Analysis of SHPRH functions in DNA repair and immunoglobulin diversification

During replication, bypass of DNA lesions is orchestrated by the Rad6 pathway. Monoubiquitination of proliferating cell nuclear antigen (PCNA) by Rad6/Rad18 leads to recruitment of translesion polymerases for direct and potentially mutagenic damage bypass. An error-free bypass pathway may be initiated via K63-linked PCNA polyubiquitination by Ubc13/Mms2 and the E3 ligase Rad5 in yeast, or HLTF/SHPRH in vertebrates. For the latter two enzymes, redundancy with a third E3 ligase and alternative functions have been reported. We have previously shown that the Rad6 pathway is involved in somatic hypermutation of immunoglobulin genes in B lymphocytes. Here, we have used knockout strategies targeting expression of the entire SHPRH protein or functionally significant domains in chicken DT40 cells that do not harbor a HLTF ortholog. We show that SHPRH is apparently redundant with another E3 ligase during DNA damage-induced PCNA modification. SHPRH plays no substantial role in cellular resistance to drugs initiating excision repair and the Rad6 pathway, but is important in survival of topoisomerase II inhibitor treatment. Removal of only the C-terminal RING domain does not interfere with this SHPRH function. SHPRH inactivation does not substantially impact on the overall efficacy of Ig diversification. Redundancy of E3 ligases in the Rad6 pathway may be linked to its different functions in genome maintenance and genetic plasticity.     

Eukaryotic DNA damage tolerance and translesion synthesis through covalent modifications of PCNA

In addition to well-defined DNA repair pathways, all living organisms have evolved mechanisms to avoid cell death caused by replication fork collapse at a site where replication is blocked due to disruptive covalent modifications of DNA. The term DNA damage tolerance （DDT） has been employed loosely to include a collection of mechanisms by which cells survive replication-blocking lesions with or without associated genomic instability. Recent genetic analyses indicate that DDT in eukaryotes, from yeast to human, consists of two parallel pathways with one being error-free and another highly mutagenic. Interestingly, in budding yeast, these two pathways are mediated by sequential modifications of the proliferating cell nuclear antigen （PCNA） by two ubiquitination complexes Rad6-Rad18 and Mms2-Ubc13-Rad5. Damage-induced monoubiquitination of PCNA by Rad6-Rad18 promotes translesion synthesis （TLS） with increased mutagenesis, while subsequent polyubiquitination of PCNA at the same K164 residue by Mms2-Ubc13-Rad5 promotes error-free lesion bypass. Data obtained from recent studies suggest that the above mechanisms are conserved in higher eukaryotes. In particular, mammals contain multiple specialized TLS polymerases. Defects in one of the TLS polymerases have been linked to genomic instability and cancer.     

SHPRH and HLTF act in a damage-specific manner to coordinate different forms of postreplication repair and prevent mutagenesis

Postreplication repair (PRR) pathways play important roles in restarting stalled replication forks and regulating mutagenesis. In yeast, Rad5-mediated damage avoidance and Rad18-mediated translesion synthesis (TLS) are two forms of PRR. Two Rad5-related proteins, SHPRH and HLTF, have been identified in mammalian cells, but their specific roles in PRR are unclear. Here, we show that HLTF and SHPRH suppress mutagenesis in a damage-specific manner, preventing mutations induced by UV and MMS, respectively. Following UV, HLTF enhances PCNA monoubiquitination and recruitment of TLS polymerase η, while also inhibiting SHPRH function. In contrast, MMS promotes the degradation of HLTF and the interactions of SHPRH with Rad18 and polymerase κ. Our data suggest not only that cells differentially utilize HLTF and SHPRH for different forms of DNA damage, but also, surprisingly, that HLTF and SHPRH may coordinate the two main branches of PRR to choose the proper bypass mechanism for minimizing mutagenesis.     

Role of yeast Rad5 and its human orthologs,HLTF and SHPRH in DNA damage tolerance

In the yeast Saccharomyces cerevisiae, the Rad6–Rad18 DNA damage tolerance pathway constitutes a major defense system against replication fork blocking DNA lesions. The Rad6–Rad18 ubiquitin-conjugating/ligase complex governs error-free and error-prone translesion synthesis by specialized DNA polymerases, as well as an error-free Rad5-dependent postreplicative repair pathway. For facilitating replication through DNA lesions, translesion synthesis polymerases copy directly from the damaged template, while the Rad5-dependent damage tolerance pathway obtains information from the newly synthesized strand of the undamaged sister duplex. Although genetic data demonstrate the importance of the Rad5-dependent pathway in tolerating DNA damages, there has been little understanding of its mechanism. Also, the conservation of the yeast Rad5-dependent pathway in higher order eukaryotic cells remained uncertain for a long time. Here we summarize findings published in recent years regarding the role of Rad5 in promoting error-free replication of damaged DNA, and we also discuss results obtained with its human orthologs, HLTF and SHPRH.     

The human RAD18 gene product interacts with HHR6A and HHR6B

During DNA replication, lesion bypass is an important cellular response to unrepaired damage in the genome. In the yeast Saccharomyces cerevisiae, Rad6 and Rad18 are required for both the error-free and error-prone lesion bypass mechanisms. Furthermore, Rad6–Rad18 interaction is thought to be critical at an early step during lesion bypass in yeast. Two closely related human homologs of yeast Rad6 have been identified as HHR6A and HHR6B. Here, we report a full-length cDNA coding for the human homolog of yeast Rad18. The human RAD18 gene codes for a protein of 484 amino acid residues with a calculated molecular weight of 54 804 Da, and the gene is localized to chromosome 3 between reference intervals D3S3591 and D3S1283. Human RAD18 protein (hRAD18) was found to interact with HHR6A and HHR6B. When co-expressed in yeast cells, stable hRAD18–HHR6A and hRAD18–HHR6B protein complexes were identified and purified to near homogeneity. Thus, through interaction and complex formation with HHR6A and HHR6B, RAD18 protein may play an important role in lesion bypass mech­anisms in humans. Consistent with its role as a fundamental lesion bypass protein, the RAD18 gene is ubiquitously expressed in various human tissues.     

DNA-damage tolerance mediated by PCNA?Ub fusions in human cells is dependent on Rev1 but not Polη

In response to replication-blocking lesions, proliferating cell nuclear antigen (PCNA) can be sequentially ubiquitinated at the K164 residue, leading to two modes of DNA-damage tolerance, namely, translesion DNA synthesis (TLS) and error-free lesion bypass. Although the majority of reported data support a model whereby monoubiquitinated PCNA enhances its affinity for TLS polymerases and hence recruits them to the damage sites, this model has also been challenged by several observations. In this study, we expressed the PCNA-164R and ubiquitin (UB) fusion genes in an inducible manner in an attempt to mimic PCNA monoubiquitination in cultured human cells. It was found that expression of both N- and C-terminal PCNA•Ub fusions conferred significant tolerance to ultraviolet (UV)-induced DNA damage. Surprisingly, depletion of Polη, a TLS polymerase dedicated to bypassing UV-induced pyrimidine dimers, did not alter tolerance conferred by PCNA•Ub. In contrast, depletion of Rev1, another TLS polymerase serving as a scaffold for the assembly of the TLS complex, completely abolished PCNA•Ub-mediated damage tolerance. Similar genetic interactions were confirmed when UV-induced monoubiquitination of endogenous PCNA is abolished by RAD18 deletion. Hence, PCNA•Ub fusions bypass the requirement for PCNA monoubiquitination, and UV damage tolerance conferred by these fusions is dependent on Rev1 but independent of Polη.     

Human SHPRH suppresses genomic instability through proliferating cell nuclear antigen polyubiquitination

Differential modifications of proliferating cell nuclear antigen (PCNA) determine DNA repair pathways at stalled replication forks. In yeast, PCNA monoubiquitination by the ubiquitin ligase (E3) yRad18 promotes translesion synthesis (TLS), whereas the lysine-63-linked polyubiquitination of PCNA by yRad5 (E3) promotes the error-free mode of bypass. The yRad5-dependent pathway is important to prevent genomic instability during replication, although its exact molecular mechanism is poorly understood. This mechanism has remained totally elusive in mammals because of the lack of apparent RAD5 homologues. We report that a putative tumor suppressor gene, SHPRH, is a human orthologue of yeast RAD5. SHPRH associates with PCNA, RAD18, and the ubiquitin-conjugating enzyme UBC13 (E2) and promotes methyl methanesulfonate (MMS)-induced PCNA polyubiquitination. The reduction of SHPRH by stable short hairpin RNA increases sensitivity to MMS and enhances genomic instability. Therefore, the yRad5/SHPRH-dependent pathway is a conserved and fundamental DNA repair mechanism that protects the genome from genotoxic stress.     

The Yeast Shu Complex Utilizes Homologous Recombination Machinery for Error-free Lesion Bypass via Physical Interaction with a Rad51 Paralogue

DNA-damage tolerance (DDT) is defined as a mechanism by which eukaryotic cells resume DNA synthesis to fill the single-stranded DNA gaps left by replication-blocking lesions. Eukaryotic cells employ two different means of DDT, namely translesion DNA synthesis (TLS) and template switching, both of which are coordinately regulated through sequential ubiquitination of PCNA at the K164 residue. In the budding yeast Saccharomyces cerevisiae, the same PCNA-K164 residue can also be sumoylated, which recruits the Srs2 helicase to prevent undesired homologous recombination (HR). While the mediation of TLS by PCNA monoubiquitination has been extensively characterized, the method by which K63-linked PCNA polyubiquitination leads to template switching remains unclear. We recently identified a yeast heterotetrameric Shu complex that couples error-free DDT to HR as a critical step of template switching. Here we report that the Csm2 subunit of Shu physically interacts with Rad55, an accessory protein involved in HR. Rad55 and Rad57 are Rad51 paralogues and form a heterodimer to promote Rad51-ssDNA filament formation by antagonizing Srs2 activity. Although Rad55-Rad57 and Shu function in the same pathway and both act to inhibit Srs2 activity, Shu appears to be dedicated to error-free DDT while the Rad55-Rad57 complex is also involved in double-strand break repair. This study reveals the detailed steps of error-free lesion bypass and also brings to light an intrinsic interplay between error-free DDT and Srs2-mediated inhibition of HR.     

The Rad5 Helicase and RING Domains Contribute to Genome Stability through their Independent Catalytic Activities

Genomic stability is compromised by DNA damage that obstructs replication. Rad5 plays a prominent role in DNA damage bypass processes that evolved to ensure the continuation of stalled replication. Like its human orthologs, the HLTF and SHPRH tumor suppressors, yeast Rad5 has a RING domain that supports ubiquitin ligase activity promoting PCNA polyubiquitylation and a helicase domain that in the case of HLTF and Rad5 was shown to exhibit an ATPase-linked replication fork reversal activity. The RING domain is embedded in the helicase domain, confusing their separate investigation and the understanding of the exact role of Rad5 in DNA damage bypass. Particularly, it is still debated whether the helicase domain plays a catalytic or a non-enzymatic role during error-free damage bypass and whether it facilitates a function separately from the RING domain. In this study, through in vivo and in vitro characterization of domain-specific mutants, we delineate the contributions of the two domains to Rad5 function. Yeast genetic experiments and whole-genome sequencing complemented with biochemical assays demonstrate that the ubiquitin ligase and the ATPase-linked activities of Rad5 exhibit independent catalytic activities in facilitating separate pathways during error-free lesion bypass. Our results also provide important insights into the mutagenic role of Rad5 and indicate its tripartite contribution to DNA damage tolerance.     

Error‐free DNA damage tolerance pathway is facilitated by the Irc5 translocase through cohesin

DNA damage tolerance (DDT) mechanisms facilitate replication resumption and completion when DNA replication is blocked by bulky DNA lesions. In budding yeast, template switching (TS) via the Rad18/Rad5 pathway is a favored DDT pathway that involves usage of the sister chromatid as a template to bypass DNA lesions in an error‐free recombination‐like process. Here, we establish that the Snf2 family translocase Irc5 is a novel factor that promotes TS and averts single‐stranded DNA persistence during replication. We demonstrate that, during replication stress, Irc5 enables replication progression by assisting enrichment of cohesin complexes, recruited in an Scc2/Scc4‐dependent fashion, near blocked replication forks. This allows efficient formation of sister chromatid junctions that are crucial for error‐free DNA lesion bypass. Our results support the notion of a key role of cohesin in the completion of DNA synthesis under replication stress and reveal that the Rad18/Rad5‐mediated DDT pathway is linked to cohesin enrichment at sites of perturbed replication via the Snf2 family translocase Irc5.     

PCNA modifications for regulation of post-replication repair pathways

Stalled DNA replication forks activate specific DNA repair mechanism called post-replication repair (PRR) pathways that simply bypass DNA damage. The bypassing of DNA damage by PRR prevents prolonged stalling of DNA replication that could result in double strand breaks (DSBs). Proliferating cell nuclear antigen (PCNA) functions to initiate and choose different bypassing pathways of PRR. In yeast, DNA replication forks stalled by DNA damage induces monoubiquitination of PCNA at K164, which is catalyzed by Rad6/Rad18 complex. PCNA monoubiquitination triggers the replacement of replicative polymerase with special translesion synthesis (TLS) polymerases that are able to replicate past DNA lesions. The PCNA interaction motif and/or the ubiquitin binding motif in most TLS polymerases seem to be important for the regulation of TLS. The TLS pathway is usually error-prone because TLS polymerases have low fidelity and no proofreading activity. PCNA can also be further polyubiquitinated by Ubc13/ Mms2/Rad5 complex, which adds an ubiquitin chain onto monoubiquitinated K164 of PCNA. PCNA polyubiquitination directs a different PRR pathway known as error-free damage avoidance, which uses the newly synthesized sister chromatid as a template to bypass DNA damage presumably through template switching mechanism. Mammalian homologues of all of the yeast PRR proteins have been identified, thus PRR is well conserved throughout evolution. Mutations of some PRR genes are associated with a higher risk for cancers in mice and human patients, strongly supporting the importance of PRR as a tumor suppressor pathway.     

Rad18 E3 ubiquitin ligase activity mediates Fanconi anemia pathway activation and cell survival following DNA topoisomerase 1 inhibition

Camptothecin (CPT) and related chemotherapeutic drugs induce formation of DNA topoisomerase I (Top1) covalent or cleavage complexes (Top1ccs) that block leading-strand DNA synthesis and elicit DNA Double Stranded Breaks (DSB) during S phase. The Fanconi Anemia (FA) pathway is implicated in tolerance of CPT-induced DNA damage yet the mechanism of FA pathway activation by Top1 poisons has not been studied. We show here that the FA core complex protein FANCA and monoubiquitinated FANCD2 (an effector of the FA pathway) are rapidly mobilized to chromatin in response to CPT treatment in several human cancer cell lines and untransformed primary human dermal fibroblasts. FANCD2 depletion using siRNA leads to impaired recovery from CPT-induced inhibition or DNA synthesis, persistence of γH2AX (a DSB marker) and reduced cell survival following CPT treatment. The E3 ubiquitin ligase Rad18 is necessary for CPT-induced recruitment of FANCA and FANCD2 to chromatin. Moreover, Rad18-depletion recapitulates the DNA synthesis and survival defects of FANCD2-deficiency in CPT-treated cells. It is well-established that Rad18 promotes FA pathway activation and DNA damage tolerance in response to bulky DNA lesions via a mechanism involving PCNA monoubiquitination. In contrast, PCNA monoubiquitination is not involved in Rad18-mediated FA pathway activation or cell survival following acquisition of CPT-induced DSB. Moreover, while Rad18 is implicated in recombinational repair of DSB via an E3 ligase-independent mechanism, we demonstrate that Rad18 E3 ligase activity is essential for appropriate FA pathway activation and DNA damage tolerance after CPT treatment. Taken together, our results define a novel pathway of Rad18-dependent DSB repair that is dissociable from known Rad18-mediated DNA repair mechanisms based on its independence from PCNA ubiquitination and requirement for E3 ligase activity.Key words: camptothecin, Rad18, topoisomerase I, double strand breaks, Fanconi anemia     

Tumour suppressor ING1b maintains genomic stability upon replication stress

The lesion bypass pathway, which is regulated by monoubiquitination of proliferating cell nuclear antigen (PCNA), is essential for resolving replication stalling due to DNA lesions. This process is important for preventing genomic instability and cancer development. Previously, it was shown that cells deficient in tumour suppressor p33ING1 (ING1b) are hypersensitive to DNA damaging agents via unknown mechanism. In this study, we demonstrated a novel tumour suppressive function of ING1b in preserving genomic stability upon replication stress through regulating PCNA monoubiquitination. We found that ING1b knockdown cells are more sensitive to UV due to defects in recovering from UV-induced replication blockage, leading to enhanced genomic instability. We revealed that ING1b is required for the E3 ligase Rad18-mediated PCNA monoubiquitination in lesion bypass. Interestingly, ING1b-mediated PCNA monoubiquitination is associated with the regulation of histone H4 acetylation. Results indicate that chromatin remodelling contributes to the stabilization of stalled replication fork and to the regulation of PCNA monoubiquitination during lesion bypass.     

Requirement of RAD5 and MMS2 for postreplication repair of UV-damaged DNA in Saccharomyces cerevisiae

UV lesions in the template strand block the DNA replication machinery. Genetic studies of the yeast Saccharomyces cerevisiae have indicated the requirement of the Rad6-Rad18 complex, which contains ubiquitin-conjugating and DNA-binding activities, in the error-free and mutagenic modes of damage bypass. Here, we examine the contributions of the REV3, RAD30, RAD5, and MMS2 genes, all of which belong to the RAD6 epistasis group, to the postreplication repair of UV-damaged DNA. Discontinuities, which are formed in DNA strands synthesized from UV-damaged templates, are not repaired in the rad5Delta and mms2Delta mutants, thus indicating the requirement of the Rad5 protein and the Mms2-Ubc13 ubiquitin-conjugating enzyme complex in this repair process. Some discontinuities accumulate in the absence of RAD30-encoded DNA polymerase eta (Poleta) but not in the absence of REV3-encoded DNA Polzeta. We concluded that replication through UV lesions in yeast is mediated by at least three separate Rad6-Rad18-dependent pathways, which include mutagenic translesion synthesis by Polzeta, error-free translesion synthesis by Poleta, and postreplication repair of discontinuities by a Rad5-dependent pathway. We suggest that newly synthesized DNA possessing discontinuities is restored to full size by a "copy choice" type of DNA synthesis which requires Rad5, a DNA-dependent ATPase, and also PCNA and Poldelta. The possible roles of the Rad6-Rad18 and the Mms2-Ubc13 enzyme complexes in Rad5-dependent damage bypass are discussed.     

Differential regulation of Rad18 through Rad6-dependent mono- and polyubiquitination

Rad18 is involved in postreplication repair mainly through monoubiquitination of proliferating cell nuclear antigen (PCNA). Here we show that Rad18 protein was detected in human cells as two major bands at 75 and 85 kDa by Western blot. The bands were identified as nonubiquitinated and monoubiquitinated forms of Rad18, respectively, by mass spectrometry. Multiple ubiquitinated bands of Rad18 were detected in vitro in the presence of E1, E2 (Rad6), and methylated ubiquitin, indicating that Rad18 was monoubiquitinated at multiple sites through autoubiquitination. Rad18 self-associates, and this interaction was abolished by replacing one of the conserved cysteine residues with phenylalanine in the zinc finger domain (C207F). In the C207F mutant Rad18, monoubiquitination of Rad18 was not observed in vivo, suggesting that self-association was critical for monoubiquitination. Monoubiquitinated Rad18 was detected mainly in the cytoplasm, whereas nonubiquitinated Rad18 was detected predominantly in the nuclei. Furthermore, Rad18 was shown to be polyubiquitinated in cells treated with proteasome inhibitors. Purified Rad18 was also polyubiquitinated in an in vitro system containing E1, E2 (Rad6), and ubiquitin, and it was degraded by the addition of proteasomes. These results suggest that the amount of Rad18 in the nucleus is regulated differentially by mono- and polyubiquitination.     

PCNA is ubiquitinated by RNF8

The ubiquitination of PCNA is an essential event in the operation of the DNA Damage Tolerance (DDT) pathway that is activated after DNA damage caused by UV or chemical agents during S-phase. This pathway allows the bypass of DNA damage by translesion synthesis that would otherwise cause replication fork stalling. PCNA is mono-ubiquitinated by Rad18-Rad6, and polyubiquitinated by Rad5-Ubc13/Uev1 in the DDT pathway. Mono-and polyubiquitination of PCNA are key processes in the translesion bypass and template switching sub-pathways of the DDT. DNA damage by IR causes DSBs, which trigger the DNA Damage Response (DDR). The ubiquitin ligase RNF8 has a critical role in the assembly of BRCA1 complexes at the DSBs in the DDR. We show that RNF8 readily mono-ubiquitinates PCNA in the presence of UbcH5c, and polyubiquitinates PCNA in the added presence of Ubc13/Uev1a. These reactions are the same as those performed by Rad18-Rad6 and Rad5-Ubc13. RNF8 depletion suppressed both UV and MNNG-stimulated mono-ubiquitination of PCNA, revealing that an RNF8-dependent pathway for PCNA ubiquitination is operative in vivo. These findings provide evidence that RNF8, a key E3 ligase in the DDR, may also play a role in the DDT.     

Role of the ubiquitin-binding domain of Polη in Rad18-independent translesion DNA synthesis in human cell extracts

In eukaryotic cells, the Rad6/Rad18-dependent monoubiquitination of the proliferating cell nuclear antigen (PCNA) plays an essential role in the switching between replication and translesion DNA synthesis (TLS). The DNA polymerase Polη binds to PCNA via a consensus C-terminal PCNA-interacting protein (PIP) motif. It also specifically interacts with monoubiquitinated PCNA thanks to a recently identified ubiquitin-binding domain (UBZ). To investigate whether the TLS activity of Polη is always coupled to PCNA monoubiquitination, we monitor the ability of cell-free extracts to perform DNA synthesis across different types of lesions. We observe that a cis-syn cyclobutane thymine dimer (TT-CPD), but not a N-2-acetylaminofluorene-guanine (G-AAF) adduct, is efficiently bypassed in extracts from Rad18-deficient cells, thus demonstrating the existence of a Polη-dependent and Rad18-independent TLS pathway. In addition, by complementing Polη-deficient cells with PIP and UBZ mutants, we show that each of these domains contributes to Polη activity. The finding that the bypass of a CPD lesion in vitro does not require Ub-PCNA but nevertheless depends on the UBZ domain of Polη, reveals that this domain may play a novel role in the TLS process that is not related to the monoubiquitination status of PCNA.     

RAD5a and REV3 function in two alternative pathways of DNA-damage tolerance in Arabidopsis

DNA-damage tolerance (DDT) in yeast is composed of two parallel pathways and mediated by sequential ubiquitinations of PCNA. While monoubiquitination of PCNA promotes translesion synthesis (TLS) that is dependent on polymerase ζ consisted of a catalytic subunit Rev3 and a regulatory subunit Rev7, polyubiquitination of PCNA by Mms2-Ubc13-Rad5 promotes error-free lesion bypass. Inactivation of these two pathways results in a synergistic effect on DNA-damage responses; however, this two-branch DDT model has not been reported in any multicellular organisms. In order to examine whether Arabidopsis thaliana possesses a two-branch DDT system, we created rad5a rev3 double mutant plant lines and compared them with the corresponding single mutants. Arabidopsis rad5a and rev3 mutations are indeed synergistic with respect to root growth inhibition induced by replication-blocking lesions, suggesting that AtRAD5a and AtREV3 are required for error-free and TLS branches of DDT, respectively. Unexpectedly this study reveals three modes of genetic interactions in response to different types of DNA damage, implying that plant RAD5 and REV3 are also involved in DNA damage responses independent of DDT. By comparing with yeast cells, it is apparent that plant TLS is a more frequently utilized means of lesion bypass than error-free DDT in plants.     

Rad18 E3 ubiquitin ligase activity mediates Fanconi anemia pathway activation and cell survival following DNA Topoisomerase 1 inhibition

Camptothecin (CPT) and related chemotherapeutic drugs induce formation of DNA Topoisomerase I (Top1) covalent or cleavage complexes (Top1ccs) that block leading-strand DNA synthesis and elicit DNA Double Stranded Breaks (DSB) during S phase. The Fanconi Anemia (FA) pathway is implicated in tolerance of CPT-induced DNA damage yet the mechanism of FA pathway activation by Top1 poisons has not been studied. We show here that the FA core complex protein FANCA and monoubiquitinated FANCD2 (an effector of the FA pathway) are rapidly mobilized to chromatin in response to CPT treatment in several human cancer cell lines and untransformed primary human dermal fibroblasts. FANCD2 depletion using siRNA leads to impaired recovery from CPT-induced inhibition or DNA synthesis, persistence of γH2AX (a DSB marker) and reduced cell survival following CPT treatment. The E3 ubiquitin ligase Rad18 is necessary for CPT-induced recruitment of FANCA and FANCD2 to chromatin. Moreover, Rad18-depletion recapitulates the DNA synthesis and survival defects of FANCD2-deficiency in CPT-treated cells. It is well-established that Rad18 promotes FA pathway activation and DNA damage tolerance in response to bulky DNA lesions via a mechanism involving PCNA monoubiquitination. In contrast, PCNA monoubiquitination is not involved in Rad18-mediated FA pathway activation or cell survival following acquisition of CPT-induced DSB. Moreover, while Rad18 is implicated in recombinational repair of DSB via an E3 ligase-independent mechanism, we demonstrate that Rad18 E3 ligase activity is essential for appropriate FA pathway activation and DNA damage tolerance after CPT treatment. Taken together, our results define a novel pathway of Rad18-dependent DSB repair that is dissociable from known Rad18-mediated DNA repair mechanisms based on its independence from PCNA ubiquitination and requirement for E3 ligase activity.     

A novel Rad18 function involved in protection of the vertebrate genome after exposure to camptothecin

In Saccharomyces cerevisiae, Rad18 functions in post-replication repair pathways, such as error-free damage bypass involving Rad30 (Poleta) and error-prone damage bypass involving Rev3/7 (Polzeta). Chicken DT40 RAD18(-/-) cells were found to be hypersensitive to camptothecin (CPT), while RAD30(-/-) and REV3(-/-) cells, which are defective in translesion DNA synthesis, were not. RAD18(-/-) cells also showed higher levels of H2AX phosphorylation and chromosomal aberrations, particularly chromosomal gaps and breaks, upon exposure to CPT. Detailed analysis by alkaline sucrose density gradient centrifugation revealed that RAD18(-/-) and wild type cells exhibited similar rates of elongation of newly synthesized DNA in the presence or absence of low concentrations of CPT but that DNA breaks frequently occurred on both parental and nascent strands within 1h after a brief exposure to an elevated concentration of CPT, with more breaks induced in RAD18(-/-) cells than in wild type cells. These data suggest a previously unanticipated role for Rad18 in dealing with replication forks upon encountering DNA lesions induced by CPT.