1-2

suggest that the mutated proteins might have different effects on

vegetative and reproductive development or that these mutations

have different effects in C24 and Columbia accessions. In both

accessions, it seems that these mutations affect the reproductive

process more than vegetative growth.

METHODS

Plant Materials and Growth Conditions

The C24 wild type, ros1mutation, and other Arabidopsis thalianamutants

(C24 background) mentioned in this article all carry the RD29A-LUC and

35S-NPTII transgenes. The T-DNA line (SALK_140231; at5g22010) was

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,

during which the seeds germinated, the plates were transferred to a

phytotron at 228C under long-day (23 h light/1 h dark) conditions.

Generally, 7-d-old seedlings were transferred to soil and cultured in a

growth room with 228C (light) or 188C (dark) under 16-h-light/8-h-dark

conditions.

Firefly Luciferase Imaging Analysis

Seedlings that had grown on MS medium for 7 d were transferred onto

filter paper saturated with 200mMNaCl. After 4-h treatment, these plants

were sprayed with D-luciferin (NanoFuels) assay buffer and immediately

subjected to CCD imaging analysis in a dark environment.

Mutation Screening, Map-Based Cloning, and

Mutant Complementation

The isolation of the rfc1-1mutant was described before (Xia et al., 2006).

The rfc1-1 ros1-1 mutant was backcrossed four times to ros1-1 before

genetic analysis. For the cloning of the RFC1 gene, rfc1-1 ros1-1 was

crossed with wild-type gl1 (Columbia accession), and 1485 plants that

showed rfc1-1 developmental phenotypes were selected from the F2

progeny. Map-based cloning was performed using simple sequence

length polymorphism markers (see Supplemental Table 1 online). The

mutated gene was mapped to chromosome 5 between BAC T10F18 and

MWD9. Sequencing analysis revealed a G-to-A mutation in gene

AT5G22010.

A full-length genomic DNA fragment (;8.8 kb, from22065 to +6755 bp

containing from the putative start codon ATG) was released from BAC

2348 The Plant Cell

clone T6G21 and subcloned into the pCAMBIA1391 binary vector (http://

www.cambia.org/daisy/cambialabs/materials.html) by restriction enzyme

sites SalI and BstEII. The strain GV3101 carrying this construct was

transformed to the rfc1-1 ros1-1 mutant (Clough and Bent, 1998). The

transgenic plants with resistance to hygromycin were selected, and F4

generation plants without any segregation were analyzed for kanamycin

resistance and compared with the growth phenotypes of wild-type plants.

RNA Isolation and Real-Time PCR

Total RNA was extracted from the seedlings under different conditions

using Trizol reagent (Invitrogen). After the RNAwas digested with RNase-

free DNase I (NEB) at 378C for 20 min, 4 mg of total RNA was reverse

transcribed byM-MLV reverse transcriptase (Promega) in a 20-mL sample

volume. The cDNA products to be subjected to quantitative real-time

PCR were diluted 20-fold. Each 20-mL reaction contained 10 mL SYBR

Green Master mix (Takara), 0.2 mM (final concentration) of each gene-

specific primer (see Supplemental Table 2 online), and 2mL cDNA. Three-

step PCR (denaturation, 10 s at 958C; annealing, 10 s at 618C; extension,

15 s at 728C) was carried out on a PTC-200 DNA engine cycler (MJ

Research) with Chromo4 Detector. The Ct values presented are the

means of three biological replicates (means +SD, n = 3). Results of qRT-

PCR were verified by at least three independent experiments.

Gene Expression and DNA Gel Blot Analysis

The transcript levels of endogenous RD29A and TSI were determined by

real-time RT-PCR. ACTIN was used as the control. The NPTII level was

determined by immunoblots. DNA methylation changes in the RD29A

promoter, 35S promoter, ribosome DNA, centromere DNA regions, and

retrotransposon Ta3 were analyzed by DNA gel blots with methylation-

sensitive restriction enzymes as previously described (Gong et al., 2002a).

Histochemical GUS Analysis

A genomic DNA fragment (;900 bp) of the RFC1 promoter upstream of

ATG was amplified by specific primers 59-GATGGGAAGCTTTGGATGG-

GAAGTG-39 and 59-TGCCCGTCTCGAGAATTCGACTAC-39 and then

cloned into the pCAMBIA1391 vector. The fused RFC1 promoter-GUS

gene construct was transformed into Arabidopsis. The transgenic T2

lines, which were resistant to hygromycin, were subjected to a GUS

activity assay as described before (Yin et al., 2009).

Localization of the RFC1-GFP Fusion Protein

The full-length cDNA of the RFC1 gene was amplified from total

cDNA from flowers (C24 accession) by the PCR primer pair: cDNA-SalI-

F (59-CGCGTCGACGTAGTCGAATTCTCGAGACGGGCAAATG-39) and

cDNA-SpeI-R (59-CGCACTAGTTCTCTTTCTCTTGGCACCAGAGCC-39).

The PCR products were directly digested by SalI and SpeI and then

cloned into the modified vector pCAMBIA1300. GFP was fused in frame

to RFC1 at its C terminus. The Agrobacterium tumefaciens strain GV3101

carrying RFC1-GFP constructs together with the P19 strain were injected

into 4-week-old tobacco (Nicotiana benthamiana) leaves using a plastic

syringe as described before (Walter et al., 2004). The transient GFP

fluorescence was imaged with a confocal laser scanning microscope

(LSM510; Carl Zeiss).

DNA Damage Assay

Two methods were used to measure the sensitivity to DNA-damaging

agents. In one method, seeds were sown directly on solid MS medium

containing the specified concentration of cisplantin or MMS, and seed-

lings were assessed and photographed after 3 weeks. In the second

method, 5-d-old seedlings were transferred from standard MS medium

(without DNA-damaging agents) to MS medium containing MMS, and

seedlings were assessed and photographed after 2 weeks.

CYCB1;1:GUS Activity Assay

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

homozygous lines carrying only the GUS reporter were used as controls.

Recombination Assays

The mutant ros1-1 rfc1-1 (accession C24) was crossed with the recom-

bination reporter line NllC4 No.651, which carries overlapping 566-bp

sequences of theGUS gene in inverted orientation (C24 ecotype) (Puchta

et al., 1995). The homozygous lines for both recombination reporter and

ros1 and/or rfc1 were selected from F3 progeny plants. To monitor the

SHR frequency, we stained different-stage seedlings with GUS as de-

scribed above. About 50 individual seedlings were analyzed for each

sample. To analyze the effect of cisplatin or MMS on SHR, we transferred

seedlings to MSmedium containing 10 mM cisplatin or 50 ppmMMS and

grew them for another 7 d before GUS staining.

ChIP

ChIP was performed as described previously (Xia et al., 2006) using 18-d-

old seedlings grown onMSmedium under long-day conditions (23 h light/

1 h dark). Immunoprecipitations were performed with anti-H3K9me2

(Upstate; 17-648) and anti-H3Ac (Upstate; 06-599) antibodies. Real-time

PCR analysis was performed using specific primers listed in Supplemen-

tal Table 2 online.

Assay for Detection of Telomere Length

Genomic DNA extracted from 3-week-old seedlings was digested

overnight with the restriction enzymes BfuCI or Hinfl. The samples of di-

gested DNA (30 mg) were subjected to DNA gel blot analysis as de-

scribed previously (Gallego et al., 2003). The telomeric repeat probe

[59-(TTTAGGG)7] was directly synthesized, and the 59 end was labeled

by [g-32P]ATP using T4 polynucleotide kinase.

Accession Numbers

Sequence data from this article can be found in the Arabidopsis Genome

Initiative or GenBank/EMBL databases under the following accession

numbers: RAD51 (at5g20850), Ku70 (at1g16970), ATGR (at3g52115),

BRCA (at4g21070), MRE11 (at5g54260), CYCB1;1 (at4g37490), TERT

(at5g16850), POT1a (at2g05210), BT2 (at3g48360), FLC (at5g10140),

RFC1 (at5g22010), and ACTIN2 (atg3g18780).

Supplemental Data

The following materials are available in the online version of this article.

Supplemental Table 1. Primer Pairs of the Markers Used for Map-

Based Cloning.

Supplemental Table 2. The Primers Used for Real-Time PCR and

ChIP.

ACKNOWLEDGMENTS

This work was supported by the National Nature Science Foundation of

China (30630004 and 30721062), the National Transgenic Research

Replication Factor C and Epigenetic Instability 2349

Project (2008ZX08009-002), and the Program of Introducing Talents of

Discipline to Universities (B06003) and Chinese Universities Scientific

Fund to Z.G. We thank Yuelin Zhang for sharing the T-DNA lines

provided by the ABRC (Columbus, OH).

Received May 4, 2010; revised June 18, 2010; accepted June 28, 2010;

published July 16, 2010.

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2352 The Plant Cell

DOI 10.1105/tpc.110.076349; originally published online July 16, 2010; 2010;22;2336-2352Plant Cell

Zhu and Zhizhong GongQian Liu, Junguo Wang, Daisuke Miki, Ran Xia, Wenxiang Yu, Junna He, Zhimin Zheng, Jian-Kang

ArabidopsisDNA Replication Factor C1 Mediates Genomic Stability and Transcriptional Gene Silencing in

 This information is current as of March 30, 2011

 

Supplemental Data http://www.plantcell.org/content/suppl/2010/07/15/tpc.110.076349.DC1.html

References http://www.plantcell.org/content/22/7/2336.full.html#ref-list-1

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