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Mutation in Rpa1 results in defective DNA double-strand break repair, chromosomal instability and cancer in mice Yuxun Wang1, Christopher D Putnam2, Michael F Kane2, Weijia Zhang3, Lisa Edelmann4, Robert Russell5, Danaise V Carrio´n3, Lynda Chin6, Raju Kucherlapati3, Richard D Kolodner2 & Winfried Edelmann1 Most cancers have multiple chromosomal rearrangements; the molecular mechanisms that generate them remain largely unknown. Mice carrying a heterozygous missense change in one of the DNA-binding domains of Rpa1 develop lymphoid tumors, and their homozygous littermates succumb to early embryonic lethality. Array comparative genomic hybridization of the tumors identified large-scale chromosomal changes as well as segmental gains and losses. The Rpa1 mutation resulted in defects in DNA double-strand break repair and precipitated chromosomal breaks as well as aneuploidy in primary heterozygous mutant mouse embryonic fibroblasts. The equivalent mutation in yeast is hypomorphic and semidominant and enhanced the formation of gross chromosomal rearrangements in multiple genetic backgrounds. These results indicate that Rpa1 functions in DNA metabolism are essential for the maintenance of chromosomal stability and tumor suppression. Replication protein A (Rpa) is a heterotrimeric single-stranded DNA binding complex consisting of subunits Rpa1, Rpa2 and Rpa3. Rpa1, the largest subunit, is conserved in eukaryotes and has essential roles in DNA replication, recombination and repair1. In yeast, the L221P missense change in one of three DNA binding domains led to gross chromosomal rearrangements (GCRs) that resembled those frequently found in human cancers, suggesting that RFA1 (Rpa1 in multicellular eukaryotes) has a crucial role in maintaining genomic stability2,3. This hypomorphic mutation also causes sensitivity to DNA-damaging agents, including those that induce double-strand breaks (DSBs); homologous recombination defects in meiosis associated with extensive resection of the 5¢ ends of meiosis-specific DSBs much like those seen in rad51, rad52, rad55 and rad57 mutants4; and checkpoint defects but no substantial defect in DNA replication2,3,5–8. To study the effect of Rpa1 mutations in mammals, we generated a mouse line carrying the corresponding mutation Rpa1 689T-C (resulting in the amino acid substitution L230P) using a knock-in gene-targeting
approach (Fig. 1a,b). We confirmed expression in the tissues of F1 offspring by RT-PCR analysis and direct sequencing (Fig. 1c,d). The mutant Rpa1 L230P protein was present in heterozygous mutant cells at levels equivalent to those of Rpa1 in wild-type cells; this is consistent with modeling studies indicating that the L230P mutation disrupts one of the three Rpa1 DNA-interacting domains but does not interfere with domain folding or stability (Fig. 1e,f and Supplementary Fig. 1 online). Matings between Rpa1689C/+ males and females produced no homozygous mutant Rpa1689C/689C mice, indicating that the 689T-C mutation caused embryonic lethality. Genotyping of mutant embryos showed that homozygous Rpa1689C/689C embryos were present at embryonic day (E) 3.5 but not at later developmental stages (Table 1). The E3.5 embryos consisted of only a few cells that were unable to proliferate in the in vitro culture, in contrast to inner cell mass cells of wild-type and heterozygous mutant E3.5 embryos (Fig. 2a,b). This observation indicates that homozygosity with respect to the Rpa1 689T-C missense mutation is lethal and is consistent with the idea that Rpa1 has an essential role in replicating cells. In contrast, the corresponding mutation rfa1 662T-C (resulting in the amino acid substitution L221P, also called rfa1-t48) is not lethal in yeast2,3,8. This difference might be caused by the homologous recombination defect, possibly in conjunction with the checkpoint defect caused by the mutation. In mice, homologous recombination defects like those caused by a Rad51 mutation also cause embryonic lethality between E3.5 and E7.5, whereas such defects do not cause lethality in yeast9–11. To study the effect of the Rpa1 689T-C mutation in heterozygous mutant mice, we monitored the status of mutant mice during a 2-y period. Rpa1689C/+ mice developed normally but had significantly lower survival than their wild-type littermates (P o 0.01, log-rank test; Fig. 2c). As early as 4 months of age, Rpa1689C/+ mice began to die, and by 12 months, 420% of them had died, whereas all wild-type mice were still alive. To determine the cause of death, we killed moribund Rpa1689C/+ mice between the ages of 4 and 14 months and
1Department of Cell Biology, Albert Einstein College of Medicine, Bronx, New York 10461, USA. 2Ludwig Institute for Cancer Research, Cancer Center and Departments of Medicine and Cellular and Molecular Medicine, University of California San Diego, La Jolla, California 92093, USA. 3Harvard Medical School-Partners Healthcare Center for Genetics and Genomics, Harvard Medical School, Boston, Massachusetts 02115, USA. 4Department of Human Genetics, Mount Sinai School of Medicine, New York University, New York, New York 10029, USA. 5Department of Pathology, Albert Einstein College of Medicine, Bronx, New York 10461, USA. 6Department of Medical Oncology, Dana-Farber Cancer Institute, Boston, Massachusetts 02115, USA. Correspondence should be addressed to R.D.K. (
[email protected]) or W.E. (
[email protected]).
Published online 19 June 2005; doi:10.1038/ng1587
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LETTERS Figure 1 Generation of Rpa1689C mutant mice. G K L F S gga aag ctt ttc tct (a) Gene-targeting scheme. The 689T-C point HincII Genomic Rpa1 locus HincII mutation (red asterisks; resulting in the amino acid substitution L230P) in exon 8 of Rpa1, as E6 E7 E8 E9 E 13 well as a silent mutation resulting in a XhoI site pRpa1689C targeting vector (at codon 233), was introduced by homologous HincII KpnI Kpn I recombination (dotted line) into embryonic stem * PGKneo cells. The Rpa1 wild-type locus, the pRpa1689C E6 E7 E8 E9 E 13 gene targeting vector and the modified Rpa1 G418 allele are shown. After germline transmission of the modified allele, the PGKneo cassette was HincII HincII Modified Rpa1 allele * deleted by Cre-loxP–mediated recombination PGKneo E6 E7 E8 E9 E 13 by mating of F1 male offspring to Zp3-Cre Cre transgenic female mice. (b) Southern-blot analysis of wild-type (+/+) and heterozygous HincII HincII Rpa1689C allele * Rpa1689Cneo/+ (+/mneo) F1 offspring showing germline transmission of the modified Rpa1 E6 E7 E8 E9 E 13 allele. (c) RT-PCR analysis of total RNA in wildtype (+/+) and heterozygous Rpa1689C/+ (+/m) HindIII Rpa1+ 121 bp mutant mice. Top, diagnostic scheme. The wild235 bp * type allele contains a HindIII restriction site, and 224 bp 132 bp m Rpa1 XhoI +/m digestion with HindIII generates fragments of 235 and 121 bp. The mutated allele has lost the M 1 2 3 4 7.6 kb HindIII restriction site and contains a newly 5.6 kb introduced XhoI site. Digestion with XhoI +/+ 356 bp 356 bp generates fragments of 224 and 132 bp. Bottom, 224 bp 235 bp 132 bp 121 bp the presence of the 689T-C mutation was verified by restriction digestion of RT-PCR Arg products in wild-type (lanes 1 and 3) and 216 30 µg 15 µg 7.5 µg 689C/+ Leu221 Rpa1 cells (lanes 2 and 4) with HindIII ssDNA (lanes 3 and 4) and XhoI (lanes 1 and 2), Trp 212 yielding diagnostic fragments of 235 and 121 bp with HindIII in wild-type cells and of 224 and Rpa1 132 bp with XhoI in Rpa1689C/+ cells. M, 100-bp Leu 221 DNA markers. (d) Sequence analysis of the Phe Arg 689C/+ β-actin RT-PCR product in Rpa1 mice confirmed OB1 238 OB2 210 the presence of the 689T-C mutation (red asterisk). (e) Comparative western-blot analysis of wild-type (+/+) and heterozygous Rpa1689C/+ (+/m) MEF cells indicates stable expression of the Rpa1 L230P protein. (f) Human Leu221 (equivalent to mouse Leu230) is in a b-ribbon that generates a hydrophobic patch at the DNA-binding interface and is illustrated from the complex of oligonucleotide binding (OB) domains 1 and 2 with single-stranded DNA (ssDNA). Prolines cannot adopt an extended conformation, and the L221P mutation would be expected to disrupt the DNA-binding interface but not the OB1 domain (Supplementary Fig. 1 online). +/
+/
+
eo
b
d
mn +/ eo + +/ mn
c
subjected them to detailed histopathological analysis. All Rpa1689C/+ mice had marked lymphoid hyperplasia as well as altered hematopoiesis in the bone marrow. Notably, most of the mice (9 of 14) developed lymphomas, and several of the lymphomas were highly disseminated, infiltrating multiple tissues (Fig. 2d and Table 2). Only one of the wild-type mice died at 14 months of age, of unknown causes but with no detectable tumors. The difference in survival between the two cohorts continued to increase, and by 2 years of age, only 40% of heterozygous mutant mice were still alive, compared with 470% of wild-type mice. To identify the genetic events associated with tumorigenesis in Rpa1689C/+ mice, we analyzed the genomic DNA of Rpa1689C/+ tumors for loss of heterozygosity at the Rpa1 locus after laser-capture microdissection. None of the six tumors analyzed showed loss of either the mutated Rpa1689C allele or the wild-type allele (Supplementary Fig. 2 online). The retention of the wild-type Rpa1 allele is consistent with the essential role of Rpa1 in DNA metabolism and suggests that the mutated Rpa1689C allele has a dominant effect. Genetic analysis of the corresponding rfa1 662T-C mutation in yeast indicates that rfa1662C is a partially dominant allele. Mutations in RFA1 in yeast, including the missense mutation 662T-C (also called rfa1-t48), cause increased accumulation of GCRs reminiscent of genome rearrangements seen in human
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cancers2,3. We therefore analyzed tumors in Rpa1689C/+ mice for chromosomal abnormalities by array comparative genomic hybridization (CGH) and found that the genomes in all tumors analyzed (n ¼ 9) contained multiple alterations (Supplementary Table 1 online). Notably, most tumors showed gains of the entire chromosomes 6 and 15 (Fig. 3a,b). We also identified segmental gains and losses of regions on other chromosomes (Fig. 3b). None of the losses involved regions known to contain tumor-suppressor genes, but the gains on chromosomes 6, 15 and 16 involved regions containing Table 1 Viability analysis of mouse embryos Genotype Developmental stage
+/+
+/m
m/m
ND
Unfertilized eggs
Total
E3.5 E7.5
8 10
15 21
7 0
12 0
12 NA
54 31
E9.5–E14.5 Live birth
6 31
10 62
0 0
0 0
NA NA
16 93
Wild-type (+/+) and heterozygous Rpa1689C/+ (+/m) mice were born at a 1:2 ratio, but no homozygous Rpa1689C/689C (m/m) mice were detected at birth. Rpa1689C/689C embryos were present at E3.5 but not at later developmental stages. NA, not applicable; ND, not determined.
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LETTERS Rpa1689C/+ mice. For each genotype, we examined several hundred metaphase spreads +/+ from three independent MEF lines for chromosome abnormalities. The frequency of Rpa1m (360 bp) +/m abnormal karyotypes was significantly greater Rpa1+ (277 bp) in Rpa1689C/+ MEFs than in wild-type MEFs m/m (P o 0.01; Fig. 3c,d and Supplementary Table 2 online). In addition to an increase 100 in aberrant metaphases containing aneuploid c d MLN (200×) Liver (400×) Kidney (100×) karyotypes (Fig. 3c and Supplementary 80 Table 2 online), we also detected chromosomal breaks in Rpa1689C/+ mutant cells that 60 were not found in wild-type cells (Fig. 3d). DNA DSBs in mammalian cells are potent 40 inducers of chromosomal instability15. The Spleen (200×) Spinal cord (100×) Brain (630×) Rpa1 689T-C mutation may result in +/+ 20 increased DSBs by causing defects in DSB +/m repair or by causing checkpoint defects that 0 result in failure to process replication errors 0 2 4 6 8 10 12 14 16 18 20 22 24 Months correctly and thus lead to mutagenic repair of spontaneous DNA damage and to GCRs Figure 2 Phenotypes of Rpa1 mutant mice. (a) Homozygous mutant Rpa1689C/689C (m/m) embryos had similar to those observed in Rpa1689C/+ severe growth retardation, and blastocyst outgrowth experiments showed that, in contrast to wild-type mutant MEFs2–8,16. To investigate this possi(+/+) and heterozygous mutant (+/m) embryos, homozygous embryos could not proliferate and further bility, we studied DSB repair after exposure degenerated in the in vitro culture. (b) Representative PCR genotyping analysis of wild-type (lanes 2, to the replication inhibitor aphidicolin. We 6 and 8), heterozygous (lanes 1, 4, 5 and 9) and homozygous (lanes 3 and 7) mutant embryos at 4 d in culture. M, 100-bp DNA markers. (c) Kaplan-Meier survival curve. The survival of wild-type (+/+; observed substantial levels of phosphorylated n ¼ 62) and heterozygous Rpa1689C/+ (+/m; n ¼ 54) mice was monitored for a period of 24 months. H2AX (g-H2AX), a marker for DSBs in Rpa1689C/+ mice had a significantly lower survival rate than did their wild-type littermates (P o 0.01, mammalian cells, in Rpa1689C/+ MEFs after log-rank test). (d) Lymphoma development in Rpa1689C/+ mice. Representative histopathological treatment with aphidicolin (Fig. 3e). In consections of disseminated lymphomas infiltrating into multiple tissues showing pronounced enlargement trast, there was little induction of g-H2AX in with the lymphomas replacing the architecture in mesenteric lymph node (MLN), liver and spleen as wild-type cells. Moreover, high levels of well as infiltration of lymphomas into kidney, spinal cord and brain. g-H2AX remained even 2 h after withdrawal of aphidicolin from the culture medium oncogenes. Although their involvement in lymphomagenesis in (Fig. 3e), indicating that DSB repair was impaired in Rpa1689C/+ MEFs. Rpa1689C/+ mice remains to be established, two of these oncogenes, To gain mechanistic insight into the development of lymphomas in Myc on chromosome 15 and Raf1 on chromosome 6, are frequently Rpa1689C/+ mice, we carried out parallel experiments in yeast. We involved in human and mouse neoplasia12–14. introduced the equivalent rfa1662C allele at the chromosomal locus or The Rpa1 689T-C mutation predisposed the mice to increased expressed the wild-type RFA1 or rfa1662C alleles under control of the tumorigenesis, with tumors bearing widespread chromosomal rear- native RFA1 promoter on either low-copy ARS CEN or high-copy rangements, but whether this mutation caused genomic instability and 2-micron plasmids in different yeast strains (Table 3). Western-blot predisposed Rpa1689C/+ mice to increased cancer susceptibility, or analysis showed that expression of Rpa1 from an ARS CEN plasmid whether the chromosomal aberrations in Rpa1689C/+ tumors were did not alter the levels of Rpa1 or Rpa2, whereas expression of Rpa1 secondary events during tumor progression, was not known. To from a 2-micron plasmid resulted in no more than a twofold increase address this issue, we analyzed the karyotypes of primary mouse of both Rpa1 and Rpa2 (data not shown; Rpa3 was not analyzed). embryonic fibroblast (MEF) strains derived from wild-type and Expression of the two different plasmid rfa1662C alleles in a wild-type +1
+3
+4
Time (d)
b
M 1 2 3 4 5 6 7 8 9
Survival (%)
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Table 2 Histopathological analysis of lymphomas in Rpa1689C/+ mice Mouse
Sex
1
F
9
+/m
Lymphoma
Bone marrow, thymus, liver, spleen, lymph nodes
2 3
F F
8.5 4
+/m +/m
Lymphoma Disseminated lymphoma
Spleen Lymph nodes, thymus, liver, kidney, brain
4 5
F F
6 8
+/m +/m
Disseminated lymphoma Disseminated lymphoma
Brain, spleen, thymus, kidney, liver, lung, lymph nodes Lymph nodes, liver
6
F
11
+/m
Disseminated lymphoma
Spleen, liver, kidney, lymph nodes, thymus, skeletal muscle, brain, spinal cord, stomach, bone marrow
7 8
M F
10 11
+/m +/m
Lymphoma Lymphoma
Lymph nodes Spleen, lymph nodes
M
14
+/m
Lymphoma
Gastric lymph nodes, mesenteric lymph nodes, spleen, bone marrow
9 +/m,
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Age (months)
Genotype
Histological type
Anatomic site
Rpa1689C/+.
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c
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Rpa1689C/+
Figure 3 Chromosome analyses of tumors and MEFs in mice. (a) Representative array CGH results showing gains (green) and losses (red) of chromosomes in individual tumors. (b) Ideograms highlighting gains (green) and losses (red) of chromosomal regions. (c) Example of an aneuploid metaphase in Rpa1689C/+ MEFs. (d) An example of chromosome breaks in Rpa1689C/+ MEFs. (e) Aphidicolin treatment results in increased induction of g-H2AX in Rpa1689C/+ MEFs. Cells were cultured in the presence or absence of 5 mM aphidicolin, and protein was isolated 0, 1, 2 and 4 h after aphidicolin treatment. Protein was also prepared from cells 2 h (+2) after aphidicolin withdrawal after the cells had been treated with aphidicolin for 4 h.
strain resulted in increased sensitivity to DNA-damaging agents including ultraviolet light, hydroxyurea, methylmethanesulfonate (high-copy plasmid only) and HO endonuclease-induced DSBs (high-copy plasmid only), as well as increased rates of accumulating GCRs, though not to the extent caused by chromosomal rfa1662C (Table 3 and Supplementary Tables 3–5 and Supplementary Fig. 3 online). When the plasmid rfa1662C alleles were expressed in strains carrying mutations that inactivate homologous recombination (mre11 or rad52), suppression of de novo telomere addition (pif1-m2) or different checkpoints (mre11, rad24, mec1 or rad53), the GCR rate increased by fivefold to more than 200-fold. The increased GCR rates
e Aphidicolin
+/m +/+ 0 1 2 4 +2 0 1 2 4 +2
Time (h) γ-H2AX β-actin
were not as great as those seen in double mutants carrying the chromosomal rfa1662C allele (Table 3). In addition, the chromosomal rfa1662C allele showed synthetic lethality when combined with mre11D and rad27D mutations, whereas the plasmid rfa1662C alleles did not17 (Table 3 and data not shown). These results support the view that the rfa1662C allele is both hypomorphic and semidominant when expressed from both low-copy and high-copy plasmids in the presence of a single copy of the wild-type RFA1 gene. The pattern of genetic interactions that we observed supports the hypothesis that the plasmid rfa1662C mutation causes partial defects in both homologous recombination4 and checkpoints5 (Supplementary Tables 3–5 online) and
Table 3 Expression of the rfa1662C allele in yeast shows partial dominance for GCR formation GCR rate (1010) (plasmid allele/chromosome allele) Genotype
Plasmid type
Vector/RFA1
Wild-type
Low
o50
mre11D
High Low
o2 1,900
rad52D
High High
2,800 56
rad57D pif1-m2
High High
o41 o31
rad24D mec1D sml1D
High Low
6.4 110
rad53D sml1D
High High
o9.6 o16
RFA1/RFA1 o0.80
rfa1662C/RFA1
Vector/rfa1662C
16
27,000
75 3,300
39,000 SL
920 o43
9,100 10,000
SL 38,600
67 22
1,200 6,200
33,000 ND
o45 o30
820 330
85,000 ND
1,700 3,000
57,000 120,000
o3.1 600
9.8 o20
GCR rates were determined for the indicated strains (10–28 cultures from at least two and an average of three independent isolates) containing the wild-type RFA1 allele or the rfa1662C allele on either low–copy number (ARS CEN, pRS415) or high–copy number (2-micron, pRS425) LEU2 plasmids or vector only. Plasmid allele is listed first; chromosome allele second. ND, not determined; SL, synthetic lethal mutation combinations. Extra copies of wild-type RFA1 seem to partially suppress the GCR rate of the mre11D mutant. All 11 rearrangements recovered from the wild-type strain containing a high–copy number plasmid with a rfa1662C allele were de novo telomere additions. All 19 mre11D spores from the cross between the mre11D and rfa1662C mutants were RFA1, indicating that mre11D and rfa1662C are synthetically lethal (P ¼ 0.00013; w2 test).
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LETTERS hence shows genetic interactions with checkpoint defects7 and homologous recombination defects, respectively18–20. The cell-cycle checkpoint and homologous recombination defects observed in yeast might underlie the chromosomal gains and rearrangements in the observed lymphomas, as such defects cause both genome rearrangements and mis-segregation of chromosomes18,20–22. But the specificity of both the induced cancers and the associated altered chromosomes could also occur because the DNA damage promoted by the Rpa1689C/+ background provides a selection for activation or inactivation of a specific set of oncogenes or tumor-suppressor genes, respectively. Human RPA1 maps to chromosome 17p13.3 (ref. 23), and loss of this chromosomal region has repeatedly been implicated in various malignancies including colorectal cancers, breast cancers, lymphomas and leukemias24–29. In addition, comparisons of CGH data suggest that human tumors with loss of 17p also have more total genomic aberrations (SKY/M-FISH and CGH database). Imbalances at a nearby locus, 17p13.1, have been reported in several types of cancers. TP53 maps to 17p13.1, and its loss is believed to be responsible for the associated cancer phenotypes25. Notably, allelic changes occur at a substantially higher frequency at 17p13.3 than at 17p13.1 in human cancers27. Although the relevance of 17p13.3 loss for tumorigenesis is not known, defects in other proteins associated with DNA metabolism underlie several human genetic disorders associated with cancer susceptibility23. Our results indicate that Rpa1 is essential for cell survival and for maintaining chromosomal stability and tumor suppression in mammalian cells. These findings may also have implications for tumor formation in humans. METHODS Generation of Rpa1689C mutant mice and preparation of MEFs. We introduced the missense mutation 689T-C (resulting in the amino acid substitution L230P) in exon 8 of Rpa1 into the mouse genome by gene targeting. We introduced the targeting vector into WW6 embryonic stem cells by electroporation, isolated G418-resistant colonies and screened them by PCR (primer sequences available on request). We identified five positive clones and confirmed the correct targeting event by digestion of genomic DNA with HincII and Southern-blot analysis. We injected three correctly targeted embryonic stem cell lines into C57BL/6J blastocysts to generate male chimeras that transmitted the Rpa1689C allele through the germ line. We then mated F1 males carrying the mutated allele to Zp3Cre transgenic females (C57BL/6J) to eliminate the resistance cassette by loxP-mediated recombination. We intercrossed male and female mice carrying the modified allele to generate Rpa1+/+, Rpa1689C/+ and Rpa1689C/689C mutant mice. To produce Rpa1+/+ and Rpa1689C/+ MEF lines, we intercrossed Rpa1689C/+ mice, isolated E13.5 embryos and prepared MEF lines from embryos by standard procedures. We cultured MEFs in Dulbecco’s modified Eagle medium with 10% fetal calf serum and genotyped them by PCR. RT-PCR. We isolated total RNA from the liver, thymus, spleen, muscle, intestine and testis of Rpa1+/+ and Rpa1689C/+ mice (RNeasy Mini kit, Qiagen) and carried out RT-PCR (primer sequences available on request; Titan One Tube RT-PCR System, Roche Diagnostics Corporation). We digested the amplified 356-bp cDNA fragment with either HindIII to detect the wild-type transcript or XhoI to detect the mutant transcript. To confirm expression of the Rpa1 689T-C mutation, we sequenced the cDNA fragment. Western-blot analysis. We isolated protein from MEFs using Trizol reagent (Invitrogen) and quantified it (BCA Protein Assay Reagent, Pierce). We created serial dilutions (30, 15 and 7.5 mg) of protein of each cell line, separated samples by 10% SDS-PAGE and transferred them onto PVDF membranes (Immobilon P, Millipore). We then incubated the membranes with antibodies directed against Rpa1 (Ab-1, Oncogene Sciences) and b-actin (ACTN05, NEOMARKERS, Inc.) as a loading control. For the DSB repair assay, we used
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the antibody to H2AX phosphorylated at Ser139 (g-H2AX; Upstate Biotechnology) at a 1:1,000 dilution in TBST containing 3% nonfat dried milk. Viability analysis and blastocyst outgrowth of Rpa1689C mutant mice. We isolated mouse embryos at stages E14.5, E9.5, E7.5 and E3.5 and genotyped them by PCR. We isolated Rpa1+/+, Rpa1689C/+ and Rpa1689C/689C blastocysts, cultured them in vitro on gelatinized tissue culture plates and monitored their growth and morphology for 4 d. Histopathology. We killed mice when they became moribund and subjected them to systematic histopathological analysis. We processed tissues in accordance with standard procedures and evaluated tumor and normal tissue paraffin sections by staining with hematoxylin and eosin. Chromosome analysis. We prepared metaphase spreads from three independent lines of wild-type and Rpa1689C/+ MEFs using standard protocols. We stained the spreads with Giemsa (BDH Laboratory Supplies) and examined them for chromosomal abnormalities. Array CGH analysis. The methods used for array CGH analysis are described in Supplementary Methods online. Yeast procedures. We generated the high-copy plasmids containing the RFA1 (pRDK1139) and rfa1662C (pRDK1140) alleles by subcloning the HindIII-SalI fragment from the low-copy plasmids pKU1 and pKU1-t48 (ref. 8) into pRS425 (ref. 30). Yeast mutants were isogenic to the wild-type strain RDKY3615 (MATa, ura3-52, his3D200, trp1D63, leu2D1, hom3-10, ade2D1, ade8, yel069::URA3)21. We generated double mutants by crossing an isogenic MATa rfa1662C strain with mutants of interest and identified mutations by sequencing PCR products amplified from genomic DNA preparations and by plating onto selective media. We used media and methods for determining GCR rates as previously described3, except that we grew liquid cultures in SCLeu medium for 2 d at 30 1C to reach saturation before plating them on GCR selection medium. We determined Rfa1 and Rfa2 levels by western blotting using antibodies provided by S. Brill (Rutgers University). URL. The SKY/M-FISH and CGH database is available at http://www. ncbi.nlm.nih.gov/sky/skyweb.cgi/. Accession numbers. GenBank: mouse Rpa1, NM_026653; yeast RFA1, M60262. PDB: single-stranded DNA-binding domain of human RPA1 bound to single-stranded DNA, 1JMC. Note: Supplementary information is available on the Nature Genetics website. ACKNOWLEDGMENTS We thank H. Hou, Jr. and B. Jin for technical assistance and S. Brill for providing antibodies. This work was supported by grants from the US National Institutes of Health (to R.K., R.D.K. and W.E.). COMPETING INTERESTS STATEMENT The authors declare that they have no competing financial interests. Received 24 August 2004; accepted 16 May 2005 Published online at http://www.nature.com/naturegenetics/
1. Wold, M.S. Replication protein A: a heterotrimeric, single-stranded DNA-binding protein required for eukaryotic DNA metabolism. Annu. Rev. Biochem. 66, 61–92 (1997). 2. Chen, C., Umezu, K. & Kolodner, R.D. Chromosomal rearrangements occur in S. cerevisiae rfa1 mutator mutants due to mutagenic lesions processed by doublestrand-break repair. Mol. Cell 2, 9–22 (1998). 3. Chen, C. & Kolodner, R.D. Gross chromosomal rearrangements in Saccharomyces cerevisiae replication and recombination defective mutants. Nat. Genet. 23, 81–85 (1999). 4. Soustelle, C., Vedel, M., Kolodner, R. & Nicolas, A. Replication protein A is required for meiotic recombination in Saccharomyces cerevisiae. Genetics 161, 535–547 (2002). 5. Kim, H.S. & Brill, S.J. Rfc4 interacts with Rpa1 and is required for both DNA replication and DNA damage checkpoints in Saccharomyces cerevisiae. Mol. Cell. Biol. 21, 3725–3737 (2001). 6. Lee, S.E. et al. Saccharomyces Ku70, mre11/rad50 and RPA proteins regulate adaptation to G2/M arrest after DNA damage. Cell 94, 399–409 (1998).
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