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Nucleic Acids Research logoLink to Nucleic Acids Research
. 2003 Dec 15;31(24):7141–7149. doi: 10.1093/nar/gkg917

A novel allele of fission yeast rad11 that causes defects in DNA repair and telomere length regulation

Yuuki Ono, Kazunori Tomita, Akira Matsuura 2, Takuro Nakagawa 3, Hisao Masukata 3, Masahiro Uritani, Takashi Ushimaru 1, Masaru Ueno *
PMCID: PMC291861  PMID: 14654689

Abstract

Replication protein A (RPA) is a heterotrimeric single-stranded DNA-binding protein involved in DNA replication, recombination and repair. In Saccharomyces cerevisiae, several mutants in the RFA1 gene encoding the large subunit of RPA have been isolated and one of the mutants with a missense allele, rfa1-D228Y, shows a synergistic reduction in telomere length when combined with a yku70 mutation. So far, only one mutant allele of the rad11+ gene encoding the large subunit of RPA has been reported in Schizosaccharomyces pombe. To study the role of S.pombe RPA in DNA repair and possibly in telomere maintenance, we constructed a rad11-D223Y mutant, which corresponds to the S.cerevisiae rfa1-D228Y mutant. rad11-D223Y cells were methylmethane sulfonate, hydroxyurea, UV and γ-ray sensitive, suggesting that rad11-D223Y cells have a defect in DNA repair activity. Unlike the S.cerevisiae rfa1-D228Y mutation, the rad11-D223Y mutation itself caused telomere shortening. Moreover, Rad11-Myc bound to telomere in a ChIP assay. These results strongly suggest that RPA is directly involved in telomere maintenance.

INTRODUCTION

Replication protein A [RPA, also known as human single-stranded DNA-binding protein (SSB) or replication factor A (RFA)] is a heterotrimeric single-stranded DNA-binding protein consisting of three subunits: RPA1 (70 kDa), RPA2 (36 kDa) and RPA3 (14 kDa) (1). RPA was originally identified as an essential factor for SV40 DNA replication in vitro (24). RPA is also required for nucleotide excision repair and mismatch repair in vitro (57). Moreover, RPA stimulates the activities of eukaryotic homologous pairing proteins in vitro (813). These biochemical studies have suggested that RPA is involved in DNA replication, recombination and repair in vivo.

Functions of RPA in vivo have been well studied in Saccharomyces cerevisiae (1417). The RFA1 gene encoding the large subunit (70 kDa) of RPA is essential for cell viability. So far, several rfa1 mutants have been isolated. One of the mutants, rfa1-44, is sensitive to UV and X-ray irradiation and is also defective in HO-endonuclease-induced plasmid-to-chromosome gene conversion (16). Another mutant, rfa1-D228Y, was isolated in a screen for suppressors of the defect in direct repeat recombination in rad1 rad52 double mutants (15). rfa1-D228Y mutants display increased levels of direct repeat recombination, decreased levels of heteroallelic recombination and UV sensitivity. Although the rfa1-D228Y mutation itself does not affect telomere length, synergistic reduction in telomere length is observed in the yku70 rfa1-D228Y double mutant, suggesting a role for RPA in telomere maintenance in the absence of Ku heterodimer (18).

Telomeres, the specialized structures at the ends of eukaryotic chromosomes, ensure chromosome stability by protecting chromosome ends from degradation and fusion (19). In Schizosaccharomyces pombe, proteins that bind to telomere ends have been shown to positively or negatively influence telomere length (2025). Telomere length is also controlled by DNA repair or DNA damage checkpoint proteins including Ku heterodimer, Rad32/Rad50/Nbs1 complex, and Rad3/Rad26 complex (2632). Ku heterodimer binds to double-strand break (DSB) ends and is required for non-homologous end-joining repair (33), while Rad32/Rad50/Nbs1 complex is required for homologous recombination (HR) repair (34). Rad3/Rad26 complex is required for replication and the DNA damage checkpoint (35).

Telomere ends acquire G-rich single-stranded overhangs in S phase in both S.cerevisiae and S.pombe (36,37). The G-rich overhang is required for telomere elongation, because it is used for binding of the RNA component in the telomerase complex (38). Schizosaccharomyces pombe Pot1 is thought to bind to the G-rich overhang in vivo (25,39). As RPA is a single-stranded DNA-binding protein, it might function on the G-rich single-stranded overhang. However, there is no direct evidence suggesting that RPA functions at telomere ends in wild-type cells.

In S.pombe, the genes encoding the three subunits of RPA, ssb1+ (p68 subunit gene), ssb2+ (p30 subunit gene) and ssb3+ (p12 subunit gene), have been cloned (40). The reconstituted SpSSB/RPA complex expressed in Escherichia coli is active in single-stranded DNA binding and the T-antigen-dependent unwinding of SV40 ori DNA (40). However, the in vivo function of S.pombe RPA is poorly understood. So far, only one rad11/ssb1 mutant allele has been reported (41). This rad11 mutant is UV and γ-ray sensitive, but the DNA damage checkpoint is intact.

To understand the function of S.pombe RPA in DNA repair and possibly in telomere maintenance, we created a rad11-D223Y mutant in which the asparagine at position 223 is mutated to tyrosine, which corresponds to the S.cerevisiae rfa1-D228Y mutant. In this work, we examined the DNA damage sensitivity and telomere length of the rad11-D223Y mutant. We provide here the first evidence suggesting that RPA is involved in telomere maintenance.

MATERIALS AND METHODS

Schizosaccharomyces pombe strains, media and genetic methods

The S.pombe strains used in this work are listed in Table 1. Cells were grown in YPAD medium (1% yeast extract, 2% polypeptone, 2% glucose, 0.04% adenine), YE medium (0.5% yeast extract, 3% glucose) or Edinburgh minimal medium (EMM) with required supplements. Standard procedures were used for propagation and genetic manipulation (42). Sensitivity to γ-rays and UV light was examined as described previously (43). Briefly, exponentially growing cells were irradiated with γ-rays from a 60Co source at a dose rate of 100–200 Gy/h or with UV light from a germicidal lamp (UVP UV-CROSSLINKER, CL-1000) at a dose rate of 50–100 J/m2/min. Duplicate samples of irradiated cells and unirradiated cells were plated on YPAD plates and incubated at 30°C for 4 days, and the colonies were counted (44). For semi-quantitative analysis of DNA repair activity, the spot assay was employed as described previously (43). Briefly, 3 µl of serial 10-fold dilutions of log-phase cells (0.5 × 107 cells/ml) were spotted onto a YPAD plate or a YPAD plate containing the indicated concentration of methylmethane sulfonate (MMS) or hydroxyurea (HU). All experiments were repeated at least twice and gave similar results.

Table 1. Schizosaccharomyces pombe strains used in this work.

Strains Genotype Source
JY746 h+ leu1-32 ura4-D18 ade6-M210 M. Yamamoto
JY741 h leu1-32 ura4-D18 ade6-M216 M. Yamamoto
YO001 h+ leu1-32 ura4-D18 ade6-M210 rad11D223Y This work
YO002 h+ leu1-32 ura4-D18 ade6-M210 rad11D223Y pku70:LEU2+ This work
YO003 h+ leu1-32 ura4-D18 ade6-M210 rad11D223Y rad50:ura4+ This work
YO004 h+ leu1-32 ura4-D18 ade6-M210 rad11:Myc:kanMX6 This work
YO005 h+ leu1-32 ura4-D18 ade6-M210 rad11D223Y:Myc:kanMX6 This work
KT120 h+ leu1-32 ura4-D18 ade6-M210 rad50::LEU2+ (31)
PKu70L h leu1-32 ura4-D18 ade6-M216 pku70::LEU2+ (44)
TNF79 h ade6D ade6B::ura4+::ade6X This work
YO006 h ade6D ade6B::ura4+::ade6X rad11D223Y This work

Construction of plasmid pT7-rad11-D223Y-ura4, which contains the rad11-D223Y mutation

Site-directed mutagenesis was carried out by using a Mutan-Super Express Km Kit (TaKaRa) according to the manufacturer’s instructions. Briefly, a DNA fragment containing a partial rad11+ gene, which was amplified by PCR with a sense primer (5′-AAATAGTGTATCGTCAGGCTA-3′) and an antisense primer, (5′-GATAAAATTGGTAATCCCG-3′) using the S.pombe genomic DNA as template, was subcloned into pT7Blue T-Vector, giving the plasmid pT7-rad11. Next the EcoRI–XbaI fragment from pT7-rad11 was inserted into the EcoRI–XbaI site in pKF18, giving the plasmid pKF18-rad11. To introduce the mutation, a DNA fragment was amplified by PCR with a mutation primer (5′-TCCCCACTTTCATAGAGTAAAT-3′) and a selection primer (provided in the Kit) using pKF18-rad11 as template, giving the plasmid pKF18-rad11-D223Y. The NheI–ClaI fragment from pKF18-rad11-D223Y was inserted into the NheI–ClaI site in pT7-rad11, giving the plasmid pT7-rad11-D223Y. Then the ura4+ cassette was inserted into the SmaI site in pT7-rad11-D223Y, giving the plasmid pT7-rad11-D223Y-ura4.

Construction of rad11-Myc cells and rad11-D223Y-Myc cells

To tag Rad11 and Rad11-D223Y with the Myc epitope at the C-terminus, a DNA fragment containing the partial rad11+ gene, which was amplified by PCR with a sense primer (5′-GGATCCGTGTTACGCTTTGGGGA-3′) and an antisense primer (5′-TTAATTAATTGAGCAGACTCAATGAAAT-3′) using the S.pombe genomic DNA as template, was cloned into the BamHI–PacI site in pFA6a-13Myc-kanMX6, giving the plasmid pFA6a-13Myc-kanMX6-rad11. pFA6a-13Myc-kanMX6 plasmid, which contains 13 copies of the Myc epitope and a kanMX6 marker, was provided by John R. Pringle (University of North Carolina) (45). The resulting plasmid pFA6a-13Myc-kanMX6-rad11 was linearized with NspV and used for transformation of JY746 and YO001.

Determination of recombination rates

The recombination rate was measured between the ade6B::ura4+::ade6X direct repeats located on chromosome III. ade6B and ade6X mutations were constructed by destroying the BamHI208 and XhoI1651 restriction sites, respectively, using Klenow fragment, resulting in a frame-shift (T. Nakagawa, N. Nitani and H. Masukata, manuscript in preparation). A single colony was inoculated into 2 ml of YE + adenine + uracil medium and incubated for 2 days, and the cells were plated onto EMM + adenine + uracil, EMM + uracil + guanine and EMM + guanine after appropriate dilutions with distilled water. Each amino acid was added at a final concentration of 225 µg/ml. Guanine was added to EMM at a final concentration of 50 µg/ml to avoid colony formation of ade cells on the minimal medium. The number of colonies was counted after 4 days of incubation at 30°C. A fluctuation test was performed to determine spontaneous recombination rates. The median value of recombination frequencies of nine cultures was used to calculate the rate of recombination (46).

Measurement of telomere length

Telomere length was measured by Southern hybridization according to the procedure described previously (21) by using an AlkPhos DirectTM kit module (Amersham Pharmacia Biotech). Briefly, chromosomal DNA, which was digested with ApaI and separated by electrophoresis on a 2% agarose gel, was probed with a 0.3 kb DNA fragment containing telomeric repeat sequences, which was derived from pNSU70 (47).

Chromatin immunoprecipitation

The chromatin immunoprecipitation (ChIP) assay described by Takahashi et al. (48) was adopted with modification. Cells grown in 100 ml of YPAD medium at 30°C were fixed with formaldehyde. For immunoprecipitation, anti-Myc antibody (Cell Signaling Technology™) and protein G-coated dynabeads (Dynal) were used. Immunoprecipitated DNA was extracted and suspended in TE buffer (10 mM Tris–HCl, 1 mM EDTA). The following primers were used in PCRs to amplify the telomeric DNA: TOP, 5′-CGGCTGACGGG TGGGGCCCAATA-3′; BOTTOM, 5′-GTGTGGAATTGAG TATGGTGAA-3′; and the eno1+ DNA: TOP, 5′-TGC CCCGGGTTTAAAACTTAGCAGCCTT-3′; BOTTOM, 5′-CTTTCAACGTCTTGAACG-3′.

RESULTS

Construction of the rad11-D223Y mutant

To construct the rad11-D223Y mutant, we used the allele replacement method as described previously (49). First, the plasmid pT7-rad11-D223Y-ura4 (see Materials and Methods) was cut with AatII and transformed into haploid strain JY746 (h+ leu1-32 ura4-D18 ade6-M210) (Fig. 1). Next, the resultant cells were grown in YPAD medium to allow deletion of the ura4+ cassette by intrachromosomal recombination between the rad11 allele and the rad11-D223Y allele. Then, ura4 cells were selected on EMM plates containing 0.2% 5-fluoroorotic acid (5-FOA). Then, the rad11-D223Y mutation was confirmed by DNA sequencing.

Figure 1.

Figure 1

Schematic illustration of construction of the rad11-D223Y mutant by allele replacement method. The plasmid shown at the top (pT7-rad11-D223Y-ura4, see Materials and Methods) contains the ura4+ cassette and the rad11-D223Y allele. The position corresponding to the D223Y mutation is denoted by a solid circle. The plasmid linearized by digestion with AatII was transformed into haploid strain JY746 (h+ leu1-32 ura4-D18 ade6-M210). The rad11-D223Y mutant was obtained by spontaneous direct-repeat recombination between the rad11+ allele and the rad11-D223Y allele.

We first examined the effect of the rad11-D223Y mutation on normal mitotic growth. The growth rate was not affected by the rad11-D223Y mutation (data not shown). Flow cytometric analysis of logarithmically growing rad11-D223Y cells showed that most of the rad11-D223Y cells had a DNA content of 2C, suggesting that rad11-D223Y cells are not arrested in G1 or S phase (data not shown). Moreover, when the structure of S.pombe chromosomes was analyzed by pulsed-field gel electrophoresis, chromosomes from rad11-D223Y cells entered the gel and were separated into three chromosomes, indicating that DNA replication is completed in the rad11-D223Y cells (data not shown). Based on these analyses, we concluded that the mitotic cell cycle is not affected by the rad11-D223Y mutation.

rad11-D223Y mutation has little effect on direct repeat recombination

The S.cerevisiae rfa1-D228Y mutant was originally isolated by screening for a mutant with a mutation that suppresses the decrease of the recombination rate in rad1 rad52 strains (15). The rfa1-D228Y mutation on its own causes a 15-fold increase in direct repeat recombination (15,50). Therefore, we examined the recombination frequency of rad11-D223Y cells. We used strains containing a non-tandem direct repeat of ade6 heteroalleles to measure the ade6+ recombination frequency (Fig. 2A). The rate of ade6+ formation was not significantly increased in rad11-D223Y cells (Fig. 2B). These results indicate that, unlike the S.cerevisiae rfa1-D228Y mutation, the rad11-D223Y mutation has little effect on the frequency of direct-repeat recombination.

Figure 2.

Figure 2

Spontaneous recombination between the ade6B::ura4+::ade6X direct repeats. (A) Schematic illustration of intrachromosomal recombination substrate and recombination products. The strain contains ade6B (solid circle) and ade6X (open circle). The ade6 repeats are separated by a 1.8 kb HindIII region containing ura4+ marker. ade6+ and ura4+ alleles are denoted by a filled-in box. ade+ ura+ recombinants are referred to as conversion-type, and ade+ ura as deletion-type recombinants. (B) Effect of rad11-D223Y mutation on the spontaneous rate of adenine prototroph formation. The rates of ade+ formation and ade+ ura+ formation were experimentally determined using isogenic strains of the wild type (TNF79) and rad11 (YO006). The rate (×10–5) of the formation of ade+ ura+ recombinants (conversion) and the rate (×10–5) of the formation of ade+ ura recombinants (deletion) are shown. The rate of ade+ ura formation was calculated by subtracting the ade+ ura+ rate from the ade+ (ura±) rate. The recombination rate per cell division was determined using the median value of the recombination frequency of nine cultures. The bar shows the mean value of three sets of experiments. Standard deviations are shown by error bars.

rad11-D223Y mutant is methylmethane sulfonate and hydroxyurea sensitive

Although the rad11-D223Y mutation does not affect the mitotic cell cycle, it may affect the viability when DNA replication is disturbed. Thus, we examined the HU and MMS sensitivity of the rad11-D223Y mutant. As shown in Figure 3A, the growth of rad11-D223Y cells was strongly inhibited in the presence of MMS or HU, indicating that rad11-D223Y cells are MMS and HU sensitive. MMS alkylates the DNA. These DNA can block DNA synthesis and stall the replication fork. Indeed, DNA replication is slowed down in the presence of 0.01% MMS in S.pombe, suggesting that the DNA damage caused by MMS causes problems during replication and is not repaired until S phase (27). HU also blocks DNA replication by depleting deoxynucleotides. Therefore, our results suggest that the rad11-D223Y mutant has a defect in the recovery from stalled replication forks.

Figure 3.

Figure 3

rad11-D223Y cells are MMS and HU sensitive. (A) The sensitivities of wild-type cells (JY746) and rad11-D223Y cells (YO001) to MMS and HU determined in a spot test. (B) Viability of the wild-type cells and the rad11-D223Y cells in YPAD medium in the presence of 20 mM HU. Standard deviations are shown by error bars. (C) Both the wild-type cells and the rad11-D223Y cells were elongated after 8 h of incubation in YPAD medium in the presence of 10 mM HU. For genotypes, see Table 1.

Next, we examined the response to HU in more detail. First, we examined the viability loss in 20 mM HU in liquid medium. In human cells and S.cerevisiae, RPA is required for recruitment of the ATR–ATRIP complex and Mec1–Ddc2 complex, which correspond to the S.pombe Rad3–Rad26 complex, to sites of DNA damage (51). Similarly, if the rad11-D223Y mutant had a defect in the recruitment of the Rad3–Rad26 complex to sites of DNA damage in S.pombe, the mutant would lose viability very quickly in the presence of HU. Only 0.1% of rad3-d cells survived after 4 h in 10 mM HU (52). In contrast, the cell viability of the rad11-D223Y mutant was still 40% after 4 h in 20 mM HU (Fig. 3B). These results suggest that the rad11-D223Y mutation does not cause a severe S-phase checkpoint defect. Consistent with these data, most of the rad11-D223Y cells became elongated after treatment with 10 mM HU for 8 h (Fig. 3C). These results suggest that cell cycle arrest mediated by the S-phase checkpoint appears to be normal in rad11-D223Y cells.

Epistasis analysis between rad11-D223Y cells and rad50-d cells or pku70-d cells for γ-ray sensitivity

The S.cerevisiae rfa1-D228Y mutant is not γ-ray sensitive. However, the S.pombe rad11-D223Y mutant might show a different phenotype in DNA repair ability. Thus, we examined the γ-ray sensitivity of rad11-D223Y cells. Unlike the S.cerevisiae rfa1-D228Y mutant, rad11-D223Y cells were more γ-ray sensitive than wild-type cells (Fig. 4A). DNA DSBs caused by γ-rays are mainly repaired by HR in S.pombe. Therefore, our result suggests that rad11-D223Y cells have a defect in HR repair ability.

Figure 4.

Figure 4

Epistasis analysis between rad11-D223Y cells and rad50-d cells or pku70-d cells for γ-ray sensitivity. (A) The sensitivities to γ-rays of wild-type cells, JY746 (diamonds), rad11-D223Y cells, YO001 (squares), rad50-d cells, KT120 (triangles) and rad11-D223Y rad50 double mutants, YO003 (circles). (B) The sensitivities to γ-rays of wild-type cells, JY746 (diamonds), rad11-D223Y cells, YO001 (squares), pku70-d cells, Pku70L (triangles), and rad11-D223Y pku70 double mutants, YO002 (circles). For genotypes, see Table 1. Standard deviations are shown by error bars.

We next examined the γ-ray sensitivity of the rad50 rad11-D223Y double mutant. If the rad11-D223Y mutation affected some DNA repair activity other than Rad50-dependent HR repair activity, rad50 rad11-D223Y double mutants should become more γ-ray sensitive than each single mutant. However, the γ-ray sensitivity of the rad50 rad11-D223Y double mutant was almost the same as that of the rad50 single mutant, indicating that the rad11-D223Y mutant has a defect in the Rad50-dependent HR repair pathway (Fig. 4A).

In S.cerevisiae, it is reported that the rfa1-t11 mutation does not affect the degradation rate of HO-induced DSB ends (53). However, it is unknown whether the S.pombe rad11-D223Y mutation affects the processing of DSB ends. We have shown that the γ-ray sensitivity of rad50-d cells is suppressed by deletion of pku70+. Based on this and other genetic data, we have concluded that the Rad50 complex is required for the processing of DSB ends; however, in the absence of the Rad50 complex, a second nuclease (Exo1) can resect DSB ends, but this nuclease activity of Exo1 is inhibited by Ku heterodimer (44). Similarly, if rad11-D223Y mutation affected the efficiency of Rad50-dependent DSB end processing, the γ-ray sensitivity of rad11-D223Y cells might be suppressed by deletion of pku70+. However, the γ-ray sensitivity of rad11-D223Y cells was not suppressed by deletion of pku70+ (Fig. 4B). This result suggests that the rad11-D223Y mutation does not affect the efficiency of Rad50-dependent DSB end processing. We assume that the rad11-D223Y mutation affects some DNA repair activities that function after DSB ends are processed by the Rad50 complex.

Epistasis analysis between rad11-D223Y cells and rad50-d cells for UV sensitivity

To further elucidate the effect of the rad11D223Y mutation on DNA repair, we examined UV sensitivity. rad11-D223Y cells were also sensitive to UV light (Fig. 5). However, unlike the situation for γ-ray sensitivity, rad11-D223Y rad50 double mutants became more UV sensitive than each single mutant (Fig. 5). These results suggest that rad11-D223Y mutants have a defect in the repair of UV-induced DNA damage that is independent of Rad50.

Figure 5.

Figure 5

Epistasis analysis between rad11-D223Y cells and rad50-d cells for UV sensitivity. The sensitivities to UV light of wild-type cells, JY746 (diamonds), rad11-D223Y cells, YO001 (squares), rad50-d cells, KT120 (triangles) and rad11-D223Y rad50 double mutants, YO003 (circles). For genotypes, see Table 1. Standard deviations are shown by error bars.

rad11-D223Y mutation causes telomere shortening

The telomere length of the S.cerevisiae rfa1-D228Y mutant itself is normal, but mutations in both yku70 and rfa1-D228Y cause synergistic telomere shortening, suggesting that RPA plays a role at telomere ends in the absence of Ku heterodimer (50). Thus, we examined the telomere length of the rad11-D223Y mutant and rad11-D223Y pku70 double mutant (Fig. 6A–C). Unlike the S.cerevisiae rfa1-D228Y mutation, the rad11-D223Y single mutation itself caused significant telomere shortening (Fig. 6B, lane 2). This result indicates that RPA is required for telomere length regulation in wild-type S.pombe cells. Mutations in both rad11+ and pku70+ caused synergistic telomere shortening (Fig. 6B, lanes 2 and 4), indicating that RPA functions independently of Ku heterodimer for telomere length regulation. We also examined the telomere length of the rad11-D223Y rad50 double mutant. Synergistic telomere shortening was not observed in the double mutant (Fig. 6C), suggesting that RPA and Rad50 are included in the same epistatic group for telomere length regulation.

Figure 6.

Figure 6

rad11+ is involved in telomere length maintenance. (A) Schematic presentation of the telomeric and telomere-associated sequences (TAS) of one chromosome arm cloned in the plasmid pNSU70 (47). The positions of telomere and telomere-associated sequences, TAS1, TAS2 and TAS3, are underlined. ApaI-digested telomere sequence was used as a probe for Southern hybridization assays. The chromosome arm is shown by the long gray bar. Restriction enzyme sites are shown above the long gray bar. (B and C) The telomere length of rad11-D223Y, rad11-D223Y pku70-d double mutants and rad11-D223Y rad50-d double mutants was evaluated by Southern hybridization. (B) Lane 1, wild-type cells (JY746); lane 2, rad11-D223Y (YO001); lane 3, pku70-d (PKU70L); lane 4, rad11-D223Y pku70-d double mutants (YO002). (C) Lane 1, wild-type cells (JY746); lane 2, rad11-D223Y (YO001); lane 3, rad50-d (KT120); lane 4, rad11-D223Y rad50-d double mutants (YO003). Telomeres are indicated by arrows. For genotypes, see Table 1. Peaks and distributions of the telomeric DNA-derived bands analyzed using NIH image 1.62 software are shown below. Telomere peaks are indicated by asterisks.

Rad11 binds to telomere ends

Requirement of rad11+ for telomere length regulation implies that RPA binds to telomere ends. Therefore we tested the binding of Rad11 to telomeres by the ChIP assay (Fig. 7A and B). We tagged the C-terminus of Rad11 and Rad11-D223Y with Myc-tag. Growth and DNA damage sensitivity were not affected by tagging of Rad11 with Myc-tag (data not shown). Anti-Myc antibody was used for immunoprecipitation and the precipitated DNA was amplified by PCR with primers for the telomeric region or eno1+as a control. Telomere DNA was significantly amplified in cells, which expressed Myc-tagged Rad11 protein from their own promoter (Fig. 7B). These results indicate that Rad11 (and probably RPA complex) binds to telomere DNA. For unknown reasons, the binding of Rad11 to telomeres was increased by rad11-D223Y mutation (Fig. 7B). Since the protein expression level of Rad11-D223Y-Myc and Rad11-Myc were almost the same (data not shown), the stronger binding of Rad11-D223Y-Myc to the telomere is not due to the increased protein level of Rad11-D223Y in rad11-D223Y cells.

Figure 7.

Figure 7

Rad11-Myc is bound to telomere DNA in the ChIP assay. (A) Schematic presentation of the location of the primer set used for the ChIP assay. The position of the primer set used for amplification of telomere DNA is shown above the long gray bar. The positions of telomere and telomere-associated sequences, TAS1, TAS2 and TAS3, are underlined. The chromosome arm is shown by the long gray bar. (B) The ChIP assay of rad11-Myc and rad11-D223Y-Myc. Untagged wild-type cells (JY746), rad11-Myc (YO004) cells and rad11-D223Y-Myc (YO005) cells were used. PCR was performed on whole-cell extracts (WCE) and on chromatin immunoprecipitates (IPs with anti-Myc) using primers to amplify telomere DNA (telomere) and primers to amplify DNA from the eno1+ gene (eno1). The relative precipitated fold enrichment is shown underneath each lane. Ratios of telomere signals and eno1+ signals were used to calculate relative precipitated fold enrichment.

DISCUSSION

RPA has been suggested to play a role in telomere maintenance in S.cerevisiae yku70-d cells. However, the role of RPA in telomere maintenance in wild-type cells remains unclear. We found that the rad11-D223Y mutation itself caused telomere shortening (Fig. 6). Moreover Rad11-Myc bound to telomere ends by ChIP assay (Fig. 7). These results strongly suggest that RPA is directly required for telomere length regulation.

How does RPA regulate telomere length? Rad11-D223Y-Myc also bound to telomeres, indicating that telomere shortening in the rad11-D223Y mutant is not due to loss of DNA-binding ability of Rad11-D223Y protein (Fig. 7). In S.pombe, G-rich single-stranded overhang is increased in S phase (37). Although the binding of Rad11 to telomere could be mediated by interaction with other telomere-binding proteins, it is plausible that RPA binds to the G-rich overhang because RPA is a single-stranded DNA-binding protein. Pot1 and Est1 are thought to bind to the G-rich overhang and these proteins are required for telomere elongation (25,20,54). Therefore, RPA might be required for the recruitment of these proteins or other proteins that are required for telomere length regulation. Another possible role of RPA on telomeres is to remove a secondary structure (possibly G-quartet) formed by the G-rich single-stranded overhang (55). Such a structure may prevent the binding of Pot1 and Est1 to telomere ends. Indeed, RPA is thought to remove secondary structure formed by 3′ single-stranded tails at DSB ends to promote the formation of a Rad51 filament (56). At this time, the exact roles of RPA on telomere are still unclear. More detailed investigation is necessary to understand the exact roles of RPA in telomere length regulation.

rad11-D223Y cells were γ-ray, UV, MMS and HU sensitive (Figs 35). Epistasis analysis suggested that rad11-D223Y cells have a defect in at least two repair pathways. rad11-D223Y cells were epistatic to rad50-d cells for γ-ray sensitivity, suggesting that the rad11-D223Y mutant has a defect in HR repair. In rad50-d cells, the efficiency of the processing of DSB ends would be very low (44,53). Since RPA is thought to bind to 3′ single-stranded overhangs at DSB ends, mutation in rad11+ would not cause a further problem if 3′ single-stranded overhangs do not exist at DSB ends in rad50-d cells. This would be the reason why the rad11-D223Y mutation does not increase the γ-ray sensitivity of rad50-d cells. This idea is consistent with our assumption that the rad11-D223Y mutation affects some DNA repair activities that function after DSBs are processed by the Rad50 complex. Since RPA stimulates the strand exchange activity of Rad51 in vitro (13), the rad11-D223Y mutation may affect the filament formation ability and/or strand exchange activity of Rhp51 (S.pombe Rad51 homolog) protein.

The second defect in DNA repair ability in rad11-D223Y cells is suggested to be independent of the HR repair pathway, because the rad11-D223Y rad50 double mutant became more UV sensitive than each single mutant. Fission yeast has at least two pathways to repair photolesions in DNA, namely, nucleotide excision repair (NER) and UV-damaged DNA endonuclease-dependent excision repair (UVER) (57). Repair downstream of the UVER pathway is divided into the Rad2 sub-pathway and recombination sub-pathway. As RPA is involved in NER repair in vitro (58), rad11-D223Y cells might have a defect in the NER pathway. However, since RPA controls Rad2 activity during Okazaki fragment processing (59), it is possible that rad11-D223Y cells have a defect in the Rad2 sub-pathway in the UVER pathway. Alternatively, if pyrimidine dimers produced by UV light were not removed, they would cause replication fork arrest. The HU sensitivity of the rad11-D223Y mutant suggests that this mutant has a defect in the recovery from stalled replication forks. Therefore, we cannot rule out the possibility that the UV sensitivity of rad11-D223Y cells could be due to a defect in the recovery from stalled replication forks.

As discussed above, the phenotypes of telomere length, direct-repeat recombination, and γ-ray sensitivity of S.pombe rad11-D223Y were very different from those of the S.cerevisiae rfa1-D228Y mutant, even though the amino acid sequences are highly conserved between S.pombe Rad11 protein and S.cerevisiae Rfa1 protein (37% identity for the full-length proteins). The amount of Rfa1 protein in rfa1-D228Y cells is reduced ∼2-fold compared with that in the wild-type cells (15,60). In contrast, the amount of Rad11-D223Y-Myc protein is almost the same as that of Rad11-Myc protein (data not shown). These differences might be the reason for the phenotypic differences between these two mutants. Indeed, the protein level of Rfa1 affects the recombination frequency and DNA repair ability in S.cerevisiae (15,60). However, the different γ-ray sensitivities of these two mutants cannot be explained by the different protein expression levels, because the S.pombe rad11-D223Y mutant is γ-ray sensitive but the protein level is not affected by the mutation. This fact suggests that the rad11-D223Y mutation affects the enzymatic activity of Rad11 protein rather than the protein expression level.

The different phenotypes in S.pombe rad11-D223Y cells and S.cerevisiae rfa1-D228Y cells emphasize the importance of the investigation of S.pombe RPA, even though extensive mutational analysis of the S.cerevisiae rfa1 gene has been performed. In S.pombe, only one rad11 mutant (rad11A) has been reported so far (41). Both rad11A cells and rad11-D223Y cells are UV and γ-ray sensitive. However, unlike rad11A cells, rad11-D223Y cells are not temperature sensitive (data not shown), indicating that these two mutations affect the function of the RPA complex in different ways. Although RPA is thought to be involved in DNA replication, recombination and repair, the exact roles of RPA in these aspects of DNA metabolism are not fully understood. Further investigation of the rad11-D223Y mutant and isolation of additional rad11 mutants will provide useful information for elucidating the roles of RPA in DNA metabolism.

Acknowledgments

ACKNOWLEDGEMENTS

We thank Keiko Umezu for the suggestion about construction of the rad11 mutant, Takeshi Saito, Shinji Yasuhira and Hiroshi Utsumi for help with the γ-ray irradiation, Kohta Takahashi, Shigeaki Saitoh and Mitsuhiro Yanagida for the ChIP assay protocol, Masayuki Yamamoto for providing strains, and John R. Pringle for providing plasmids. This work was supported by Grants-in-Aid for Scientific Research on Priority Areas from the Ministry of Education, Science, Sports and Culture of Japan to A.M and M.U., and by a grant from the Yokohama City Collaboration of Regional Entities for the Advancement of Technological Excellence, JST, to M.U. T.N. and H.M. were supported by Grants-in-Aid for Cancer Research and Scientific Research, respectively, from the Ministry of Education, Science, Sports and Culture of Japan. Part of this work was performed by using facilities of the Research Reactor Institute, Kyoto University.

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