APOBEC3B and AID have similar nuclear import mechanisms

Members of the APOBEC (apolipoprotein B mRNA editing enzyme, catalytic polypeptide-like) protein family catalyze DNA cytosine deamination and underpin a variety of immune defenses. For instance, several family members, including APOBEC3B (A3B), elicit strong retrotransposon and retrovirus restriction activities. However, unlike the other proteins, A3B is the only family member with steady-state nuclear localization. Here, we show that A3B nuclear import is an active process requiring at least one amino acid (Val54) within an N-terminal motif analogous to the nuclear localization determinant of the antibody gene diversification enzyme AID (activation-induced cytosine deaminase). Mechanistic conservation with AID is further suggested by A3B's capacity to interact with the same subset of importin proteins. Despite these mechanistic similarities, enforced A3B expression cannot substitute for AID-dependent antibody gene diversification by class switch recombination. Regulatory differences between A3B and AID are also visible during cell cycle progression. Our studies suggest that the present-day A3B enzyme retained the nuclear import mechanism of an ancestral AID protein during the expansion of the APOBEC3 locus in primates. Our studies also highlight the likelihood that, after nuclear import, specialized mechanisms exist to guide these enzymes to their respective physiological substrates and prevent gratuitous chromosomal DNA damage.     

APOBEC3 proteins and genomic stability: The high cost of a good defense

The human APOBEC3 family of cytidine deaminases constitutes a cellular intrinsic defense mechanism that is effective against a range of viruses and retro-elements. While it is well-established that these enzymes are powerful mutators of viral DNA, the possibility that their activity could threaten the integrity of the host genome has only recently begun to be investigated. Here, we discuss the implications of new evidence suggesting that APOBEC3 proteins can mediate the deamination of cellular DNA. The maintenance of genomic integrity in the face of this potential off-target activity must require high-fidelity DNA repair and strict regulation of APOBEC3 gene expression and enzyme activity. Conversely, the ability of specific members of the APOBEC3 family to activate DNA damage signaling pathways might also reflect another way that these proteins contribute to the host immune response.Key words: APOBEC3, cytidine deaminase, AID, uracil-DNA glycosylase, DNA damage, hypermutation, genomic instability, cancer     

Cell Cycle Regulates Nuclear Stability of AID and Determines the Cellular Response to AID

AID (Activation Induced Deaminase) deaminates cytosines in DNA to initiate immunoglobulin gene diversification and to reprogram CpG methylation in early development. AID is potentially highly mutagenic, and it causes genomic instability evident as translocations in B cell malignancies. Here we show that AID is cell cycle regulated. By high content screening microscopy, we demonstrate that AID undergoes nuclear degradation more slowly in G1 phase than in S or G2-M phase, and that mutations that affect regulatory phosphorylation or catalytic activity can alter AID stability and abundance. We directly test the role of cell cycle regulation by fusing AID to tags that destabilize nuclear protein outside of G1 or S-G2/M phases. We show that enforced nuclear localization of AID in G1 phase accelerates somatic hypermutation and class switch recombination, and is well-tolerated; while nuclear AID compromises viability in S-G2/M phase cells. We identify AID derivatives that accelerate somatic hypermutation with minimal impact on viability, which will be useful tools for engineering genes and proteins by iterative mutagenesis and selection. Our results further suggest that use of cell cycle tags to regulate nuclear stability may be generally applicable to studying DNA repair and to engineering the genome.     

Microtubule stress modifies intra-nuclear location of Msh2 in mouse embryonic fibroblasts

The maintenance of genomic stability in mitotic and meiotic cycles through mismatch repair (MMR) demands the coordination of MMR functions with multiple processes including cell cycle traverse, linked changes in microtubule dynamics, protein translocation at chromatin sites and checkpoint activation. We have studied changes in the intracellular location of the MMR protein Msh2 in response to mitosis, microtubule disruption by colcemid and DNA damage induction by cis-platin in mouse embryonic fibroblasts (MEFs). Image analysis indicated that MEFs have a normally high nuclear retention of Msh2 during interphase with a precipitous dispersal of protein from chromatin sites into the cytoplasm at mitosis. Dispersal was also observed in cisplatin- and colcemid-treated interphase MEFs without any change in the overall Msh2 levels throughout the cell cycle. There was no evidence of co-localization of the punctate cytoplasmic Msh2 foci with any microtubule structures and knockout of Msh2 altered neither the extent of microtubule disruption nor the functional activation of the spindle assembly checkpoint by colcemid. Critically, extra-nuclear relocation of protein did not alter the ability to mount an Msh2-dependent G2 checkpoint delay in response to cisplatin-induced DNA damage. Depletion of the nuclear pool of Msh2 protein in cells undergoing dispersal was found to involve a rapid relocation of protein from AT-rich chromatin sites as defined by coassociation studies exploiting a newly-characterized base-pair preference of the fluorescent DNA binding probe DRAQ5. The study reveals the unexpected mobility of MMR protein pools during the MEF cell cycle and in response to different stress-inducing agents. The results link for the first time microtubule-integrity with intra-nuclear Msh2 protein dynamics. The high nuclear retention of Msh2 in interphase MEFs is in contrast to human tumor cells while the observations on protein dispersal suggest that only low levels of nuclear-located Msh2 are needed for G2 checkpoint activation by DNA damage.     

Microtubule Stress Modifies Intra-Nuclear Location of Msh2 in Mouse Embryonic Fibroblasts

The maintenance of genomic stability in mitotic and meiotic cycles through mismatch repair (MMR) demands the co-ordination of MMR functions with multiple processes including cell cycle traverse, linked changes in microtubule dynamics, protein translocation at chromatin sites and checkpoint activation. We have studied changes in the intracellular location of the MMR protein Msh2 in response to mitosis, microtubule disruption by colcemid and DNA damage induction by cis-platin in mouse embryonic fibroblasts (MEFs). Image analysis indicated that MEFs have a normally high nuclear retention of Msh2 during interphase with a precipitous dispersal of protein from chromatin sites into the cytoplasm at mitosis. Dispersal was also observed in cisplatin- and colcemid-treated interphase MEFs without any change in the overall Msh2 levels throughout the cell cycle. There was no evidence of co-localisation of the punctate cytoplasmic Msh2 foci with any microtubule structures and knockout of Msh2 altered neither the extent of microtubule disruption nor the functional activation of the spindle assembly checkpoint by colcemid. Critically, extra-nuclear relocation of protein did not alter the ability to mount an Msh2-dependent G2 checkpoint delay in response to cisplatin-induced DNA damage. Depletion of the nuclear pool of Msh2 protein in cells undergoing dispersal was found to involve a rapid relocation of protein from AT-rich chromatin sites as defined by co-association studies exploiting a newly-characterised base-pair preference of the fluorescent DNA binding probe DRAQ5. The study reveals the unexpected mobility of MMR protein pools during the MEF cell cycle and in response to different stress-inducing agents. The results link for the first time microtubule-integrity with intra-nuclear Msh2 protein dynamics. The high nuclear retention of Msh2 in interphase MEFs is in contrast to human tumour cells while the observations on protein dispersal suggest that only low levels of nuclear-located Msh2 are needed for G2 checkpoint activation by DNA damage.     

Creative deaminases, self-inflicted damage, and genome evolution

Organisms minimize genetic damage through complex pathways of DNA repair. Yet a gene family-the AID/APOBECs-has evolved in vertebrates with the sole purpose of producing targeted damage in DNA/RNA molecules through cytosine deamination. They likely originated from deaminases involved in A>I editing in tRNAs. AID, the archetypal AID/APOBEC, is the trigger of the somatic diversification processes of the antibody genes. Its homologs may have been associated with the immune system even before the evolution of the antibody genes. The APOBEC3s, arising from duplication of AID, are involved in the restriction of exogenous/endogenous threats such as retroviruses and mobile elements. Another family member, APOBEC1, has (re)acquired the ability to target RNA while maintaining its ability to act on DNA. The AID/APOBECs have shaped the evolution of vertebrate genomes, but their ability to mutate nucleic acids is a double-edged sword: AID is a key player in lymphoproliferative diseases by triggering mutations and chromosomal translocations in B cells, and there is increasing evidence suggesting that other AID/APOBECs could be involved in cancer development as well.     

Fanconi anemia proteins localize to chromatin and the nuclear matrix in a DNA damage- and cell cycle-regulated manner

Fanconi anemia (FA) is a genetic disease characterized by congenital defects, bone marrow failure, and cancer susceptibility. Cells from patients with FA exhibit genomic instability and hypersensitivity to DNA cross linking agents such as mitomycin C. Despite the identification of seven complementation groups and the cloning of six genes, the function of the encoded gene products remains elusive. The FancA (Fanconi anemia complementation group A), FancC, and FancG proteins have been detected within a nuclear complex, but no change in level, binding, or localization has been reported as a result of drug treatment or cell cycle. We show that in immunofluorescence studies, FancA appears as a non-nucleolar nuclear protein that is excluded from condensed, mitotic chromosomes. Biochemical fractionation reveals that the FA proteins are found in nuclear matrix and chromatin and that treatment with mitomycin C results in increase of the FA proteins in nuclear matrix and chromatin fractions. This induction occurs in wild-type cells and mutant FA-D (Fanconi complementation group D) cells but not in mutant FA-A cells. Immunoprecipitation of FancA protein in chromatin demonstrates the coprecipitation of FancA, FancC, and FancG, showing that the FA proteins move together as a complex. Also, fractionation of mitotic cells confirms the lack of FA proteins in chromatin or the nuclear matrix. Furthermore, phosphorylation of FancG was found to be temporally correlated with exit of the FA complex from chromosomes at mitosis. Taken together, these findings suggest a role for FA proteins in chromatin and nuclear matrix.     

Cdc25B and Cdc25C differ markedly in their properties as initiators of mitosis.

We have used time-lapse fluorescence microscopy to study the properties of the Cdc25B and Cdc25C phosphatases that have both been implicated as initiators of mitosis in human cells. To differentiate between the functions of the two proteins, we have microinjected expression constructs encoding Cdc25B or Cdc25C or their GFP-chimeras into synchronized tissue culture cells. This assay allows us to express the proteins at defined points in the cell cycle. We have followed the microinjected cells by time-lapse microscopy, in the presence or absence of DNA synthesis inhibitors, and assayed whether they enter mitosis prematurely or at the correct time. We find that overexpressing Cdc25B alone rapidly causes S phase and G2 phase cells to enter mitosis, whether or not DNA replication is complete, whereas overexpressing Cdc25C does not cause premature mitosis. Overexpressing Cdc25C together with cyclin B1 does shorten the G2 phase and can override the unreplicated DNA checkpoint, but much less efficiently than overexpressing Cdc25B. These results suggest that Cdc25B and Cdc25C do not respond identically to the same cell cycle checkpoints. This difference may be related to the differential localization of the proteins; Cdc25C is nuclear throughout interphase, whereas Cdc25B is nuclear in the G1 phase and cytoplasmic in the S and G2 phases. We have found that the change in subcellular localization of Cdc25B is due to nuclear export and that this is dependent on cyclin B1. Our data suggest that although both Cdc25B and Cdc25C can promote mitosis, they are likely to have distinct roles in the controlling the initiation of mitosis.     

Human APOBEC3A Isoforms Translocate to the Nucleus and Induce DNA Double Strand Breaks Leading to Cell Stress and Death

Human APOBEC3 enzymes deaminate single stranded DNA. At least five can deaminate mitochondrial DNA in the cytoplasm, while three can deaminate viral DNA in the nucleus. However, only one, APOBEC3A, can hypermutate genomic DNA. We analysed the distribution and function of the two APOBEC3A isoforms p1 and p2 in transfected cell lines. Both can translocate to the nucleus and hypermutate CMYC DNA and induce DNA double strand breaks as visualized by the detection of ©H2AX or Chk2. APOBEC3A induced G1 phase cell cycle arrest and triggered several members of the intrinsic apoptosis pathway. Activation of purified human CD4+ T lymphocytes with PHA, IL2 and interferon α resulted in C->T hypermutation of genomic DNA and double stranded breaks suggesting a role for APOBEC3A in pro-inflammatory conditions. As chronic inflammation underlies many diseases including numerous cancers, it is possible that APOBEC3A induction may generate many of the lesions typical of a cancer genome.     

癌症新型的诊疗靶点－APOBEC

APOBEC（“载脂蛋白质B mRNA编辑催化多肽”）是一类进化保守的胞苷脱氨酶家族。在人体内,已知含有保守的DNA胞嘧啶脱氨酶结构域的基因共有11种,包括AID、APOBEC1、APOBEC2、APOBEC3基因家族APOBEC3A、APOBEC3B、APOBEC3C、APOBEC3DE、APOBEC3F、APOBEC3G、APOBEC3H（分别称为A3A、A3B、A3C、A3D、A3F、A3G和A3H）和APOBEC4。APOBEC利用其脱氨酶活性通过与RNA和/或DNA结合,催化mRNA或使DNA中的胞嘧啶核苷酸转变为尿嘧啶,或者胞嘧啶核苷酸转变为胸腺嘧啶核苷酸,进而完成各自不同的功能。目前研究发现,AID及APOBEC3（A3s）的7种脱氨酶在人类的天然免疫和适应性免疫防御过程中发挥重要的作用,且在口腔癌,肺癌（腺癌和鳞状细胞癌）,结直肠癌和乳腺癌等的诊疗过程中具有重要的潜在应用价值。AID可以通过将胞嘧啶脱氨基成尿嘧啶,来启动SHM (体细胞超突变)和CSR (类别转换重组),进而在抗体多样性方面发挥作用。它的异常表达能够使B细胞淋巴瘤等恶性肿瘤的发病频率显著增加。而A3A、A3B通过胞嘧啶到尿嘧啶转换,以及自身表达量上调而在乳腺癌和肺癌诊疗中起作用。A3G通过APOBEC3G/miR 29/MMP2为了解结直肠癌肝转移和开发治疗晚期结肠癌的有效疗法开辟了新的途径。综上所述,本文将以AID,A3A,A3B,A3G为例子,对APOBEC在癌症诊断和治疗方面的应用进行综述,以期为进一步药物研究和临床应用等提供参考。     

癌症新型的诊疗靶点－APOBEC

APOBEC（“载脂蛋白质B mRNA编辑催化多肽”）是一类进化保守的胞苷脱氨酶家族。在人体内,已知含有保守的DNA胞嘧啶脱氨酶结构域的基因共有11种,包括AID、APOBEC1、APOBEC2、APOBEC3基因家族APOBEC3A、APOBEC3B、APOBEC3C、APOBEC3DE、APOBEC3F、APOBEC3G、APOBEC3H（分别称为A3A、A3B、A3C、A3D、A3F、A3G和A3H）和APOBEC4。APOBEC利用其脱氨酶活性通过与RNA和/或DNA结合,催化mRNA或使DNA中的胞嘧啶核苷酸转变为尿嘧啶,或者胞嘧啶核苷酸转变为胸腺嘧啶核苷酸,进而完成各自不同的功能。目前研究发现,AID及APOBEC3（A3s）的7种脱氨酶在人类的天然免疫和适应性免疫防御过程中发挥重要的作用,且在口腔癌,肺癌（腺癌和鳞状细胞癌）,结直肠癌和乳腺癌等的诊疗过程中具有重要的潜在应用价值。AID可以通过将胞嘧啶脱氨基成尿嘧啶,来启动SHM (体细胞超突变)和CSR (类别转换重组),进而在抗体多样性方面发挥作用。它的异常表达能够使B细胞淋巴瘤等恶性肿瘤的发病频率显著增加。而A3A、A3B通过胞嘧啶到尿嘧啶转换,以及自身表达量上调而在乳腺癌和肺癌诊疗中起作用。A3G通过APOBEC3G/miR 29/MMP2为了解结直肠癌肝转移和开发治疗晚期结肠癌的有效疗法开辟了新的途径。综上所述,本文将以AID,A3A,A3B,A3G为例子,对APOBEC在癌症诊断和治疗方面的应用进行综述,以期为进一步药物研究和临床应用等提供参考。     

LINE-1 Retroelements Complexed and Inhibited by Activation Induced Cytidine Deaminase

LINE-1 (abbreviated L1) is a major class of retroelements in humans and mice. If unrestricted, retroelements accumulate in the cytoplasm and insert their DNA into the host genome, with the potential to cause autoimmune disease and cancer. Retroviruses and other retroelements are inhibited by proteins of the APOBEC family, of which activation-induced cytidine deaminase (AID) is a member. Although AID is mainly known for being a DNA mutator shaping the antibody repertoire in B lymphocytes, we found that AID also restricts de novo L1 integrations in B- and non-B-cell lines. It does so by decreasing the protein level of open reading frame 1 (ORF1) of both exogenous and endogenous L1. In activated B lymphocytes, AID deficiency increased L1 mRNA 1.6-fold and murine leukemia virus (MLV) mRNA 2.7-fold. In cell lines and activated B lymphocytes, AID forms cytoplasmic high-molecular-mass complexes with L1 mRNA, which may contribute to L1 restriction. Because AID-deficient activated B lymphocytes do not express ORF1 protein, we suggest that ORF1 protein expression is inhibited by additional restriction factors in these cells. The greater increase in MLV compared to L1 mRNA in AID-deficient activated B lymphocytes may indicate less strict surveillance of retrovirus.     

Evolution of the AID/APOBEC family of polynucleotide (deoxy)cytidine deaminases

The AID/APOBEC family (comprising AID, APOBEC1, APOBEC2, and APOBEC3 subgroups) contains members that can deaminate cytidine in RNA and/or DNA and exhibit diverse physiological functions (AID and APOBEC3 deaminating DNA to trigger pathways in adaptive and innate immunity; APOBEC1 mediating apolipoprotein B RNA editing). The founder member APOBEC1, which has been used as a paradigm, is an RNA-editing enzyme with proposed antecedents in yeast. Here, we have undertaken phylogenetic analysis to glean insight into the primary physiological function of the AID/APOBEC family. We find that although the family forms part of a larger superfamily of deaminases distributed throughout the biological world, the AID/APOBEC family itself is restricted to vertebrates with homologs of AID (a DNA deaminase that triggers antibody gene diversification) and of APOBEC2 (unknown function) identifiable in sequence databases from bony fish, birds, amphibians, and mammals. The cloning of an AID homolog from dogfish reveals that AID extends at least as far back as cartilaginous fish. Like mammalian AID, the pufferfish AID homolog can trigger deoxycytidine deamination in DNA but, consistent with its cold-blooded origin, is thermolabile. The fine specificity of its mutator activity and the biased codon usage in pufferfish IgV genes appear broadly similar to that of their mammalian counterparts, consistent with a coevolution of the antibody mutator and its substrate for the optimal targeting of somatic mutation during antibody maturation. By contrast, APOBEC1 and APOBEC3 are later evolutionary arrivals with orthologs not found in pufferfish (although synteny with mammals is maintained in respect of the flanking loci). We conclude that AID and APOBEC2 are likely to be the ancestral members of the AID/APOBEC family (going back to the beginning of vertebrate speciation) with both APOBEC1 and APOBEC3 being mammal-specific derivatives of AID and a complex set of domain shuffling underpinning the expansion and evolution of the primate APOBEC3s.     

Unpicking neurodegeneration in a dish with human pluripotent stem cells

Eukaryotic cell division is an orderly and timely process involving the error-free segregation of chromosomes and cytoplasmic components to give rise to two separate daughter cells. Defects in genome maintenance mechanisms such as cell cycle checkpoints and DNA repair can impact the segregation of the genome during mitosis leading to multiple chromosomal imbalances. In mammals, the DNA damage checkpoint effector Checkpoint Kinase 1 (Chk1) is essential for responses to DNA replication errors, external DNA damage, and chromatin breaks. We reported recently that Chk1 also was essential for chromosome segregation and completion of cytokinesis to prevent genomic instability. Our studies demonstrated that Chk1 deficiency in mitotic cells causes chromosome mis-alignment, lagging chromosomes, chromosome mis- segregation, cytokinetic regression, and binucleation. In addition, abrogation of Chk1 resulted in aberrant localization of mitotic Aurora B kinase at the metaphase plate, anaphase spindle midzone, and cytokinetic midbody as studied both in various cell lines and in a mouse model. Therefore, inappropriate regulation of Chk1 levels during cell cycle progression will result in failed cell division and enhanced genomic instability.     

Differential subcellular localization of functionally divergent survivin splice variants

Survivin is an inhibitor of apoptosis protein (IAP) that is markedly overexpressed in most cancers. We identified two novel functionally divergent splice variants, i.e. non-antiapoptotic survivin-2B and antiapoptotic survivin-deltaEx3. Because survivin-2B might be a naturally occurring antagonist of antiapoptotic survivin variants, we analyzed the subcellular distribution of these proteins. PSORT II analysis predicted a preferential cytoplasmic localization of survivin and survivin-2B, but a preferential nuclear localization of survivin-deltaEx3. GFP-tagged survivin variants confirmed the predicted subcellular localization and additionally revealed a cell cycle-dependent nuclear accumulation of survivin-deltaEx3. Moreover, a bipartite nuclear localization signal found exclusively in survivin-deltaEx3 may support cytoplasmic clearance of survivin-deltaEx3. In contrast to the known association between survivin and microtubules or centromeres during mitosis, no corresponding co-localization became evident for survivin-deltaEx3 or survivin-2B. In conclusion, our study provided data on a differential subcellular localization of functionally divergent survivin variants, suggesting that survivin isoforms may perform different functions in distinct subcellular compartments and distinct phases of the cell cycle.     

The role of innate APOBEC3G and adaptive AID immune responses in HLA-HIV/SIV immunized SHIV infected macaques

The AID/APOBEC family (activation induced deaminase/apolipoprotein B mRNA editing cytokine deaminase) in B cells play important roles in adaptive and innate immunity. Whereas APOBEC3G has been studied in CD4+ T cells and myeloid cells its functional potential in B cells has received little attention. AID combines two critical functions of antibodies, class switching and affinity maturation and may serve as a functional surrogate of protection. These functions were studied following systemic immunization of rhesus macaques with recombinant HLA constructs, linked with HIV and SIV antigens and HSP70 to dextran. The results showed significant upregulation of AID in CD20+ B cells, APOBEC 3G in CD27+ memory B cells and CD4+ effector memory T cells. After immunization the upregulated APOBEC 3G and AID were directly correlated in B cells (p<0.0001). Following challenge with SHIV SF162.P4 the viral load was inversely correlated with AID in B cells and APOBEC 3G in B and T cells, suggesting that both deaminases may have protective functions. Investigation of major interactions between DC, T cells and B cells showed significant increase in membrane associated IL-15 in DC and CD40L in CD4+ T cells. IL-15 binds the IL-15 receptor complex in CD4+ T and B cells, which may reactivate the DC, T and B cell interactions. The overall results are consistent with AID inhibiting pre-entry SHIV by eliciting IgG and IgA antibodies, whereas APOBEC 3G may contribute to the post-entry control of SHIV replication and cellular spread.     

Genomic Uracil Homeostasis during Normal B Cell Maturation and Loss of This Balance during B Cell Cancer Development

Activation-induced deaminase (AID) converts DNA cytosines to uracils in immunoglobulin genes, creating antibody diversification. It also causes mutations and translocations that promote cancer. We examined the interplay between uracil creation by AID and its removal by UNG2 glycosylase in splenocytes undergoing maturation and in B cell cancers. The genomic uracil levels remain unchanged in normal stimulated B cells, demonstrating a balance between uracil generation and removal. In stimulated UNG^−/− cells, uracil levels increase by 11- to 60-fold during the first 3 days. In wild-type B cells, UNG2 gene expression and enzymatic activity rise and fall with AID levels, suggesting that UNG2 expression is coordinated with uracil creation by AID. Remarkably, a murine lymphoma cell line, several human B cell cancer lines, and human B cell tumors expressing AID at high levels have genomic uracils comparable to those seen with stimulated UNG^−/−splenocytes. However, cancer cells express UNG2 gene at levels similar to or higher than those seen with peripheral B cells and have nuclear uracil excision activity comparable to that seen with stimulated wild-type B cells. We propose that more uracils are created during B cell cancer development than are removed from the genome but that the uracil creation/excision balance is restored during establishment of cell lines, fixing the genomic uracil load at high levels.     

Suppression of gross chromosomal rearrangements by a new alternative replication factor C complex

Defects in DNA replication fidelity lead to genomic instability. Gross chromosomal rearrangement (GCR), a type of genomic instability, is highly enhanced by various initial mutations affecting DNA replication. Frequent observations of GCRs in many cancers strongly argue the importance of maintaining high fidelity of DNA replication to suppress carcinogenesis. Recent genome wide screens in Saccharomyces cerevisiae identified a new GCR suppressor gene, ELG1, enhanced level of genome instability gene 1. Its physical interaction with proliferating cell nuclear antigen (PCNA) and complex formation with Rfc2-5p proteins suggest that Elg1 functions to load/unload PCNA onto DNA during a certain DNA metabolism. High level of DNA damage accumulation and enhanced phenotypes with mutations in genes involved in cell cycle checkpoints, homologous recombination (HR), or chromatin assembly in the elg1 strain suggest that Elg1p-Rfc2-5p functions in a fundamental DNA metabolism to suppress genomic instability.