![[Feedback]](/icons/banner/feedbackACT.gif){kind=icon}
![[For Subscribers]](/icons/banner/subscriberACT.gif){kind=icon}
![[Archive]](/icons/banner/archiveACT.gif){kind=icon}
![[Search]](/icons/banner/searchACT.gif){kind=icon}
![[Contents]](/icons/banner/tocACT.gif){kind=icon}
Previous Article | Next Article 
Molecular and Cellular Biology, July 2005, p. 6103-6111, Vol. 25, No. 14
0270-7306/05/$08.00+0 doi:10.1128/MCB.25.14.6103-6111.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.
,
Eiichiro Sonoda,1,
Michio Yoshimura,1,3
Yoshiaki Kawano,4
Hideyuki Saya,4
Masaoki Kohzaki,1,5 and
Shunichi Takeda1*
Department of Radiation Genetics, Kyoto University Graduate School of Medicine, Kyoto 606-8501, Japan,1 Departments of Urology,2 Therapeutic Radiology & Oncology, Kyoto University Graduate School of Medicine, Kyoto 606-8507, Japan,3 Department of Oncology, Kumamoto University School of Medicine, Kumamoto 862-0811, Japan,4 Department of Radiology and Radiation Biology, Nagasaki University Graduate School of Biomedical Sciences, Nagasaki 852-8521, Japan5
Received 8 September 2004/ Returned for modification 24 November 2004/ Accepted 5 April 2005
 |
ABSTRACT |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
(Rev3-Rev7)-independent role of Rev1 in vertebrate cells. |
INTRODUCTION |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
In yeast, the major components of PRR are divided roughly into two groups: translesion DNA synthesis (TLS) involving the Rad6-Rad18 epistasis group and HR composed of the Rad52 epistasis group (reviewed in references 4 and 10). TLS allows tolerance of DNA damage, employing a number of specialized DNA polymerases that are able to synthesize directly across template DNA lesions (reviewed in references 13 and 17). Vertebrate TLS polymerases have been reported to have biochemical functions basically similar to those of their yeast homologs. However, the biological role of the TLS polymerases is complicated and poorly characterized in vertebrate cells. Similarly, DNA polymerases involved in HR are only poorly understood even in yeast (reviewed in reference 16).
REV genes were originally identified as responsible genes for reversible mutation of UV light-induced mutagenesis in the yeast Saccharomyces cerevisiae (21, 23). Longstanding work in yeast and recent studies in vertebrates have revealed that Rev1 is one of the Y-family DNA polymerases (34), having deoxycytidyl transferase activity (31), and that Rev3 and Rev7 are the catalytic and regulatory subunits of DNA polymerase (Pol) capable of bypassing a UV-induced thymidine dimer (32; reviewed in references 20 and 30). The three yeast rev mutants and their triple mutant show very similar sensitivity to various genotoxic treatments. However, there is also a distinct phenotypic difference between the yeast rev1 mutant and the other rev mutants; for example, UV-induced frameshift mutagenesis depends much more on Rev3 and Rev7 than on Rev1 in yeast (22).
The vertebrate Rev1 and Rev3 molecules are significantly larger than their yeast counterparts, suggesting that the vertebrate enzymes may have additional functions despite similarities in their basic biochemical behavior (12, 25). Indeed, biochemical studies indicate that mammalian Rev1 can physically interact with Rev7 (26, 29) or with other Y-family polymerases, such as Pol
, Pol
, and Pol
(14) via a C-terminal region that is lacking in yeast Rev1. Another difference between vertebrate and yeast Rev functions is that the contribution of Pol to tolerance of DNA damage appears to be significantly greater in vertebrates than in budding yeast. Although mutation of yeast Rev3 has comparatively little impact on viability or sensitivity to genotoxic agents, disruption of Rev3 in mice causes embryonic lethality (2, 9, 46) and significant genome instability and extreme hypersensitivity to killing by a variety of genotoxic agents, particularly, cisplatin in chicken DT40 (42). Vertebrate Rev7 has homology with vertebrate Mad2 to a similar extent with yeast Rev7 (7, 28), also suggesting that it might act as a spindle checkpoint protein (8, 36).
The chicken B-lymphocyte line DT40 is characterized by a high efficiency of gene targeting and phenotypic stability (6, 41), providing us with a unique opportunity of dissecting the mechanism of HR by comparing the phenotypes of a variety of HR mutants (3, 40, 45). As suggested from yeast and vertebrate studies mentioned above, the vertebrate Rev molecules are supposed to have distinct as well as collaborative function. To understand the genetic interrelationship among the three Rev molecules in the higher eukaryote, we generated REV7 gene-disrupted cells (rev7 cells) and triple mutants of the three REV genes (rev1 rev3 rev7) in DT40. In this study, we reveal that the three Rev molecules may act as a functional unit in cellular tolerance to a variety of genotoxic stresses, including IR, but that Rev1 acts independently of Rev3-Rev7 in HR-dependent diversification of the Ig gene variable (Ig V) region: i.e., Ig gene conversion. We also present the nonredundant, overlapping role of these Rev molecules in the HR-mediated PRR: i.e., sister chromatid exchange (SCE), in vertebrate cells.
|
MATERIALS AND METHODS |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Chicken REV7 partial cDNA sequences were also obtained by chicken BLAST search with human REV7 sequence. cDNA fragments containing undetermined 5' sequences were amplified from LambdaZAPII chicken testis cDNA library by reverse transcription-PCR with XL polymerase (Perkin-Elmer) using the M13R primer and the primer 5'-GGGTGATTTCGAAGACAAATC-3' designed from the obtained sequences. Eight amplified PCR products were cloned into the pGEM-T Easy vector (Promega), and the complete sequences were determined.
Chicken REV7 genomic DNAs were amplified from the EMBL3 chicken genomic DNA library (Clontech) with LA-Taq (Takara) using the primers designed from the cDNA sequence. The positions of exons and introns were determined by base sequencing.
Plasmid construction. Three REV7 disruption constructs, REV7-hisD, REV7-bsr, and REV7-puro, were generated from genomic PCR products combined with hisD, bsr, and puro selection marker cassettes. Genomic DNA sequences were amplified from DT40 genomic DNA and inserted unique restriction sites near the 5' and 3' ends using the following primers: for the left arm (0.6 kb), 5'-AGCTTCTAGACTGGAGGCGTTTAAGGACAT-3' (denoted XbaI site with underlined) and 5'-AGCTGGATCCTTAAGGTCCTGTCGTGTGAG-3' (BamHI site); and for the right arm (3.5 kb), 5'-AGCTGGATCCTGCACTGTGTAAAGCCACTG-3' (BamHI site) and 5'-AGCTGGTACCTACCTCAGGATTGTGACCCG-3' (KpnI site). Amplified PCR products for each arm were cloned into pGEM-T Easy vector (Promega), and SalI-BamHI fragment containing the left arm was inserted into the right arm vector, and then each selection marker cassette flanked by BamHI site was inserted. REV1 disruption constructs REV1-neo and REV1-puro and REV3 disruption constructs REV3-his/loxP and REV3-bsr/loxP were generated as described previously (39, 42). Modified REV1 disruption constructs REV1-hisD, REV1-bsr, and REV1-hygro were made from REV1-neo by replacing the 1-kb short arm with a new 3.5-kb genomic fragment in order to improve targeting efficiency and by alternating each selection marker cassette. The new 3.5-kb fragments were obtained by PCR with primers 5'-GGGGGATCCAAAGCAGGCAGGTCTGCACTTAAAACTG-3' (BamHI site) and 5'-GGGCTCGAGTCTGGTAATATTGCCATTCAAGCTGGG-3' (XhoI site). The human REV7 expression construct, p345, was generated from pEGFP-N2 (Clontech), in the EcoRI site of which a human REV7 cDNA (containing stop codon) was inserted. The chicken REV1 expression construct, pCR3-loxP-cREV1/IRES-EGFP-loxP (named p667 in short), in which chicken REV1 and eukaryotic green fluorescent protein (EGFP) genes are flanked by the loxP sequences, was made by inserting a chicken REV1 cDNA into the BamHI-XhoI site of pCR3-loxP-MCS/IRES-EGFP-loxP (11, 47). The plasmids were linearized prior to transfection into DT40 cells; REV7-hisD, REV7-bsr, REV7-puro, p345, and p667 were digested with KpnI, ScaI, KpnI, NotI, and PvuI, respectively. REV1-hisD, REV1-bsr, and REV1-hygro were all linearized with NotI.
Cell culture and DNA transfection. The conditions for cell culture, selection, and DNA transfections have been described previously (42, 44).
Generation of mutant cells. CL18 is a wild-type (WT) DT40 clone which is negative in surface IgM (sIgM) and is characterized its sequence at the IgL locus (5). CL18 cells were sequentially transfected with REV1-hisD and REV1-bsr targeting constructs to obtain new rev1 clones (Fig. 1A). Also CL18 cells and rev3/CRE cells without a drug-resistant cassette (42) were sequentially transfected with REV7-hisD and REV7-bsr constructs to generate rev7 and rev3 rev7 cells, respectively. rev7+hREV7 cells were obtained from rev7 cells by transfection with the p345 expression vector. rev1 rev3 rev7 cells were derived from rev3 rev7 cells by successive transfection with REV1-puro and REV1-neo constructs (39). rev1 and rev1 rev3 rev7 cells were transfected with p667, resulting in rev1+cREV1 and rev1 rev3 rev7+cREV1 cells, respectively (Fig. 1A). ATM–/– (atm) and rad18 cells were described previously (27, 47). All the mutant DT40 clones described in this paper are deposited in the RIKEN Cell Bank (RIKEN, Wako, Japan).
|
Colony formation assay. The colony formation assay was performed as described previously (35), with the following modification in exposure to UV and hydrogen peroxide. UV (wavelength = 254 nm) irradiation was performed without medium exchange for PBS since the treatment of rev mutants with PBS causes the significant reduction of cell viability. UV dose applied to the cells within complete medium was estimated and calibrated accordingly. Hydrogen peroxide (H2O2; 30% [wt/vol]; Santoku, Japan) was appropriately diluted with distilled water just before use. For treatment of cells with H2O2, 1 x 105 cells were incubated at 39.5°C in 1 ml of complete medium containing a pertinent concentration of H2O2 for 1 h. Then cells were serially diluted and plated in triplicate onto six-well plates with 5 ml/well of 1.5% (wt/vol) methylcellulose (Aldrich) containing Dulbecco's modified Eagle's medium-F-12 (Gibco-BRL), 15% fetal calf serum (Equitech-Bio), 1.5% chicken serum (Sigma), and 10 µM ß-mercaptoethanol and were incubated at 39.5°C for 6 to 7 days (wild-type cells) or 10 to 14 days (mutant cells) for the estimation of surviving colonies.
Chromosomal aberration analysis. Preparation of chromosome spreads and karyotype analysis were performed as described previously (35, 40). For the enrichment of mitotic cells, Colcemid was added to the last 3 h of incubation.
Measurement of SCE level. Measurement of SCE level was carried out as described previously (35, 47). Cells were treated with Colcemid for the last 2 h of incubation to enrich mitotic cells. To compare the SCE results, nonparametric statistical analysis (Mann-Whitney U test) was performed using StatView5.0 software.
Measurement of gene targeting frequency. Targeted gene integration frequency was measured as described previously (44).
Measurement of Ig gene conversion rate. Measurement of the Ig gene conversion rate using surface IgM detection was performed as described previously (3, 5, 38).
Antibody. Anti-Rev7 antibody was raised against glutathione S-transferase (GST)-human Rev7 and purified by a maltose birding protein (MBP)-human Rev7 column.
Nucleotide sequence accession numbers. The chicken REV1 and REV7 cDNA sequences have been submitted to the GenBank database under accession no. AY675169 and AY675170, respectively.
|
RESULTS |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
joint (CL18) (5), in order to accurately measure the rate of Ig gene conversion. The growth properties of these two rev7 clones were monitored using growth curves and cell cycle analysis. The rev7 clones exhibited growth properties very similar to those of rev1 and rev3 clones, including retarded growth kinetics (Fig. 2A) and higher fractions of dead cells (Fig. 2B). Complementation of the rev7 cells with human Rev7 resulted in growth kinetics indistinguishable from those of WT cells (Fig. 2A). The significant homology between vertebrate Mad2 and Rev7 (see Fig. S1 in the supplemental material) suggested that Rev7 might be involved in the spindle assembly checkpoint similarly to Mad2 (24). To address this question, we measured the mitotic index with time after addition of Colcemid, an inhibitor of microtubule formation. rev7 cells were normally arrested before entering metaphase in the presence of Colcemid, indicating that disruption of Rev7 has no effect on the spindle checkpoint (Fig. 2C). Similarly, suppression of DNA synthesis after IR irradiation and a delay in G2/M transition after exposure to IR or UV were observed in Rev7-deficient cells as well as in wild-type cells, also suggesting that disruption of Rev7 has no effect on DNA damage checkpoints (see Fig. S2 in the supplemental material).
|
|
rays; 137Cs), cisplatin [cis-diaminedichloroplatinum (II); CDDP], H2O2, and methyl methanesulfonate (MMS), as do rev1 and rev3 cells (Fig. 3B to E). The sensitivity of each rev single mutant to these agents is also similar.
|
appears to be composed of the Rev3 and Rev7 subunits (28, 32). Subsequently, we generated two rev1 rev3 rev7 clones from one rev3 rev7 clone. rev1 rev3 rev7 cells showed a phenotype similar to that of single rev3 and rev7 mutants, which includes proliferation rate (Fig. 2A), the level of spontaneous chromosomal aberrations (Table 1), and sensitivity to killing by various genotoxic stresses (Fig. 3). The overall similarity in the phenotypes of the rev single mutants and the rev1 rev3 rev7 triple-mutant cells supports the notion that the three Rev molecules might operate as a single functional entity in the repair or tolerance of DNA damage. The three rev mutants have indistinguishable phenotypes in HR-mediated DSB repair while exhibiting variant phenotypes in sister chromatid exchange. We previously showed that the significant increase in IR sensitivity in rev3 cells is presumably caused by a defect in HR-mediated DSB repair as well as in TLS (42). The observed hypersensitivity of rev1 and rev7 mutants to IR and cisplatin led us to address further assessment of role for Rev molecules in HR. To investigate the role for Rev1 and Rev7 in HR, we studied HR-dependent DSB repair, gene targeting frequency, SCE, and Ig gene conversion in the rev single and triple mutants.
To specifically evaluate the contribution of HR-dependent DSB repair in rev1 and rev7 cells, asynchronous populations of cells were exposed to 2 Gy of IR and induced chromosome breaks were measured at 3 h after the irradiation. The majority of the cells that enter mitosis within 3 h after irradiation should have been irradiated in the G2 phase but not in the late S phase (42). Thus, the number of induced chromosomal breaks in metaphase spreads should reflect DSB repair capability during the G2 phase, mainly mediated by HR (44). Following this protocol, rev1 and rev7 cells also showed a marked increase in the level of chromosomal aberrations (Fig. 3F). A similar increase in IR-induced aberrations was seen in the triple mutants. This observation suggests that Rev1 and Rev7, as well as Rev3 (42), are essential for efficient DSB repair following DNA damage, most likely during the DNA synthesis step of the HR reaction.
On the contrary, the levels of SCEs, which are thought to reflect HR-mediated PRR, tended to be different among the rev mutants (Fig. 4). We assessed SCE levels of two clones at least for each single and triple rev mutant and confirmed the tendency that the levels of SCEs are the same among the identical genotypic clones (data not shown). Since the distribution of the number of SCEs per cell in each mutant does not follow the normal distribution, we employed nonparametric statistical analysis, the Mann-Whitney U test. According to this test, we obtained statistical orders that WT < rev1 rev3 rev7 < rev1 = rev7 = rev3 < rev1 rev3 rev7+cREV1 in spontaneous SCE level, and that WT = rev1 rev3 rev7 < rev3 = rev1 = rev7 = rev1 rev3 rev7+cREV1 in 4-nitroquinolone oxide (4-NQO)-induced SCE level (=, not significantly different; <, significantly lower [P < 0.002]). As expected, rev7 cells showed the same level of increase in SCE events, as did rev3 cells, both with and without 4-NQO treatment. rev1 cells also showed a significant increase in the level of SCE, which is similar to those of rev3 and rev7 cells. This observation does not differ much from Sale's original work, as the level of spontaneous SCE in their rev1 mutant appeared to be indeed higher than that of wild-type cells (39). Remarkably, rev1 rev3 rev7 triple mutants show nearly a wild-type level of SCE even in the induced condition. Expression of chicken REV1 cDNA in rev1 rev3 rev7 cells increased SCE events to a level similar to that of rev3 and rev7 cells (Fig. 4).
|
|
segments following completion of V(D)J rearrangement. This DT40 assay allows for the determination of gene conversion events as well as the identification of aberrant events. To assess the rate of Ig gene conversion, we monitored the gain of sIgM expression from sIgM-negative cells that carried the same frameshift mutation in the rearranged VJ joint (CL18) (5). The gain of sIgM reflects the repair of the frameshift mutation through superimposed gene conversion events. We previously showed that a defect in Rev3 has no impact on the Ig gene conversion in chicken DT40 (42). In the current study, we newly made three rev1 clones from CL18 (Fig. 1A; see Materials and Methods) and analyzed Ig gene conversion in the cells together with rev1 rev3 rev7 cells (Fig. 5). Analyses of fluctuations in sIgM gain were performed on 30 to 40 subclones expanded from single sIgM-negative clones following a 3-week culture. Remarkably, rev1 and rev1 rev3 rev7 cells exhibited about a 3.5-fold decrease in appearance of sIgM-positive revertant subclones, while rev3 cells showed a wild-type level (Fig. 5A). The expression of chicken REV1 transgene in rev1 cells normalized the level of sIgM gain to a wild-type level. This observation suggests that Rev1, but neither Rev3 nor Rev7, is required for efficient Ig gene conversion.
|
segments in sIgM-positive revertants from the three independent rev1 clones. On the whole, revertant subclones derived from rev1 mutants showed rare somatic mutation events occurred independently of gene conversion events, as observed in wild-type cells (Fig. 5B). The sequences around the parental frameshift mutation were indeed replaced by pseudo-V sequences in sIgM-positive revertant subclones in rev1 mutants, as observed in wild-type cells (see Fig. S3 in the supplemental material). In rev1 cells, the average length of the gene conversion tract and the selection of putative pseudo-V donor were comparable to those of wild-type cells (Fig. 5B; average tract length is 92.8 bp in the wild type versus 98.2, 76.9, and 78.7 bp in three rev1 mutants). These results suggest that Rev1, acting independently of Rev3 and Rev7, apparently influences the rate of Ig gene conversion but not the length or fidelity of conversion tract or the choice of donor segments. |
DISCUSSION |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
. In marked contrast, Rev1 may act in two different manners, as an additional component of Pol complex and in a role independent of Pol. rev1 cells and the other rev mutants exhibited similar phenotypes as follows: defective growth properties with spontaneous chromosomal aberrations, elevated sensitivity to a variety of DNA-damaging agents, and induced chromosomal breaks after IR in the G2 phase; indicating that the similarity in phenotypes reflects the cooperative function of the three Rev molecules in these circumstances. On the other hand, the rev1 and rev1 rev3 rev7 clones, but not rev3 or rev7 clones, exhibited reduced Ig gene conversion, suggesting that Rev1 may be required for the intragenic HR independently of Pol or presumably by associating with other unknown DNA polymerase(s). In summary, the current study demonstrates that Rev1 plays Rev3-Rev7-dependent and independent roles in the cellular tolerance of DNA-damaging agents and HR.
Cooperative function of the Rev1, Rev3, and Rev7 proteins in DNA damage response.
The current study showed that the three rev single mutants displayed similar sensitivities to a variety of DNA damage. This phenotypic similarity can be interpreted in two different ways. First, all the Rev proteins may be equally required for efficient TLS past a variety of DNA damage. Alternatively, although Rev1 may have another contribution to cellular tolerance of DNA damage, as suggested from a biochemical study indicating that Rev1 interacts with another TLS polymerase (14), the contribution might be little in DT40 cells, resulting in rev1 cells lacking a more prominent phenotype than rev3 or rev7 cells. We favor the former idea, because the triple mutant also exhibited the same level of sensitivities as did all the single mutants, and because an essential role for Rev1 in Pol-dependent TLS is unlikely at least in DT40, from our observation that rev3 and pol double mutations caused an additive increase in UV sensitivity (manuscript in preparation). Thus, it is unlikely that some Rev molecules can act independently of the other Rev molecules in tolerance to exogenous DNA damage.
We previously demonstrated that Rev3 is involved in not only TLS but also HR-dependent DSB repair after IR (42). Since Rev3 is relatively indifferent to structural distortions at DSBs caused by IR, Rev3 may play an important role in DNA synthesis of HR-mediated DSB repair, particularly after IR. Interestingly, rev1, rev7, and rev1 rev3 rev7 mutants as well as rev3 cells all exhibited very similar levels of chromosomal breaks after IR irradiation (Fig. 3F). We, therefore, conclude that the three Rev molecules may participate in HR-mediated DSB repair as a functional unit, though their involvement in other unknown DSB repair pathways is not completely eliminated.
Rev1 and Rev3-Rev7 may contribute nonredundantly to SCE. It is thought that SCE is a result of PRR events mediated by HR resulting in crossover events in higher eukaryotic cells (19). We have shown that TLS mutant DT40 cells display elevated level of SCE, while HR mutant DT40s show reduced SCE level (16). The level of SCE should be determined by the balance of two major factors of PRR: TLS and HR. A defect in TLS may result an increase in the number of unfilled gap, which may activate HR-mediated PRR (i.e., SCE), whereas a defect in HR should directly reduce SCE events. To assess the role of each Rev molecule in HR-mediated PRR, we measured the levels of SCE comparing the three rev single mutants as well as the triple mutant. Remarkably, two clones of rev1 rev3 rev7 cells exhibited suppressed levels of spontaneous or induced SCE events, although each rev single mutant consistently exhibited a significant increase in spontaneous SCE (Fig. 4). Furthermore, reconstitution of the triple mutant with REV1 cDNA increased in the level of SCE to those of the three rev single mutants.
It is unclear how SCE levels in rev triple mutants are suppressed, although those of rev single mutants are elevated. The suppressed SCE levels in rev triple mutants are reminiscent of HR-deficient DT40 mutants that demonstrate SCE is a PRR mediated by HR (41). It is likely that SCE in eukaryotic cells reflects only a proportion of total HR events which result in crossing over. Furthermore, it is difficult to know what is leading to the reduced level of SCEs in rev triple mutants, since a reduction in SCEs may reflect a decreased number of lesions being processed by recombination and/or a decreased number of crossover recombination events.
Rev1 may act independently of Rev3-Rev7 proteins in Ig gene conversion.
In this study, we found rev1 cells displayed a significant decrease in the kinetics of Ig gene conversion, although the previous study did not find such reduction in their rev1 clone using a distinctly different assay system (39). They examine the gene conversion events that inactivate an IgV gene and thereby lead to loss of sIgM expression. This assay is suitable for detection of single-nucleotide substitutions, as they wished to examine, but does not necessarily provide reproducible data for counting the number of HRs, because the vast majority of gene conversion events do not inactivate an IgV gene and their rate should be substantially affected by the nucleotide sequences of the IgV gene. To solve these problems, we created for this study three additional rev1-deficient clones as well as the other rev mutants from the wild-type cells that carry the defined IgV sequences, including a specific frameshift mutation (5), and examined in all genotypes analyzed the number of Ig gene conversion events that occurred around identical sequences, including the frameshift mutation. Indeed, we have obtained consistent result from each genotype, including wild-type cells, two rev1 clones, two clones reconstituted with Rev1 cDNA, and two clones of rev1 rev3 rev7 triple mutants. We, therefore, conclude that a defect in Rev1 indeed had an impact on the Ig gene conversion rate, whereas neither Rev3 nor Rev7 appears to participate in this intragenic HR reaction. Thus, Rev1 may play a role in HR even in the absence of Pol. There are two possible mechanisms of action of Rev1. First, the nontemplate nucleotide insertion activity of Rev1 might contribute to the initiation of DNA synthesis in gene conversion. However, this possibility may be unlikely, because yeast Rev1 does not require its conserved deoxycytidyl transferase activity for at least TLS (1, 33). Alternatively, Rev1 associates with other unknown DNA polymerase(s) and may play a regulatory role in DNA synthesis, as suggested from the biochemical study (14). Our observation that the defect in Rev1 significantly reduced the rate of Ig gene conversion but had no impact on the length of gene conversion tract suggests a certain regulatory role of Rev1 in Ig gene conversion, which agrees with the latter idea. This model is open to further testing, and we need to identify the DNA polymerases involved. We assume that this task should be performed by TLS polymerases that can extend from mismatched 3' end, such as Pol, as well as Pol and Pol, because substantial sequence divergence is present between donor pseudo-V and recipient VJ segments in Ig gene conversion. Presumably, the DNA synthesis in gene conversion may be achieved by the combination of at least two DNA polymerases: One may require Rev1, whereas the other polymerase can act without Rev1 and might have extensive processivity.
In conclusion, the present data suggest two types of Rev1 action in HR; Rev1 is involved in HR-mediated repair of IR-induced DSBs as a component of the Pol complex and also participates in Ig gene conversion without interacting with Pol. From a view of DNA repair, the three Rev molecules may form a functional unit to undergo TLS past a wide range of DNA lesions and also achieve the repair of IR-induced DSBs. Furthermore, Rev1 and Rev3-Rev7 might act independently of each other and be required for HR-mediated PRR of DNA damage (Fig. 6). As reviewed in a recent report concerning genes involved in chromosome fragility syndromes (43), further study of the REV genes and other HR-related genes disrupted in DT40 clones would help in dissecting the complex and independent roles of each Rev molecule as well as revealing unknown genetic interaction with other molecules in a variety of DNA repair and HR with the related pathways.
|
|
ACKNOWLEDGMENTS |
|---|
Financial support was provided in part by a Core Research for Evolutional Science and Technology grant from Japan Science and Technology Corporation; by the Center of Excellence (COE) grant for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology of the Japanese Government; and by grants from the Uehara Memorial Foundation and the Naito Foundation.
|
FOOTNOTES |
|---|
Supplemental material for this article may be found at http://mcb.asm.org/.
T.O. and E.S. contributed equally to this work.
Present address: Molecular Biology Program, Memorial Sloan-Kettering Cancer Center, New York, NY 10021.
|
REFERENCES |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
2. Bemark, M., A. A. Khamlichi, S. L. Davies, and M. S. Neuberger. 2000. Disruption of mouse polymerase zeta (Rev3) leads to embryonic lethality and impairs blastocyst development in vitro. Curr. Biol. 10:1213-1216.[CrossRef][Medline]
3. Bezzubova, O., A. Silbergleit, Y. Yamaguchi-Iwai, S. Takeda, and J. M. Buerstedde. 1997. Reduced X-ray resistance and homologous recombination frequencies in a RAD54–/– mutant of the chicken DT40 cell line. Cell 89:185-193.[CrossRef][Medline]
4. Broomfield, S., T. Hryciw, and W. Xiao. 2001. DNA postreplication repair and mutagenesis in Saccharomyces cerevisiae. Mutat. Res. 486:167-184.[Medline]
5. Buerstedde, J. M., C. A. Reynaud, E. H. Humphries, W. Olson, D. L. Ewert, and J. C. Weill. 1990. Light chain gene conversion continues at high rate in an ALV-induced cell line. EMBO J. 9:921-927.[Medline]
6. Buerstedde, J. M., and S. Takeda. 1991. Increased ratio of targeted to random integration after transfection of chicken B cell lines. Cell 67:179-188.[CrossRef][Medline]
7. Cahill, D. P., L. T. da Costa, E. B. Carson-Walter, K. W. Kinzler, B. Vogelstein, and C. Lengauer. 1999. Characterization of MAD2B and other mitotic spindle checkpoint genes. Genomics 58:181-187.[CrossRef][Medline]
8. Chen, J., and G. Fang. 2001. MAD2B is an inhibitor of the anaphase-promoting complex. Genes Dev. 15:1765-1770.
9. Esposito, G., I. Godindagger, U. Klein, M. L. Yaspo, A. Cumano, and K. Rajewsky. 2000. Disruption of the Rev3l-encoded catalytic subunit of polymerase zeta in mice results in early embryonic lethality. Curr. Biol. 10:1221-1224.[CrossRef][Medline]
10. Friedberg, E. C., G. C. Walker, and W. Siede. 1995. DNA repair and mutagenesis. ASM Press, Washington, D.C.
11. Fujimori, A., S. Tachiiri, E. Sonoda, L. H. Thompson, P. K. Dhar, M. Hiraoka, S. Takeda, Y. Zhang, M. Reth, and M. Takata. 2001. Rad52 partially substitutes for the Rad51 paralog XRCC3 in maintaining chromosomal integrity in vertebrate cells. EMBO J. 20:5513-5520.[CrossRef][Medline]
12. Gibbs, P. E., W. G. McGregor, V. M. Maher, P. Nisson, and C. W. Lawrence. 1998. A human homolog of the Saccharomyces cerevisiae REV3 gene, which encodes the catalytic subunit of DNA polymerase zeta. Proc. Natl. Acad. Sci. USA 95:6876-6880.
13. Goodman, M. F., and B. Tippin. 2000. The expanding polymerase universe. Nat. Rev. Mol. Cell. Biol. 1:101-109.[CrossRef][Medline]
14. Guo, C., P. L. Fischhaber, M. J. Luk-Paszyc, Y. Masuda, J. Zhou, K. Kamiya, C. Kisker, and E. C. Friedberg. 2003. Mouse Rev1 protein interacts with multiple DNA polymerases involved in translesion DNA synthesis. EMBO J. 22:6621-6630.[CrossRef][Medline]
15. Haber, J. E. 1992. Mating-type gene switching in Saccharomyces cerevisiae. Trends Genet. 8:446-452.[Medline]
16. Hochegger, H., E. Sonoda, and S. Takeda. 2004. Post-replication repair in DT40 cells: translesion polymerases versus recombinases. Bioessays 26:151-158.[CrossRef][Medline]
17. Hubscher, U., H. P. Nasheuer, and J. E. Syvaoja. 2000. Eukaryotic DNA polymerases, a growing family. Trends Biochem. Sci. 25:143-147.[CrossRef][Medline]
18. Keeney, S. 2001. Mechanism and control of meiotic recombination initiation. Curr. Top. Dev. Biol. 52:1-53.[Medline]
19. Latt, S. A. 1981. Sister chromatid exchange formation. Annu. Rev. Genet. 15:11-55.[CrossRef][Medline]
20. Lawrence, C. W. 2002. Cellular roles of DNA polymerase and Rev1 protein. DNA Repair 1:425-435.[CrossRef][Medline]
21. Lawrence, C. W., and R. B. Christensen. 1976. UV mutagenesis in radiation-sensitive strains of yeast. Genetics 82:207-232.
22. Lawrence, C. W., and R. B. Christensen. 1979. Ultraviolet-induced reversion of cyc1 alleles in radiation-sensitive strains of yeast III rev3 mutant strains. Genetics 92:397-408.
23. Lemontt, J. F. 1971. Mutants of yeast defective in mutation induced by ultraviolet light. Genetics 68:21-33.
24. Li, Y. and R. Benezra. 196. Identification of a human mitotic checkpoint gene: hsMAD2. Science 274:246-248.
25. Lin, W., H. Xin, Y. Zhang, X. Wu, F. Yuan, and Z. Wang. 1999. The human REV1 gene codes for a DNA template-dependent dCMP transferase. Nucleic Acids Res. 27:4468-4475.
26. Masuda, Y., M. Ohmae, K. Masuda, and K. Kamiya. 2003. Structure and enzymatic properties of a stable complex of the human REV1 and REV7 proteins. J. Biol. Chem. 278:12356-12360.
27. Morrison, C., E. Sonoda, N. Takao, A. Shinohara, K. Yamamoto, and S. Takeda. 2000. The controlling role of ATM in homologous recombinational repair of DNA damage. EMBO J. 19:463-471.[CrossRef][Medline]
28. Murakumo, Y., T. Roth, H. Ishii, D. Rasio, S. Numata, C. M. Croce, and R. Fishel. 2000. A human REV7 homolog that interacts with the polymerase zeta catalytic subunit hREV3 and the spindle assembly checkpoint protein hMAD2. J. Biol. Chem. 275:4391-4397.
29. Murakumo, Y., Y. Ogura, H. Ishii, S. Numata, M. Ichihara, C. M. Croce, R. Fishel, and M. Takahashi. 2001. Interactions in the error-prone postreplication repair proteins hREV1, hREV3, and hREV7. J. Biol. Chem. 276:35644-35651.
30. Murakumo, Y. 2002. The property of DNA polymerase zeta: REV7 is a putative protein involved in translesion DNA synthesis and cell cycle control. Mutat. Res. 510:37-44.[Medline]
31. Nelson, J. R., C. W. Lawrence, and D. C. Hinkle. 1996. Deoxycytidyl transferase activity of yeast Rev1 protein. Nature 382:729-731.[CrossRef][Medline]
32. Nelson, J. R., C. W. Lawrence, and D. C. Hinkle. 1996. Thymine-thymine dimer bypass by yeast DNA polymerase zeta. Science 272:1646-1649.[Abstract]
33. Nelson, J. R., P. E. Gibbs, A. M. Nowicka, D. C. Hinkle, and C. W. Lawrence. 2000. Evidence for a second function for Saccharomyces cerevisiae Rev1p. Mol. Microbiol. 37:549-554.[CrossRef][Medline]
34. Ohmori, H., E. C. Friedberg, R. P. Fuchs, M. F. Goodman, F. Hanaoka, D. Hinkle, T. A. Kunkel, C. W. Lawrence, Z. Livneh, T. Nohmi, L. Prakash, S. Prakash, T. Todo, G. C. Walker, Z. Wang, and R. Woodgate. 2001. The Y-family of DNA polymerases. Mol. Cell 8:7-8.[CrossRef][Medline]
35. Okada, T., E. Sonoda, Y. M. Yamashita, S. Koyoshi, S. Tateishi, M. Yamaizumi, M. Takata, O. Ogawa, and S. Takeda. 2002. Involvement of vertebrate Pol in Rad18-independent postreplication repair of UV damage. J. Biol. Chem. 277:48690-48695.
36. Pfleger, C. M., A. Salic, E. Lee, and M. W. Kirschner. 2001. Inhibition of Cdh1-APC by the MAD2-related protein MAD2L2: a novel mechanism for regulating Cdh1. Genes Dev. 15:1759-1764.
37. Reynaud, C. A., B. Bertocci, A. Dahan, and J. C. Weill. 1994. Formation of the chicken B-cell repertoire: ontogenesis, regulation of Ig gene rearrangement, and diversification by gene conversion. Adv. Immunol. 57:353-378.[Medline]
38. Sale, J. E., D. M. Calandrini, M. Takata, S. Takeda, and M. S. Neuberger. 2001. Ablation of XRCC2/3 transforms immunoglobulin V gene conversion into somatic hypermutation. Nature 412:921-926.[CrossRef][Medline]
39. Simpson, L. J., and J. E. Sale. 2003. Rev1 is essential for DNA damage tolerance and non-templated immunoglobulin gene mutation in a vertebrate cell line. EMBO J. 22:1654-1664.[CrossRef][Medline]
40. Sonoda, E., M. S. Sasaki, J. M. Buerstedde, O. Bezzubova, A. Shinohara, H. Ogawa, M. Takata, Y. Yamaguchi-Iwai, and S. Takeda. 1998. Rad51-deficient vertebrate cells accumulate chromosomal breaks prior to cell death. EMBO J. 17:598-608.[CrossRef][Medline]
41. Sonoda, E., M. Takata, Y. M. Yamashita, C. Morrison, and S. Takeda. 2001. Homologous DNA recombination in vertebrate cells. Proc. Natl. Acad. Sci. USA 98:8388-8394.
42. Sonoda, E., T. Okada, G.-Y. Zhao, S. Tateishi, M. Yamaizumi, T. Yagi, N. S. Verkaik, D. C. van Gent, M. Takata, and S. Takeda. 2003. Multiple roles of Rev3, the catalytic subunit of pol in maintaining genome stability in vertebrate. EMBO J. 22:3188-3197.[CrossRef][Medline]
43. Surralles, J., S. P. Jackson, M. Jasin, M. B. Kastan, S. C. West, and H. Joenje. 2004. Molecular cross-talk among chromosome fragility syndromes. Genes Dev. 18:1359-1370.
44. Takata, M., M. S. Sasaki, E. Sonoda, C. Morrison, M. Hashimoto, H. Utsumi, Y. Yamaguchi-Iwai, A. Shinohara, and S. Takeda. 1998. Homologous recombination and non-homologous end-joining pathways of DNA double-strand break repair have overlapping roles in the maintenance of chromosomal integrity in vertebrate cells. EMBO J. 17:5497-5508.[CrossRef][Medline]
45. Takata, M., M. S. Sasaki, S. Tachiiri, T. Fukushima, E. Sonoda, D. Schild, L. Thompson, and S. Takeda. 2001. Chromosome instability and defective recombinational repair in knockout mutants of the five Rad51 paralogs. Mol. Cell. Biol. 21:2858-2866.
46. Wittschieben, J., M. K. Shivji, E. Lalani, M. A. Jacobs, F. Marini, P. J. Gearhart, I. Rosewell, G. Stamp, and R. D. Wood. 2000. Disruption of the developmentally regulated Rev3l gene causes embryonic lethality. Curr. Biol. 10:1217-1220.[CrossRef][Medline]
47. Yamashita, Y. M., T. Okada, T. Matsusaka, E. Sonoda, G-Y. Zhao, K. Araki, S. Tateishi, M. Yamaizumi, and S. Takeda. 2002. RAD18 and RAD54 cooperatively contribute to maintenance of genomic stability in vertebrate cells. EMBO J. 21:5558-5566.[CrossRef][Medline]
This article has been cited by other articles:
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| J. Bacteriol. | J. Virol. | Eukaryot. Cell |
|---|
S phase), G1 cells, G2/M cells, and sub-G1 cells, respectively. Numbers show the percentage of cells falling in each gate. (C) Normal spindle assembly checkpoint in rev7 cells. Cells in mitosis were identified by costaining with PI and antibody to phosphorylated histone H3, and the percentages of the mitotic cells are shown as the mitotic index. The period of chase indicates time after addition of 0.1 µg/ml of Colcemid.
