Polyubiquitination Of Proliferating Cell Nuclear Antigen Research Articles

Genetic analysis of DNA-damage tolerance pathways in Arabidopsis.

Genetic analysis revealed a two-branch DNA-damage tolerance mechanism in Arabidopsis, namely translesion DNA synthesis and error-free lesion bypass, represented by Rev3 and Rad5a-Uev1C/D, respectively. DNA-damage tolerance (DDT) is a mechanism by which cells complete replication in the presence of replication-blocking lesions. In budding yeast, DDT is achieved through Rad6-Rad18-mediated monoubiquitination of proliferating cell nuclear antigen (PCNA), which promotes translesion DNA synthesis (TLS) and is followed by Ubc13-Mms2-Rad5 mediated K63-linked PCNA polyubiquitination that promotes error-free lesion bypass. Arabidopsis and other known plant genomes contain all of the above homologous genes except RAD18, and whether plants possess an intact DDT mechanism is unclear. In this study, we created Arabidopsis UEV1 (homologous to yeast MMS2) gene mutations and obtained two sets of double mutant lines Atuev1ab and Atuev1cd. It turned out that the Atuev1cd, but not the Atuev1ab mutant, was sensitive to DNA damage. Genetic analyses revealed that AtUEV1C/D and AtRAD5a function in the same pathway, while TLS represented by AtREV3 functions in a separate pathway in response to replication-blocking lesions. Furthermore, unlike budding yeast RAD5 that also functions in the TLS pathway, AtRAD5a is not required for TLS. Observations in this study collectively establish a two-branch DDT model in plants with similarity to and difference from the yeast DDT.

Human Germline HLTF E259K Mutation Identified in Familial MDS Patients Accumulates DNA Damage through Impaired PCNA Polyubiquitination

Although several causal genes of familial myelodysplastic syndromes (MDS) have been discovered, the genetic landscape and the molecular pathogenesis still remain unclear. To explore novel causal genetic alterations and their pathogenetic significance, we performed whole-exome sequencing analysis using four samples from a Japanese familial MDS pedigree, two affected and two unaffected as controls. In total, 16889 non-synonymous germline substitutions were obtained. Among them, twelve candidate single-nucleotide variations (ANK1, C9orf43, CYP7B1, EFHB, ENTPD7, FAM160B2, HELZ2, HLTF, INPP5J, ITPKB, RYK, TTN) were elucidated. Of the candidates, we elucidated HLTF, encoding a member of the SWItch/Sucrose Non-Fermenting (SWI/SNF) family, as a putative associated gene. HLTF works as an E3-ubiquitin ligase and binds with Methyl Methanesulfonate Sensitive 2 (MMS2)/Ubiquitin Conjugating Enzyme E2N (UBC13) complex. This complex is known to promote the Lys-63 linked polyubiquitination of proliferating cell nuclear antigen (PCNA), followed by an error-free postreplication DNA repair by arranging template switching (Mailand et al. Nat Rev Mol Cell Biol 2013). The obtained germline mutation of HLTF was p.Glu259Lys (E259K), heterozygous mutation.To study the functional role of HLTF, we firstly evaluated the effects of Hltf silencing on hematopoiesis by in vitro colony-formation assay with Hltf-knocked down mouse primary bone marrow cells, and found that these cells have enhanced in vitro colony-formation capacity. In agreement with the acquisition of an immature phenotype, the cells expressing c-kit, a hematopoietic stem/ progenitor cell marker, were increased in Hltf shRNA-expressing colonies after the first round. We then focused on DNA damage in HLTF knockdown HEL cells according to the function of HLTF post-replication DNA repair. We examined the phosphorylation of gamma H2AX foci by immunofluorescent staining and found that the number of foci was increased in HLTF knockdown cells compared with control cells, suggesting that DNA damage was accumulated in HLTF knockdown cells.To investigate the effects of exogenous expression of HLTF E259K mutant protein, we evaluated in vitro apoptosis status with HEL cells transduced with empty vector (EV), HLTF wild type (WT), and HLTF E259K mutant (MT), respectively. In apoptosis assay, annexin V positive cells were significantly increased in MT-overexpressed HEL cells compared with WT-overexpressed HEL cells. In addition, we found that the number of gamma H2AX foci was increased in HLTF MT-overexpressed HEL cells, suggesting that DNA double-strand breaks were accumulated in MT-overexpressed cells. To explore the mechanism of accumulated DNA double-strand breaks by mutant HLTF, we evaluated the binding ability of HLTF mutant protein with PCNA, and MMS2/UBC13, all of which play a critical role in the DNA double-strand break repair process. We found that binding of HLTF MT to MMS2 or UBC13 was significantly attenuated compared with HLTF WT although binding to PCNA remained unchanged, suggesting HLTF MT cannot efficiently form a complex that induces polyubiquitination of PCNA. In line with this, PCNA polyubiquitination was impaired in HLTF MT-overexpressed cells compared with HLTF WT-overexpressed cells.Finally, we searched somatic HLTF mutation in sporadic MDS bone marrow samples. Whole HLTF coding regions of 40 samples were sequenced. As a result, a novel HLTF missense heterozygous mutation (p.Thr50Ala, T50A) was identified in one sample (1/40 = 2.5%). These data suggested the possibility that HLTF mutation might contribute to the pathogenesis not only in familial MDS but also in sporadic MDS.In conclusion, our results indicated that HLTF E259K germ line mutation accumulates DNA damage through impaired PCNA polyubiquitination. The aberrant function of HLTF mutant might play a role in the pathogenesis of familial MDS. DisclosuresMasamoto:Astellas Pharma Inc.: Research Funding; Nippon Shinyaku Co., Ltd.: Other: Scholarship donations for the laboratory; Pfizer Inc.: Research Funding. Arai:Bristol-Myers Squibb: Research Funding. Suzuki:Bristol-Myers K.K: Honoraria; Novarltis: Honoraria; Celgen: Honoraria; Ono: Honoraria; Fujimoto: Honoraria; Takeda: Honoraria. Harada:NIPPON SHINYAKU CO.: Speakers Bureau; NOVARTIS: Research Funding; Celgene: Research Funding. Kurokawa:Bristol-Myers Squibb: Speakers Bureau; Nippon Shinyaku Co., Ltd.: Other: Scholarship donations for the laboratory, Speakers Bureau; Astellas Pharma Inc.: Research Funding; Takeda Pharmaceutical Company Limited.: Research Funding, Speakers Bureau; Pfizer Inc.: Research Funding; Celgene Corporation: Speakers Bureau.

DNA-damage tolerance through PCNA ubiquitination and sumoylation.

DNA-damage tolerance (DDT) is employed by eukaryotic cells to bypass replication-blocking lesions induced by DNA-damaging agents. In budding yeast Saccharomyces cerevisiae, DDT is mediated by RAD6 epistatic group genes and the central event for DDT is sequential ubiquitination of proliferating cell nuclear antigen (PCNA), a DNA clamp required for replication and DNA repair. DDT consists of two parallel pathways: error-prone DDT is mediated by PCNA monoubiquitination, which recruits translesion synthesis DNA polymerases to bypass lesions with decreased fidelity; and error-free DDT is mediated by K63-linked polyubiquitination of PCNA at the same residue of monoubiquitination, which facilitates homologous recombination-mediated template switch. Interestingly, the same PCNA residue is also subjected to sumoylation, which leads to inhibition of unwanted recombination at replication forks. All three types of PCNA posttranslational modifications require dedicated conjugating and ligation enzymes, and these enzymes are highly conserved in eukaryotes, from yeast to human.

Fork Cleavage-Religation Cycle and Active Transcription Mediate Replication Restart after Fork Stalling at Co-transcriptional R-Loops.

Formation of co-transcriptional R-loops underlies replication fork stalling upon head-on transcription-replication encounters. Here, we demonstrate that RAD51-dependent replication fork reversal induced by R-loops is followed by the restart of semiconservative DNA replication mediated by RECQ1 and RECQ5 helicases, MUS81/EME1 endonuclease, RAD52 strand-annealing factor, the DNA ligase IV (LIG4)/XRCC4 complex, and the non-catalytic subunit of DNA polymerase δ, POLD3. RECQ5 disrupts RAD51 filaments assembled on stalled forks after RECQ1-mediated reverse branch migration, preventing a new round of fork reversal and facilitating fork cleavage by MUS81/EME1. MUS81-dependent DNA breaks accumulate in cells lacking RAD52 or LIG4 upon induction of R-loop formation, suggesting that RAD52 acts in concert with LIG4/XRCC4 to catalyze fork religation, thereby mediating replication restart. The resumption of DNA synthesis after R-loop-associated fork stalling also requires active transcription, the restoration of which depends onMUS81, RAD52, LIG4, and the transcription elongation factor ELL. These findings provide mechanistic insights into transcription-replication conflict resolution.

TonEBP Regulates PCNA Polyubiquitination in Response to DNA Damage through Interaction with SHPRH and USP1.

SummaryPolyubiquitination of proliferating cell nuclear antigen (PCNA) regulates the error-free template-switching mechanism for the bypass of DNA lesions during DNA replication. PCNA polyubiquitination is critical for the maintenance of genomic integrity; however, the underlying mechanism is poorly understood. Here, we demonstrate that tonicity-responsive enhancer-binding protein (TonEBP) regulates PCNA polyubiquitination in response to DNA damage. TonEBP was recruited to DNA damage sites with bulky adducts and sequentially recruited E3 ubiquitin ligase SHPRH, followed by deubiquitinase USP1, to DNA damage sites, in correlation with the dynamics of PCNA polyubiquitination. Similarly, TonEBP was found to be required for replication fork protection in response to DNA damage. The Rel-homology domain of TonEBP, which encircles DNA, was essential for the interaction with SHPRH and USP1, PCNA polyubiquitination, and cell survival after DNA damage. The present findings suggest that TonEBP is an upstream regulator of PCNA polyubiquitination and of the DNA damage bypass pathway.

A germline HLTF mutation in familial MDS induces DNA damage accumulation through impaired PCNA polyubiquitination

Although several causal genes of familial myelodysplastic syndromes (MDS) have been identified, the genetic landscape and the molecular pathogenesis are not totally understood. To explore novel driver genes and their pathogenetic significance, we performed whole-exome sequence analysis of four individuals from a familial MDS pedigree and 10 candidate single-nucleotide variants (C9orf43, CYP7B1, EFHB, ENTPD7, FAM160B2, HELZ2, HLTF, INPP5J, ITPKB, and RYK) were identified. Knockdown screening revealed that Hltf downregulation enhanced colony-forming capacity of primary murine bone marrow (BM) stem/progenitor cells. γH2AX immunofluorescent staining assay revealed increased DNA damage in a human acute myeloid leukemia (AML) cell line ectopically expressing HLTF E259K, which was not observed in cells expressing wild-type HLTF. Silencing of HLTF in human AML cells also led to DNA damage, indicating that HLTF E259K is a loss-of-function mutation. Molecularly, we found that an E259K mutation reduced the binding capacity of HLTF with ubiquitin-conjugating enzymes, methanesulfonate sensitive 2 and ubiquitin-conjugating enzyme E2N, resulting in impaired polyubiquitination of proliferating cell nuclear antigen (PCNA) in HLTF E259K-transduced cells. In summary, our results indicate that a familial MDS-associated HLTF E259K germline mutation induces accumulation of DNA double-strand breaks, possibly through impaired PCNA polyubiquitination.

Regulation of HLTF-mediated PCNA polyubiquitination by RFC and PCNA monoubiquitination levels determines choice of damage tolerance pathway.

DNA-damage tolerance protects cells via at least two sub-pathways regulated by proliferating cell nuclear antigen (PCNA) ubiquitination in eukaryotes: translesion DNA synthesis (TLS) and template switching (TS), which are stimulated by mono- and polyubiquitination, respectively. However, how cells choose between the two pathways remains unclear. The regulation of ubiquitin ligases catalyzing polyubiquitination, such as helicase-like transcription factor (HLTF), could play a role in the choice of pathway. Here, we demonstrate that the ligase activity of HLTF is stimulated by double-stranded DNA via HIRAN domain-dependent recruitment to stalled primer ends. Replication factor C (RFC) and PCNA located at primer ends, however, suppress en bloc polyubiquitination in the complex, redirecting toward sequential chain elongation. When PCNA in the complex is monoubiquitinated by RAD6-RAD18, the resulting ubiquitin moiety is immediately polyubiquitinated by coexisting HLTF, indicating a coupling reaction between mono- and polyubiquitination. By contrast, when PCNA was monoubiquitinated in the absence of HLTF, it was not polyubiquitinated by subsequently recruited HLTF unless all three-subunits of PCNA were monoubiquitinated, indicating that the uncoupling reaction specifically occurs on three-subunit-monoubiquitinated PCNA. We discuss the physiological relevance of the different modes of the polyubiquitination to the choice of cells between TLS and TS under different conditions.

Abstract B16: The nuclear IGF-1R regulates DNA damage tolerance through tyrosine phosphorylation of PCNA in human embryonic stem cells

Abstract Background: The type 1 insulin-like growth factor receptor (IGF-1R) and its signaling components promote cell proliferation, survival, and development of the malignant phenotype. Recently, the IGF-1R has been shown to translocate to the nucleus; however its nuclear functions are not fully elucidated. The proliferating cell nuclear antigen (PCNA) is a nuclear protein that coordinates DNA replication, and is involved in DNA damage repair and in the DNA damage tolerance (DDT) pathways. DDT allows the cell to replicate over polymerase-blocking lesions. DDT mechanisms include translesion DNA synthesis (TLS), and template switching, and the ubiquitination status of PCNA is crucial in these processes. Mono-ubiquitination of PCNA by Rad6 (E2)-Rad18 (E3) activates TLS. The mono-ubiquitin can be further extended though K63-linkages and the non degradative K63 polyubiquitination of PCNA by Ubc13 (E2)-SHPRH/HLTF (E3) leads to template switching. Purpose: The purpose of the present work was to study the functions of nIGF-1R in human embryonic stem cells (hESCs) and in the IGF-1R knockout (igf1r -/-) (R-) and the R- cells overexpressing human IGF1R (R+) mouse embryonic fibroblasts (MEFs), using a mass-spectrometry based approach. Methods and Results: Using subcellular protein fractionation (Qiagen Qproteome Cell Compartment Kit, Qiagen, Germany) and western blot (WB), as well as immunofluorescence, we detected the expression of the nIGF-1R in hESCs, human fibroblasts, MEFs, and in cancer cell lines. To investigate potential functions of nIGF-1R in hESCs, nIGF-1R binding partners were identified using co-immunoprecipitation (Co-IP) and mass spectrometry (MS) (hybrid LTQ-Orbitrap Velos mass spectrometer, Thermo Scientific). Multiple protein candidates were identified including PCNA. IGF-1R-PCNA association was confirmed by Co-IP/immunoblot and by in situ proximity ligation assay (PLA) (OLINK Bioscience, Uppsala, Sweden) to morphologically demonstrate the nuclear localization of the complex. The association of nIGF-1R with PCNA could only be detected in hESCs, MEFs and in human fibroblast cell lines, but not in any of the seven cancer cell lines used. Using a phospho-tyrosine antibody we detected the phosphorylation of PCNA immunoprecipitated from hESCs and R+ but not from R- MEFs or cancer cell lines. Using an in vitro kinase assay, recombinant IGF-1R kinase domain and His-tag recombinant PCNA, we showed that IGF-1R directly phosphorylates PCNA. Using LC-MS/MS and site directed mutagenesis, 3 tyrosine phosphorylation sites (Y60,133,250) were identified. Although PCNA ubiquitination was detected by WB of PCNA immunoprecipitated from R+ MEFs, PCNA protein stability was not affected. The ubiquitination pattern resembled the K63-linked polyubiquitination. Using Co-IP/WB, Ubc13 and SHPRH/HLTF were found to be associated with PCNA in R+ but not in R- cells. Similar results were also achieved for the mono-ubiquitin Rad18 E3 ligase and the main TLS polymerase DNA polymerase. Results were confirmed in hESCs but not in cancer cell lines. Further, PCNA ubiquitination was increased after DNA damage using UV irradiation in R+ MEFs and DNA replication was delayed after methyl methanesulfonate (MMS) treatment, while replication fork stalling in R- cells was highly significant (DNA-fiber labeling assay). Inhibition or knockout of IGF-1R abrogated PCNA phosphorylation, ubiquitination, progress through S-phase of cell cycle, or restart of DNA synthesis after inducing DNA damage. Conclusions: Our results demonstrate a novel role of nIGF-1R in regulating the DNA damage tolerance pathway through PCNA phosphorylation and ubiquitination, potentially maintaining genomic stability in in hESC and in normal fibroblasts. In contrast, nIGF-1R does not interact with PCNA in cancer cell lines. Citation Format: Ahmed Waraky, Yingbo Lin, Eiman Aleem, Olle Larsson. The nuclear IGF-1R regulates DNA damage tolerance through tyrosine phosphorylation of PCNA in human embryonic stem cells. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Cancer Cell Cycle - Tumor Progression and Therapeutic Response; Feb 28-Mar 2, 2016; Orlando, FL. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(11_Suppl):Abstract nr B16.

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.

PHD domain from human SHPRH

SHPRH (SNF2, histone linker, PHD, RING, helicase) is a SWI2/SNF2-family ATP-dependent chromatin remodeling factor, and one of E3 ubiquitin ligases responsible for Ubc13-Mms2-dependent K63 poly-ubiquitination of PCNA (proliferating cell nuclear antigen) that promotes error-free DNA damage tolerance in eukaryotes. In contrast to its functional homologues, S. cerevisiae Rad5 and human HLTF (helicase like transcription factor), SHPRH contains a PHD (plant homeodomain) finger embedded in the ‘minor’ insert region of the core helicase-like domain. PHD fingers are often found in proteins involved in chromatin remodeling and transcription regulation, and are generally considered as ‘readers’ of methylation state of histone tails, primarily the lysine 4 (K4) residue of histone H3 (H3K4). Here we report the solution NMR structure of the SHPRH PHD domain and investigate whether this domain is capable of recognizing H3K4 modifications. The domain adopts a canonical PHD-finger fold with a central two-stranded anti-parallel β-sheet flanked on both sides by the two interleaved zinc-binding sites. Despite the presence of a subset of aromatic residues characteristic for PHD-fingers that preferentially bind methylated H3K4, NMR titration experiments reveal that SHPRH PHD does not specifically interact with the H3-derived peptides irrespective of K4 methylation. This result suggests that the SHPRH PHD domain might have evolved a different function other than recognizing histone modifications.

Regulatory Role of Ubiquitin in Eukaryotic DNA Translesion Synthesis

Although often associated with proteasome-mediated protein degradation, ubiquitin plays essential nondegradative roles in a myriad of cellular processes, including chromatin dynamics, membrane trafficking, innate immunity, and DNA damage response. The recent progress in understanding DNA translesion synthesis (TLS), an important branch of DNA damage response, has largely been stimulated by the finding that ubiquitination of an essential nuclear protein, proliferating cell nuclear antigen (PCNA), controls precisely how eukaryotic cells respond to DNA damage. Despite the remarkable activity of the TLS polymerases in synthesizing past the damaged nucleotides, they are intrinsically error-prone on the normal DNA template. Therefore, a stringent regulation of the TLS polymerases is essential for the faithful replication of the DNA genome. Here we review the structure and function of the Y-family TLS polymerases and their interactions with ubiquitin and monoubiquitinated PCNA (Ub-PCNA). Driven by the need for monoubiquitinated PCNA in a sufficient quantity and purity, researchers developed both chemical and enzymatic methods for PCNA monoubiquitination, which have propelled our understanding of the structure of Ub-PCNA by X-ray crystallography and small-angle X-ray scattering. Together with studies using a reconstituted polymerase switching assay, these investigations revealed a surprising conformational flexibility of ubiquitin as a modifier on PCNA. Although the molecular details of TLS in cells still need to be deciphered, two working models, polymerase switching and postreplicative gap filling, have been proposed and tested in both in vitro and cellular systems. Evidence for both models is discussed herein. Compared to PCNA monoubiquitination, polyubiquitination of PCNA in DNA damage response is much less well understood and will be the subject of a future investigation. Given the close connection of DNA damage response and anticancer therapy, an in-depth understanding of the eukaryotic translesion synthesis and its regulation by ubiquitin will likely provide new opportunities for therapeutic intervention.

USP7 regulates the stability and function of HLTF through deubiquitination

Human helicase-like transcription factor (HLTF) is a functional homologue of yeast Rad5 that regulates error-free replication through DNA lesions. HLTF promotes the Lys-63-linked polyubiquitination of proliferating cell nuclear antigen (PCNA) that is required for maintaining genomic stability. Here, we identified the deubiquitylating enzyme ubiquitin-specific protease 7 (USP7) as a novel regulator of HLTF stability. We found that USP7 interacted with and stabilized HLTF after genotoxic stress. Furthermore, USP7 mediated deubiquitination significantly prolonged the half-life of HLTF, which in turn increased PCNA polyubiquitination. More intriguingly, silencing of USP7 rendered A549 cells highly sensitive to DNA damage and over-expression of HLTF attenuated this sensitivity. Thus, our results delineate a previously unknown USP7-HLTF-PCNA molecular network controlling DNA damage response.

HLTF and SHPRH are not essential for PCNA polyubiquitination, survival and somatic hypermutation: Existence of an alternative E3 ligase

DNA damage tolerance is regulated at least in part at the level of proliferating cell nuclear antigen (PCNA) ubiquitination. Monoubiquitination (PCNA-Ub) at lysine residue 164 (K164) stimulates error-prone translesion synthesis (TLS), Rad5-dependent polyubiquitination (PCNA-Ub n) stimulates error-free template switching (TS). To generate high affinity antibodies by somatic hypermutation (SHM), B cells profit from error-prone TLS polymerases. Consistent with the role of PCNA-Ub in stimulating TLS, hypermutated B cells of PCNA K164R mutant mice display a defect in generating selective point mutations. Two Rad5 orthologs, HLTF and SHPRH have been identified as alternative E3 ligases generating PCNA-Ub n in mammals. As PCNA-Ub and PCNA-Ub n both make use of K164, error-free PCNA-Ub n-dependent TS may suppress error-prone PCNA-Ub-dependent TLS. To determine a regulatory role of Shprh and Hltf in SHM, we generated Shprh/ Hltf double mutant mice. Interestingly, while the formation of PCNA-Ub and PCNA-Ub n is prohibited in PCNA K164R MEFs, the formation of PCNA-Ub n is not abolished in Shprh/Hltf mutant MEFs. In line with these observations Shprh/Hltf double mutant B cells were not hypersensitive to DNA damage. Furthermore, SHM was normal in Shprh/Hltf mutant B cells. These data suggest the existence of an alternative E3 ligase in the generation of PCNA-Ub n.

381 Functional studies of gene products and signal transduction mechanisms involved in cancer progression and metastasis – influence of extracellular S100A4

DNA damage tolerance pathways, which include translesion DNA synthesis (TLS) and template switching, are crucial for prevention of DNA replication arrest and maintenance of genomic stability. However, these pathways utilize error-prone DNA polymerases or template exchange between sister DNA strands, and consequently have the potential to induce mutations or chromosomal rearrangements.Post-translational modifications of proliferating cell nuclear antigen (PCNA) play important roles in controlling these pathways. For example, TLS is mediated by mono-ubiquitination of PCNA at lysine 164, for which RAD6–RAD18 is the primary E2–E3 complex. Elaborate protein–protein interactions between mono-ubiquitinated PCNA and Y-family DNA polymerases constitute the core of the TLS regulatory system, and enhancers of PCNA mono-ubiquitination and de-ubiquitinating enzymes finely regulate TLS and suppress TLS-mediated mutagenesis. The template switching pathway is promoted by K63-linked poly-ubiquitination of PCNA at lysine 164. Poly-ubiquitination is achieved by a coupled reaction mediated by two sets of E2–E3 complexes, RAD6–RAD18 and MMS2–UBC13–HTLF/SHPRH. In addition to these mono- and poly-ubiquitinations, simultaneous mono-ubiquitinations on multiple units of the PCNA homotrimeric ring promote an unidentified damage tolerance mechanism that remains to be fully characterized. Furthermore, SUMOylation of PCNA in mammalian cells can negatively regulate recombination. Other modifications, including ISGylation, acetylation, methylation, or phosphorylation, may also play roles in DNA damage tolerance and control of genomic stability.

Ubiquitin-dependent regulation of translesion polymerases

In response to DNA damage, TLS (translesion synthesis) allows replicative bypass of various DNA lesions, which stall normal replication. TLS is achieved by low-fidelity polymerases harbouring less stringent active sites. In humans, Y-family polymerases together with Pol zeta (polymerase zeta) are responsible for TLS across different types of damage. Protein-protein interaction contributes significantly to the regulation of TLS. REV1 plays a central role in TLS because it interacts with all other Y-family members and Pol zeta. Ubiquitin-dependent regulatory mechanisms also play important roles in TLS. Ubiquitin-binding domains have been found in TLS polymerases and they might be required for TLS activity. Mono-ubiquitination of PCNA (proliferating-cell nuclear antigen), the central scaffold of TLS polymerases, is thought to promote TLS. In addition, both non-proteolytic and proteolytic polyubiquitination of PCNA and TLS polymerases has been demonstrated. Owing to their low fidelity, the recruitment of TLS polymerases is strictly restricted to stalled replication forks.

Polyubiquitination of proliferating cell nuclear antigen by HLTF and SHPRH prevents genomic instability from stalled replication forks

Chronic stalling of DNA replication forks caused by DNA damage can lead to genomic instability. Cells have evolved lesion bypass pathways such as postreplication repair (PRR) to resolve these arrested forks. In yeast, one branch of PRR involves proliferating cell nuclear antigen (PCNA) polyubiquitination mediated by the Rad5-Ubc13-Mms2 complex that allows bypass of DNA lesion by a template-switching mechanism. Previously, we identified human SHPRH as a functional homologue of yeast Rad5 and revealed the existence of RAD5-like pathway in human cells. Here we report the identification of HLTF as a second RAD5 homologue in human cells. HLTF, like SHPRH, shares a unique domain architecture with Rad5 and promotes lysine 63-linked polyubiquitination of PCNA. Similar to yeast Rad5, HLTF is able to interact with UBC13 and PCNA, as well as SHPRH; and the reduction of either SHPRH or HLTF expression enhances spontaneous mutagenesis. Moreover, Hltf-deficient mouse embryonic fibroblasts show elevated chromosome breaks and fusions after methyl methane sulfonate treatment. Our results suggest that HLTF and SHPRH are functional homologues of yeast Rad5 that cooperatively mediate PCNA polyubiquitination and maintain genomic stability.

The RAD5-dependent Postreplication Repair Pathway is Important to Suppress Gross Chromosomal Rearrangements

Genome instability is characteristic of cancer cells. Although it frequently occurs during carcinogenesis, the mechanism underlying genome instability is not clearly understood. Recent extensive genetic analyses from different organisms have begun to reveal mechanisms for the suppression of genome instability in general DNA metabolisms including DNA replication, recombination, DNA repair, and signal transduction. One DNA repair pathway called postreplication repair (also known as DNA damage bypass) has been highlighted for its role in genome stability. Central to DNA damage bypass, proliferating cell nuclear antigen (PCNA) directs different pathways through its mono- or polyubiquitination and sumoylation. In this review, we will discuss template switching dictated by the PCNA polyubiquitination and its roles in the suppression of genome instabilities.

Human HLTF functions as a ubiquitin ligase for proliferating cell nuclear antigen polyubiquitination

Human helicase-like transcription factor (HLTF) is frequently inactivated in colorectal and gastric cancers. Here, we show that HLTF is a functional homologue of yeast Rad5 that promotes error-free replication through DNA lesions. HLTF and Rad5 share the same unique structural features, including a RING domain embedded within a SWI/SNF helicase domain and an HIRAN domain. We find that inactivation of HLTF renders human cells sensitive to UV and other DNA-damaging agents and that HLTF complements the UV sensitivity of a rad5Delta yeast strain. Also, similar to Rad5, HLTF physically interacts with the Rad6-Rad18 and Mms2-Ubc13 ubiquitin-conjugating enzyme complexes and promotes the Lys-63-linked polyubiquitination of proliferating cell nuclear antigen at its Lys-164 residue. A requirement of HLTF for error-free postreplication repair of damaged DNA is in keeping with its cancer-suppression role.

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.

Conservation of DNA damage tolerance pathways from yeast to humans

Damage tolerance mechanisms, which allow the bypass of DNA lesions during replication, are controlled in eukaryotic cells by mono- and poly-ubiquitination of the DNA polymerase cofactor PCNA (proliferating-cell nuclear antigen). In the present review, I will summarize our current knowledge of the enzymatic machinery for ubiquitination of PCNA and the way in which the modifications affect PCNA function during replication and lesion bypass in different organisms. Using the budding yeast as a reference model, I will highlight some of the species-specific differences, but also point out the common principles that emerge from the genetic and biochemical studies of damage tolerance in a range of experimental systems.