Download DNA Replication Factor C1 Mediates Genomic

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 239556900, 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 methylationindependent 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 recombination 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 DNA primer using an RNA primer. 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 ringshaped 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).
1 These
authors contributed equally to this work.
correspondence to [email protected].
The author responsible for distribution of materials integral to the
findings presented in this article in accordance with the policy described
in the Instructions for Authors (www.plantcell.org) is: Zhizhong Gong
([email protected]).
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www.plantcell.org/cgi/doi/10.1105/tpc.110.076349
2 Address
The formation of the DNA replication fork can be stalled or
arrested during DNA replication if the DNA 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 polymerase a, suggesting that the replication machinery regulates
telomere length (Adams and Holm, 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 structure from the parent strands. The transmission of heterochromatin 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 and Martienssen, 2008;
Replication Factor C and Epigenetic Instability
Martienssen et al., 2008; Probst et al., 2009). Furthermore,
epigenetic markers can be changed after the replication machinery has passed the DNA replication fork during cell differentiation and development. In fission yeast, DNA polymerase
a interacts with the heterochromatin protein Swi6, a homolog
of HETEROCHROMATIN PROTEIN1 (HP1), an important determinant 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 DNA methylation (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 heterochromatin and accounts for ;30% of the total DNA methylation 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.
ROS1 mutations lead to TGS of both RD29A-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 independent 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/
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DNA polymerase « was isolated in a root-sensitive-to-abscisic
acid (ABA) screen and is also involved in TGS in an RdDMindependent manner (Yin et al., 2009). In this study, we characterized another ros1 suppressor, RFC1, the largest subunit of the
RFC complex in Arabidopsis. Mutations in RFC1 lead to developmental 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 rfc1 Mutation Releases the TGS of 35S-NPTII in the
ros1 Mutant
The accession used in this study, C24 RD29A-LUC, carries a T-DNA
locus that contains active (i.e., unsilenced) RD29A-LUC and 35SNPTII 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 kanamycin indicated that the rfc1-1 mutation was caused by a single
recessive nuclear gene (Figure 1A). We tested the kanamycinsensitive 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 C24 wild 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 the C24 wild type (see 350 mg/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 expression of RD29A-LUC and the endogenous RD29A gene, we
compared their expression after treatment with 50 mM ABA for 4 h
(for RD29A expression) or 200 mM NaCl treatment for 4 h (for
LUC image). As shown in Figure 1D, ros1-1 or 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 rfc1-1 but was silenced in both 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 transcriptional 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
the C24 wild 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 TSIs were expressed at a higher
level than in the wild type. These results indicate that the mutations
in RFC1 release the TGS in some genomic loci.
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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 RD29A Promoter or at Other DNA Repeat Regions
To test whether the rfc1 mutation affects DNA methylation levels, 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 retrotransposon 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 retrotransposon are not controlled by RdDM (Chan et al., 2006). As
reported previously, MluI (AmCGCGT) and BstUI (CGmCG) enzymes 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 difference 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) and MspI (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 rfc1-1 Mutation 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
Replication Factor C and Epigenetic Instability
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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
Lys 9 (H3K9me2), which is usually an epigenetic marker for heterochromatin. 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 total RD29A (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).
rfc1-1 Mutants 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 supplemented with 125 ppm MMS (or without MMS 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 to MMS, 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-diamminedichloroplatinum or cis-DDP), which is used as an anticancer
chemical, coordinates to DNA mainly 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 predominantly 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 rfc1-1 mutants were much more 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 constitutively expressed in the abo4 mutant (Yin et al., 2009). We
determined the expression of the following DNA repair and/or
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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, 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 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 protein 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, rfc1-1 mutants express
higher levels of GR1, RAD51, KU70, and BRCA1 than the C24
wild type (Figure 3E). Expression of MRE11 did not clearly differ
between rfc1-1 and the C24 wild type (Figure 3E). MMS treatment 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 to MMS did
not clearly differ between rfc1-1 and the wild type. These
results indicate that DNA repair-related genes were spontaneously 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
Replication Factor C and Epigenetic Instability
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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 (qRTPCR) 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
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.
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 ppm MMS and 10 mM cisplatin because higher
concentrations of these two agents will greatly inhibit seedling
growth, making comparison of seedling growth difficult. As
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 another 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
were compared between the wild type and the rfc1-1 mutant. 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
ros1-1 mutants, especially in the cotyledons of 7-, 15-, and 20-dold 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).
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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.
The rfc1-1 Mutation 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, indicating 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
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
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.
ros1 Mutation Increases the Telomere Length and rfc1
Mutation Decreases the Telomere Length and Reduces
Expression of Telomerase
Previous studies suggest that the DNA replication machinery 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 the wild 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 increased SHR, we tested whether the RFC1 mutation
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 a mutants were
only a little shorter than in the wild type. The telomeres of the pol
a ros1-1 mutant 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 ros1-1 mutants 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.
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, 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 ros1-1 mutants. 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.
Telomerase reverse transcriptase (TERT) is the catalytic subunit 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 TAZ domain 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 expression 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
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.
rfc1-1 Mutation Causes Hypomorphic Growth Phenotypes
and Earlier Flowering
rfc1-1 ros1-1 or rcf1-1 mutants were smaller and flowered earlier
than the C24 wild type or ros1-1 when 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 ros1-1 was crossed with
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 the RFC1 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
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, and rfc1-1 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
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 determine whether the rfc1-1 mutation led to specific phenotypes, we
constructed a vector containing the full-length genomic DNA of
AT5G22010, including the wild-type AT5G22010 gene containing 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 transgenic 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 the wild type (Figure 8J). We were unable to test the effect
of the rfc2-1 mutation on silenced 35S-NPTII in the ros1-1 mutant
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 analyze 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 substantial in young inflorescence tissues and older leaves. GUS expression 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 localization signals that were predicted with help of the psort program
(http://psort.nibb.ac.jp/). We examined the subcellular localization 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 overlapping 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.
Replication Factor C and Epigenetic Instability
which loads the PCNA-like protein complex Rad17-Ddc1-Mec3
around DNA (Majka and Burgers, 2003) for DNA damage checkpoint; 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«/ABO4 was 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
PCNA and primase 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 synthesize DNA. Except for Pold, whose effect on TGS has not been
tested yet, all of the other four genes affect TGS in an siRNAdirected 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 mutants, the RD29A promoter is targeted by siRNAs and hypermethylated (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 interaction 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 between Pol a 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-
2347
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 rfc1-1 mutant
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 a mutants 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 d mutation does not result in increased expression 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 rfc1-1 mutant 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 d mutant 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 required 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
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The Plant Cell
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 key mediator that loads PCNA, which recruits both laggingstrand 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 and DSBs 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 seedlings. The meristem is the most active site for DNA replication
and cell division, while older tissues support active DNA endoreduplication 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 SHR greatly 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 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
by telomerase activity rather than by recombination (Gallego
et al., 2003). The results suggest that the lengthening or shortening 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 the wild
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
once DNA 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 telangiectasiamutated) and ATR (ataxia telangiectasia-mutated and Rad3related), 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
ATR do 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, ros1 mutation, and other Arabidopsis thaliana mutants
(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 200 mM NaCl. 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 rfc1-1 mutant 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, from 22065 to +6755 bp
containing from the putative start codon ATG) was released from BAC
Replication Factor C and Epigenetic Instability
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 RNA was digested with RNasefree DNase I (NEB) at 378C for 20 min, 4 mg of total RNA was reverse
transcribed by M-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 genespecific primer (see Supplemental Table 2 online), and 2 mL cDNA. Threestep 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 qRTPCR 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 methylationsensitive restriction enzymes as previously described (Gong et al., 2002a).
2349
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 recombination reporter line NllC4 No.651, which carries overlapping 566-bp
sequences of the GUS 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 described above. About 50 individual seedlings were analyzed for each
sample. To analyze the effect of cisplatin or MMS on SHR, we transferred
seedlings to MS medium containing 10 mM cisplatin or 50 ppm MMS and
grew them for another 7 d before GUS staining.
ChIP
ChIP was performed as described previously (Xia et al., 2006) using 18-dold seedlings grown on MS medium 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 Supplemental Table 2 online.
Assay for Detection of Telomere Length
Histochemical GUS Analysis
A genomic DNA fragment (;900 bp) of the RFC1 promoter upstream of
ATG was amplified by specific primers 59-GATGGGAAGCTTTGGATGGGAAGTG-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-SalIF (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).
Genomic DNA extracted from 3-week-old seedlings was digested
overnight with the restriction enzymes BfuCI or Hinfl. The samples of digested DNA (30 mg) were subjected to DNA gel blot analysis as described 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 MapBased Cloning.
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
agents. In one method, seeds were sown directly on solid MS medium
containing the specified concentration of cisplantin or MMS, and seedlings were assessed and photographed after 3 weeks. In the second
ACKNOWLEDGMENTS
This work was supported by the National Nature Science Foundation of
China (30630004 and 30721062), the National Transgenic Research
2350
The Plant Cell
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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Plant Cell 2010;22;2336-2352; originally published online July 16, 2010;
DOI 10.1105/tpc.110.076349
This information is current as of June 16, 2017
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