CDC7/DBF4 functions in the translesion synthesis branch of the RAD6 epistasis group in Saccharomyces cerevisiae

CDC7 and DBF4 encode the essential Cdc7-Dbf4 protein kinase required for DNA replication in eukaryotes from yeast to human. Cdc7-Dbf4 is also required for DNA damage-induced mutagenesis, one of several postreplicational DNA damage tolerance mechanisms mediated by the RAD6 epistasis group. Several genes have been determined to function in separate branches within this group, including RAD5, REV3/REV7 (Pol zeta), RAD30 (Pol eta), and POL30 (PCNA). An extensive genetic analysis of the interactions between CDC7 and REV3, RAD30, RAD5, or POL30 in response to DNA damage was done to determine its role in the RAD6 pathway. CDC7, RAD5, POL30, and RAD30 were found to constitute four separate branches of the RAD6 epistasis group in response to UV and MMS exposure. CDC7 is also shown to function separately from REV3 in response to MMS. However, they belong in the same pathway in response to UV. We propose that the Cdc7-Dbf4 kinase associates with components of the translesion synthesis pathway and that this interaction is dependent upon the type of DNA damage. Finally, activation of the DNA damage checkpoint and the resulting cell cycle delay is intact in cdc7Delta mcm5-bob1 cells, suggesting a direct role for CDC7 in DNA repair/damage tolerance.     

REV1 protein interacts with PCNA: significance of the REV1 BRCT domain in vitro and in vivo

REV1 protein, a eukaryotic member of the Y family of DNA polymerases, is involved in the tolerance of DNA damage by translesion DNA synthesis. It is unclear how REV1 is recruited to replication foci in cells. Here, we report that mouse REV1 can bind directly to PCNA and that monoubiquitylation of PCNA enhances this interaction. The interaction between REV1 protein and PCNA requires a functional BRCT domain located near the N terminus of the former protein. Deletion or mutational inactivation of the BRCT domain abolishes the targeting of REV1 to replication foci in unirradiated cells, but not in UV-irradiated cells. In vivo studies in both chicken DT40 cells and yeast directly support the requirement of the BRCT domain of REV1 for cell survival and DNA damage-induced mutagenesis.     

NEK8 regulates DNA damage-induced RAD51 foci formation and replication fork protection

Proteins essential for homologous recombination play a pivotal role in the repair of DNA double strand breaks, DNA inter-strand crosslinks and replication fork stability. Defects in homologous recombination also play a critical role in the development of cancer and the sensitivity of these cancers to chemotherapy. RAD51, an essential factor for homologous recombination and replication fork protection, accumulates and forms immunocytochemically detectable nuclear foci at sites of DNA damage. To identify kinases that may regulate RAD51 localization to sites of DNA damage, we performed a human kinome siRNA library screen, using DNA damage-induced RAD51 foci formation as readout. We found that NEK8, a NIMA family kinase member, is required for efficient DNA damage-induced RAD51 foci formation. Interestingly, knockout of Nek8 in murine embryonic fibroblasts led to cellular sensitivity to the replication inhibitor, hydroxyurea, and inhibition of the ATR kinase. Furthermore, NEK8 was required for proper replication fork protection following replication stall with hydroxyurea. Loading of RAD51 to chromatin was decreased in NEK8-depleted cells and Nek8-knockout cells. Single-molecule DNA fiber analyses revealed that nascent DNA tracts were degraded in the absence of NEK8 following treatment with hydroxyurea. Consistent with this, Nek8-knockout cells showed increased chromosome breaks following treatment with hydroxyurea. Thus, NEK8 plays a critical role in replication fork stability through its regulation of the DNA repair and replication fork protection protein RAD51.     

FANCD2 and REV1 cooperate in the protection of nascent DNA strands in response to replication stress

REV1 is a eukaryotic member of the Y-family of DNA polymerases involved in translesion DNA synthesis and genome mutagenesis. Recently, REV1 is also found to function in homologous recombination. However, it remains unclear how REV1 is recruited to the sites where homologous recombination is processed. Here, we report that loss of mammalian REV1 results in a specific defect in replication-associated gene conversion. We found that REV1 is targeted to laser-induced DNA damage stripes in a manner dependent on its ubiquitin-binding motifs, on RAD18, and on monoubiquitinated FANCD2 (FANCD2-mUb) that associates with REV1. Expression of a FANCD2-Ub chimeric protein in RAD18-depleted cells enhances REV1 assembly at laser-damaged sites, suggesting that FANCD2-mUb functions downstream of RAD18 to recruit REV1 to DNA breaks. Consistent with this suggestion we found that REV1 and FANCD2 are epistatic with respect to sensitivity to the double-strand break-inducer camptothecin. REV1 enrichment at DNA damage stripes also partially depends on BRCA1 and BRCA2, components of the FANCD2/BRCA supercomplex. Intriguingly, analogous to FANCD2-mUb and BRCA1/BRCA2, REV1 plays an unexpected role in protecting nascent replication tracts from degradation by stabilizing RAD51 filaments. Collectively these data suggest that REV1 plays multiple roles at stalled replication forks in response to replication stress.     

The Saccharomyces Cerevisiae Rad30 Gene, a Homologue of Escherichia Coli Dinb and Umuc, Is DNA Damage Inducible and Functions in a Novel Error-Free Postreplication Repair Mechanism

Damage-inducible mutagenesis in prokaryotes is largely dependent upon the activity of the UmuD'C-like proteins. Since many DNA repair processes are structurally and/or functionally conserved between prokaryotes and eukaryotes, we investigated the role of RAD30, a previously uncharacterized Saccharomyces cerevisiae DNA repair gene related to the Escherichia coli dinB, umuC and S. cerevisiae REV1 genes, in UV resistance and UV-induced mutagenesis. Similar to its prokaryotic homologues, RAD30 was found to be damage inducible. Like many S. cerevisiae genes involved in error-prone DNA repair, epistasis analysis clearly places RAD30 in the RAD6 group and rad30 mutants display moderate UV sensitivity reminiscent of rev mutants. However, unlike rev mutants, no defect in UV-induced reversion was seen in rad30 strains. While rad6 and rad18 are both epistatic to rad30, no epistasis was observed with rev1, rev3, rev7 or rad5, all of which are members of the RAD6 epistasis group. These findings suggest that RAD30 participates in a novel error-free repair pathway dependent on RAD6 and RAD18, but independent of REV1, REV3, REV7 and RAD5.     

Ubiquitin-binding motifs in REV1 protein are required for its role in the tolerance of DNA damage

REV1 protein is a eukaryotic member of the Y family of DNA polymerases involved in the tolerance of DNA damage by replicative bypass. The precise role(s) of REV1 in this process is not known. Here we show, by using the yeast two-hybrid assay and the glutathione S-transferase pull-down assay, that mouse REV1 can physically interact with ubiquitin. The association of REV1 with ubiquitin requires the ubiquitin-binding motifs (UBMs) located at the C terminus of REV1. The UBMs also mediate the enhanced association between monoubiquitylated PCNA and REV1. In cells exposed to UV radiation, the association of REV1 with replication foci is dependent on functional UBMs. The UBMs of REV1 are shown to contribute to DNA damage tolerance and damage-induced mutagenesis in vivo.     

DNA damage-induced mutation: tolerance via translesion synthesis

Translesion synthesis (TLS) appears to be required for most damage-induced mutagenesis in the yeast Saccharomyces cerevisiae, whether the damage arises from endogenous or exogenous sources. Thus, the production of such mutations seems to occur primarily as a consequence of the tolerance of DNA lesions rather than an error-prone repair mechanism. Tolerance via TLS in yeast involves proteins encoded by members of the RAD6 epistasis group for the repair of ultraviolet (UV) photoproducts, in particular two non-essential DNA polymerases that catalyse error-free or error-prone TLS. Homologues of these RAD6 group proteins have recently been discovered in rodent and/or human cells. Furthermore, the operation of error-free TLS in humans has been linked to a reduced risk of UV-induced skin cancer, whereas mutations generated by error-prone TLS may increase the risk of cancer. In this article, we review and link the evidence for translesion synthesis in yeast, and the involvement of nonreplicative DNA polymerases, to recent findings in mammalian cells.     

The property of DNA polymerase zeta: REV7 is a putative protein involved in translesion DNA synthesis and cell cycle control

Translesion DNA synthesis (TLS) is an important damage tolerance system which rescues cells from severe injuries caused by DNA damage. Specialized low fidelity DNA polymerases in this system synthesize DNA past lesions on the template DNA strand, that replicative DNA polymerases are usually unable to pass through. However, in compensation for cell survival, most polymerases in this system are potentially mutagenic and sometimes introduce mutations in the next generation. In yeast Saccharomyces cerevisiae (S. cerevisiae), DNA polymerase ζ, which consists of Rev3 and Rev7 proteins, and Rev1 are known to be involved in most damage-induced and spontaneous mutations. The human homologs of S. cerevisiae REV1, REV3, and REV7 were identified, and it is revealed that the human REV proteins have similar functions to their yeast counterparts, however, a large part of the mechanisms of mutagenesis employing REV proteins are still unclear. Recently, the new findings about REV proteins were reported, which showed that REV7 interacts not only with REV3 but also with REV1 in human and that REV7 is involved in cell cycle control in Xenopus. These findings give us a new point of view for further investigation about REV proteins. Recent studies of REV proteins are summarized and several points are discussed.     

Alteration of ultraviolet-induced mutagenesis in yeast through molecular modulation of the REV3 and REV7 gene expression

DNA damage can lead to mutations during replication. The damage-induced mutagenesis pathway is an important mechanism that fixes DNA lesions into mutations. DNA polymerase zeta (Pol zeta), formed by Rev3 and Rev7 protein complex, and Rev1 are components of the damage-induced mutagenesis pathway. Since mutagenesis is an important factor during the initiation and progression of human cancer, we postulate that this mutagenesis pathway may provide an inhibiting target for cancer prevention and therapy. In this study, we tested if UV-induced mutagenesis can be altered by molecular modulation of Rev3 enzyme levels using the yeast Saccharomyces cerevisiae as a eukaryotic model system. Reducing the REV3 expression in yeast cells through molecular techniques was employed to mimic Pol zeta inhibition. Lower levels of Pol zeta significantly decreased UV-induced mutation frequency, thus achieving inhibition of mutagenesis. In contrast, elevating the Pol zeta level by enhanced expression of both REV3 and REV7 genes led to a approximately 3-fold increase in UV-induced mutagenesis as determined by the arg4-17 mutation reversion assays. In vivo, UV lesion bypass by Pol zeta requires the Rev1 protein. Even overexpression of Pol zeta could not alleviate the defective UV mutagenesis in the rev1 mutant cells. These observations provide evidence that the mutagenesis pathway could be used as a target for inhibiting damage-induced mutations.     

Dynamic localization of human RAD18 during the cell cycle and a functional connection with DNA double-strand break repair

The ubiquitin ligase RAD18 is involved in different DNA repair processes. Here, we show that in G1 phase, human RAD18 accumulates in a few relatively large spontaneous foci that contain proteins involved in double-strand break (DSB) repair. These foci persist until cells enter S phase, when numerous small foci appear. At these sites, only 20% of RAD18 colocalizes with PCNA, a known RAD18 substrate. In late G2 phase, RAD18 relocates to nucleoli. After UVC irradiation, PCNA accumulates at the damaged site, followed by RAD18, independent of the cell cycle phase. After induction of DSBs, using low-power multi-photon laser, RAD18 accumulated at the DSB sites, but no PCNA accumulation was observed. Our data show that RAD18 accumulates on DSBs independent of the cell cycle phase. DSBs marked by RAD18 and RAD51 are also positive for RPA in G1 phase, and these DSBs persist until S phase. In addition, we show that DSBs generated in G2 phase are not all repaired, and are observed again in the next G1 phase. We conclude that repair of induced and spontaneous DSBs that accumulate RAD18 and RAD51 in G1 phase cells is delayed until S phase.     

Domains required for dimerization of yeast Rad6 ubiquitin-conjugating enzyme and Rad18 DNA binding protein.

The RAD6 gene of Saccharomyces cerevisiae encodes a ubiquitin-conjugating enzyme required for postreplicational repair of UV-damaged DNA and for damage-induced mutagenesis. In addition, Rad6 functions in the N end rule pathway of protein degradation. Rad6 mediates its DNA repair role via its association with Rad18, whose DNA binding activity may target the Rad6-Rad18 complex to damaged sites in DNA. In its role in N end-dependent protein degradation, Rad6 interacts with the UBR1-encoded ubiquitin protein ligase (E3) enzyme. Previous studies have indicated the involvement of N-terminal and C-terminal regions of Rad6 in interactions with Ubr1. Here, we identify the regions of Rad6 and Rad18 that are involved in the dimerization of these two proteins. We show that a region of 40 amino acids towards the C terminus of Rad18 (residues 371 to 410) is sufficient for interaction with Rad6. This region of Rad18 contains a number of nonpolar residues that have been conserved in helix-loop-helix motifs of other proteins. Our studies indicate the requirement for residues 141 to 149 at the C terminus, and suggest the involvement of residues 10 to 22 at the N terminus of Rad6, in the interaction with Rad18. Each of these regions of Rad6 is indicated to form an amphipathic helix.     

Dissection of the functions of the Saccharomyces cerevisiae RAD6 postreplicative repair group in mutagenesis and UV sensitivity.

The RAD6 postreplicative repair group participates in various processes of DNA metabolism. To elucidate the contribution of RAD6 to starvation-associated mutagenesis, which occurs in nongrowing cells cultivated under selective conditions, we analyzed the phenotype of strains expressing various alleles of the RAD6 gene and single and multiple mutants of the RAD6, RAD5, RAD18, REV3, and MMS2 genes from the RAD6 repair group. Our results show that the RAD6 repair pathway is also active in starving cells and its contribution to starvation-associated mutagenesis is similar to that of spontaneous mutagenesis. Epistatic analysis based on both spontaneous and starvation-associated mutagenesis and UV sensitivity showed that the RAD6 repair group consists of distinct repair pathways of different relative importance requiring, besides the presence of Rad6, also either Rad18 or Rad5 or both. We postulate the existence of four pathways: (1) nonmutagenic Rad5/Rad6/Rad18, (2) mutagenic Rad5/Rad6 /Rev3, (3) mutagenic Rad6/Rad18/Rev3, and (4) Rad6/Rad18/Rad30. Furthermore, we show that the high mutation rate observed in rad6 mutants is caused by a mutator different from Rev3. From our data and data previously published, we suggest a role for Rad6 in DNA repair and mutagenesis and propose a model for the RAD6 postreplicative repair group.     

DNA damage-induced RPA focalization is independent of gamma-H2AX and RPA hyper-phosphorylation

Replication protein A (RPA) is the major eukaryotic single stranded DNA binding protein that plays a central role in DNA replication, repair and recombination. Like many DNA repair proteins RPA is heavily phosphorylated (specifically on its 32 kDa subunit) in response to DNA damage. Phosphorylation of many repair proteins has been shown to be important for their recruitment to DNA damage-induced intra-nuclear foci. Further, phosphorylation of H2AX (gamma-H2AX) has been shown to be important for either the recruitment or stable retention of DNA repair proteins to these intra-nuclear foci. We address here the relationship between DNA damage-induced hyper-phosphorylation of RPA and its intra-nuclear focalization, and whether gamma-H2AX is required for RPA's presence at these foci. Using GFP-conjugated RPA, we demonstrate the formation of extraction-resistant RPA foci induced by DNA damage or stalled replication forks. The strong DNA damage-induced RPA foci appear after phosphorylated histone H2AX and Chk1, but earlier than the appearance of hyper-phosphorylated RPA. We demonstrate that while the functions of phosphoinositol-3-kinase-related protein kinases are essential for DNA damage-induced H2AX phosphorylation and RPA hyper-phosphorylation, they are dispensable for the induction of extraction-resistant RPA and RPA foci. Furthermore, in mouse cells genetically devoid of H2AX, DNA damage-induced extraction-resistant RPA appears with the same kinetics as in normal mouse cells. These results demonstrate that neither RPA hyper-phosphorylation nor H2AX are required for the formation in RPA intra-nuclear foci in response to DNA damage/replicational stress and are consistent with a role for RPA as a DNA damage sensor involved in the initial recognition of damaged DNA or blocked replication forks.     

RAD51AP1-deficiency in vertebrate cells impairs DNA replication

RAD51-associated protein 1 (RAD51AP1) is critical for homologous recombination (HR) by interacting with and stimulating the activities of the RAD51 and DMC1 recombinases. In human somatic cells, knockdown of RAD51AP1 results in increased sensitivity to DNA damaging agents and to impaired HR, but the formation of DNA damage-induced RAD51 foci is unaffected. Here, we generated a genetic model system, based on chicken DT40 cells, to assess the phenotype of fully inactivated RAD51AP1 in vertebrate cells. Targeted inactivation of both RAD51AP1 alleles has no effect on either viability or doubling-time in undamaged cells, but leads to increased levels of cytotoxicity after exposure to cisplatin or to ionizing radiation. Interestingly, ectopic expression of GgRAD51AP1, but not of HsRAD51AP1 is able to fully complement in cell survival assays. Notably, in RAD51AP1-deficient DT40 cells the resolution of DNA damage-induced RAD51 foci is greatly slowed down, while their formation is not impaired. We also identify, for the first time, an important role for RAD51AP1 in counteracting both spontaneous and DNA damage-induced replication stress. In human and in chicken cells, RAD51AP1 is required to maintain wild type speed of replication fork progression, and both RAD51AP1-depleted human cells and RAD51AP1-deficient DT40 cells respond to replication stress by a slow-down of replication fork elongation rates. However, increased firing of replication origins occurs in RAD51AP1-/- DT40 cells, likely to ensure the timely duplication of the entire genome. Taken together, our results may explain why RAD51AP1 commonly is overexpressed in tumor cells and tissues, and we speculate that the disruption of RAD51AP1 function could be a promising approach in targeted tumor therapy.     

The Rad51 paralog Rad51B promotes homologous recombinational repair

The highly conserved Saccharomyces cerevisiae Rad51 protein plays a central role in both mitotic and meiotic homologous DNA recombination. Seven members of the Rad51 family have been identified in vertebrate cells, including Rad51, Dmc1, and five Rad51-related proteins referred to as Rad51 paralogs, which share 20 to 30% sequence identity with Rad51. In chicken B lymphocyte DT40 cells, we generated a mutant with RAD51B/RAD51L1, a member of the Rad51 family, knocked out. RAD51B(-/-) cells are viable, although spontaneous chromosomal aberrations kill about 20% of the cells in each cell cycle. Rad51B deficiency impairs homologous recombinational repair (HRR), as measured by targeted integration, sister chromatid exchange, and intragenic recombination at the immunoglobulin locus. RAD51B(-/-) cells are quite sensitive to the cross-linking agents cisplatin and mitomycin C and mildly sensitive to gamma-rays. The formation of damage-induced Rad51 nuclear foci is much reduced in RAD51B(-/-) cells, suggesting that Rad51B promotes the assembly of Rad51 nucleoprotein filaments during HRR. These findings show that Rad51B is important for repairing various types of DNA lesions and maintaining chromosome integrity.     

RAD51 foci formation in response to DNA damage is modulated by TIP49

Chromatin modification plays an important role in modulating the access of homologous recombination proteins to the sites of DNA damage. TIP49 is highly conserved component of chromatin modification/remodeling complexes, but its involvement in homologous recombination repair in mammalian cells has not been examined in details. In the present communication we studied the role of TIP49 in recruitment of the key homologous recombination protein RAD51 to sites of DNA damage. RAD51 redistribution to chromatin and nuclear foci formation induced by double-strand breaks and interstrand crosslinks were followed under conditions of TIP49 depletion by RNA interference. TIP49 silencing reduced RAD51 recruitment to chromatin and nuclear foci formation to about 50% of that of the control. Silencing of TIP48, which is closely related to TIP49, induced a similar reduction in RAD51 foci formation. RAD51 foci reduction in TIP49-silenced cells was not a result of defective DNA damage checkpoint signaling as judged by the normal histone H2AX phosphorylation and cell cycle distribution. Treatment with the histone deacetylase inhibitor sodium butyrate restored RAD51 foci formation in the TIP49-depleted cells. The results suggest that as a constituent of chromatin modification complexes TIP49 may facilitate the access of the repair machinery to the sites of DNA damage.     

Dynamics of some postreplication DNA repair proteins in carcinogen-damaged mammalian cells

Many types of DNA lesions in template strands block DNA replication and lead to a stalling of replication forks. This block can be overcome (bypassed) by special DNA polymerases (for example, DNA polymerase eta, Pol eta) that perform translesion synthesis on damaged template DNA. The phenomenon of completing DNA replication, while DNA lesions remain in the template strands, has been named post-replication repair (PRR). In yeast Saccharomyces cerevisiae, PRR includes mutagenic and error-free pathways under the regulation of the RAD6/RAD18 complex, which induces ubiquitylation of PCNA. In mammalian cells, Pol eta accumulates in replication foci but the mechanism of this accumulation is not known. Pol eta possesses a conserved PCNA binding motif at the C terminal and phosphorylation of this motif might be essential for its interaction with PCNA. We have shown previously that staurosporine, an inhibitor of protein kinases, inhibits PRR in human cells. In this study we examined whether the accumulation of Pol eta in replication foci after DNA damage is dependent on phosphorylation of the PCNA binding motif. We also studied DNA damage-induced phosphorylation of GFP-tagged human Rad18 (hRad18) and its accumulation in replication foci. Our data indicate that (1) Pol eta is not phosphorylated in response to UV irradiation or MMS treatment, but its diffusional mobility is slightly decreased, and (2) hRad18 accumulates in MMS-treated cells, and considerable amount of the protein co-localizes with detergent insoluble PCNA in replication foci; these responses are sensitive to staurosporine. Our data suggest that hRad18 phosphorylation is the staurosporine-sensitive PRR step.     

Interaction with DNA polymerase η is required for nuclear accumulation of REV1 and suppression of spontaneous mutations in human cells

Defects in the gene encoding human Polη result in xeroderma pigmentosum variant (XP-V), an inherited cancer-prone syndrome. Polη catalyzes efficient and accurate translesion DNA synthesis (TLS) past UV-induced lesions. In addition to Polη, human cells have multiple TLS polymerases such as Polι, Polκ, Polζ and REV1. REV1 physically interacts with other TLS polymerases, but the physiological relevance of the interaction remains unclear. Here we developed an antibody that detects the endogenous REV1 protein and found that human cells contain about 60,000 of REV1 molecules per cell as well as Polη. In un-irradiated cells, formation of nuclear foci by ectopically expressed REV1 was enhanced by the co-expression of Polη. Importantly, the endogenous REV1 protein accumulated at the UV-irradiated areas of nuclei in Polη-expressing cells but not in Polη-deficient XP-V cells. UV-irradiation induced nuclear foci of REV1 and Polη proteins in both S-phase and G1 cells, suggesting that these proteins may function both during and outside S phase. We reconstituted XP-V cells with wild-type Polη or with Polη mutants harboring substitutions in phenylalanine residues critical for interaction with REV1. The REV1-interaction-deficient Polη mutant failed to promote REV1 accumulation at sites of UV-irradiation, yet (similar to wild-type Polη) corrected the UV sensitivity of XP-V cells and suppressed UV-induced mutations. Interestingly however, spontaneous mutations of XP-V cells were only partially suppressed by the REV1-interaction deficient mutant of Polη. Thus, Polη–REV1 interactions prevent spontaneous mutations, probably by promoting accurate TLS past endogenous DNA lesions, while the interaction is dispensable for accurate Polη-mediated TLS of UV-induced lesions.     

The Saccharomyces cerevisiae RAD6 group is composed of an error-prone and two error-free postreplication repair pathways

The RAD6 postreplication repair and mutagenesis pathway is the only major radiation repair pathway yet to be extensively characterized. It has been previously speculated that the RAD6 pathway consists of two parallel subpathways, one error free and another error prone (mutagenic). Here we show that the RAD6 group genes can be exclusively divided into three rather than two independent subpathways represented by the RAD5, POL30, and REV3 genes; the REV3 pathway is largely mutagenic, whereas the RAD5 and the POL30 pathways are deemed error free. Mutants carrying characteristic mutations in each of the three subpathways are phenotypically indistinguishable from a single mutant such as rad18, which is defective in the entire RAD6 postreplication repair/tolerance pathway. Furthermore, the rad18 mutation is epistatic to all single or combined mutations in any of the above three subpathways. Our data also suggest that MMS2 and UBC13 play a key role in coordinating the response of the error-free subpathways; Mms2 and Ubc13 form a complex required for a novel polyubiquitin chain assembly, which probably serves as a signal transducer to promote both RAD5 and POL30 error-free postreplication repair pathways. The model established by this study will facilitate further research into the molecular mechanisms of postreplication repair and translesion DNA synthesis. In view of the high degree of sequence conservation of the RAD6 pathway genes among all eukaryotes, the model presented in this study may also apply to mammalian cells and predicts links to human diseases.     

RAD51调控REV1参与DNA双链断裂修复

REV1是跨损伤聚合酶Y家族的重要成员之一,它不仅作为支架蛋白介导Y家族聚合酶招募至损伤位点完成跨损伤DNA合成(translesion DNA synthesis, TLS),还可利用自身的dCMP转移酶活性在一些损伤位点对侧整合dCMP参与TLS。此外,REV1也被报导参与调控同源重组修复。为进一步探讨REV1互作蛋白RAD51和RAD51C在其参与的同源重组修复通路中的调控作用,本研究采用脉冲氮激光微辐射实验,发现RAD51可调控REV1到双链断裂位点的募集。同时,免疫荧光实验结果证明REV1也反过来影响RAD51应答CPT损伤。然而敲低RAD51C并不影响REV1到DNA双链断裂位点的招募。结果表明,REV1和RAD51在HR通路中存在彼此相互调控的关系。