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1.
Trans-lesion DNA synthesis (TLS) is a DNA damage-tolerance mechanism that uses low-fidelity DNA polymerases to replicate damaged DNA. The inherited cancer-propensity syndrome xeroderma pigmentosum variant (XPV) results from error-prone TLS of UV-damaged DNA. TLS is initiated when the Rad6/Rad18 complex monoubiquitinates proliferating cell nuclear antigen (PCNA), but the basis for recruitment of Rad18 to PCNA is not completely understood. Here, we show that Rad18 is targeted to PCNA by DNA polymerase eta (Polη), the XPV gene product that is mutated in XPV patients. The C-terminal domain of Polη binds to both Rad18 and PCNA and promotes PCNA monoubiquitination, a function unique to Polη among Y-family TLS polymerases and dissociable from its catalytic activity. Importantly, XPV cells expressing full-length catalytically-inactive Polη exhibit increased recruitment of other error-prone TLS polymerases (Polκ and Polι) after UV irradiation. These results define a novel non-catalytic role for Polη in promoting PCNA monoubiquitination and provide a new potential mechanism for mutagenesis and genome instability in XPV individuals. 相似文献
2.
c-Jun N-terminal kinase-mediated Rad18 phosphorylation facilitates Polη recruitment to stalled replication forks 总被引:1,自引:0,他引:1
Barkley LR Palle K Durando M Day TA Gurkar A Kakusho N Li J Masai H Vaziri C 《Molecular biology of the cell》2012,23(10):1943-1954
The E3 ubiquitin ligase Rad18 chaperones DNA polymerase η (Polη) to sites of UV-induced DNA damage and monoubiquitinates proliferating cell nuclear antigen (PCNA), facilitating engagement of Polη with stalled replication forks and promoting translesion synthesis (TLS). It is unclear how Rad18 activities are coordinated with other elements of the DNA damage response. We show here that Ser-409 residing in the Polη-binding motif of Rad18 is phosphorylated in a checkpoint kinase 1-dependent manner in genotoxin-treated cells. Recombinant Rad18 was phosphorylated specifically at S409 by c-Jun N-terminal kinase (JNK) in vitro. In UV-treated cells, Rad18 S409 phosphorylation was inhibited by a pharmacological JNK inhibitor. Conversely, ectopic expression of JNK and its upstream kinase mitogen-activated protein kinase kinase 4 led to DNA damage-independent Rad18 S409 phosphorylation. These results identify Rad18 as a novel JNK substrate. A Rad18 mutant harboring a Ser → Ala substitution at S409 was compromised for Polη association and did not redistribute Polη to nuclear foci or promote Polη-PCNA interaction efficiently relative to wild-type Rad18. Rad18 S409A also failed to fully complement the UV sensitivity of Rad18-depleted cells. Taken together, these results show that Rad18 phosphorylation by JNK represents a novel mechanism for promoting TLS and DNA damage tolerance. 相似文献
3.
PCNA is monoubiquitinated in response to DNA damage and fork stalling and then initiates recruitment of specialized polymerases in the DNA damage tolerance pathway, translesion synthesis (TLS). Since PCNA is reported to associate with Epstein-Barr virus (EBV) DNA during its replication, we investigated whether the EBV deubiquitinating (DUB) enzyme encoded by BPLF1 targets ubiquitinated PCNA and disrupts TLS. An N-terminal BPLF1 fragment (a BPLF1 construct containing the first 246 amino acids [BPLF1 1-246]) associated with PCNA and attenuated its ubiquitination in response to fork-stalling agents UV and hydroxyurea in cultured cells. Moreover, monoubiquitinated PCNA was deubiquitinated after incubation with purified BPLF1 1-246 in vitro. BPLF1 1-246 dysregulated TLS by reducing recruitment of the specialized repair polymerase polymerase η (Polη) to the detergent-resistant chromatin compartment and virtually abolished localization of Polη to nuclear repair foci, both hallmarks of TLS. Expression of BPLF1 1-246 decreased viability of UV-treated cells and led to cell death, presumably through deubiquitination of PCNA and the inability to repair damaged DNA. Importantly, deubiquitination of PCNA could be detected endogenously in EBV-infected cells in comparison with samples expressing short hairpin RNA (shRNA) against BPLF1. Further, the specificity of the interaction between BPLF1 and PCNA was dependent upon a PCNA-interacting peptide (PIP) domain within the N-terminal region of BPLF1. Both DUB activity and PIP sequence are conserved in the members of the family Herpesviridae. Thus, deubiquitination of PCNA, normally deubiquitinated by cellular USP1, by the viral DUB can disrupt repair of DNA damage by compromising recruitment of TLS polymerase to stalled replication forks. PCNA is the first cellular target identified for BPLF1 and its deubiquitinating activity. 相似文献
4.
Human Rev1 is a translesion synthesis (TLS) DNA polymerase involved in bypass replication across sites of DNA damage and postreplicational gap-filling. Rev1 plays an essential structural role in TLS by providing a binding platform for other TLS polymerases that insert nucleotides across DNA lesions (polη, polι, polκ) and extend the distorted primer-terminus (pol?). We use NMR spectroscopy to demonstrate that the Rev1 C-terminal domain utilizes independent interaction interfaces to simultaneously bind a fragment of the ’inserter’ polη and Rev7 subunit of the ’extender’ pol?, thereby serving as a cassette that may accommodate several polymerases making them instantaneously available for TLS.
Structured summary of protein interactions
REV1, REV3 and REV7physically interact by nuclear magnetic resonance (View interaction), molecular sieving (View interaction) and isothermal titration calorimetry (View interaction).REV3 and REV7bind by molecular sieving (View interaction)REV1 and Polη-RIR peptidebind by nuclear magnetic resonance (View interaction)REV1, REV3, REV7 and Polη-RIR peptidephysically interact by nuclear magnetic resonance (View interaction). 相似文献5.
Translesion synthesis polymerases (TLS Pols) are required to tolerate DNA lesions that would otherwise cause replication arrest and cell death. Aberrant expression of these specialized Pols may be responsible for increased mutagenesis and loss of genome integrity in human cancers. The molecular events that control the usage of TLS Pols in non-pathological conditions remain largely unknown. Here, we show that aberrant recruitment of TLS Polκ to replication forks results in genomic instability and can be mediated through the loss of the deubiquitinase USP1. Moreover, artificial tethering of Polκ to proliferating cell nuclear antigen (PCNA) circumvents the need for its ubiquitin-binding domain in the promotion of genomic instability. Finally, we show that the loss of USP1 leads to a dramatic reduction of replication fork speed in a Polκ-dependent manner. We propose a mechanism whereby reversible ubiquitination of PCNA can prevent spurious TLS Pol recruitment and regulate replication fork speed to ensure the maintenance of genome integrity. 相似文献
6.
The balance between exonuclease and polymerase activities promotes DNA synthesis over degradation when nucleotides are correctly added to the new strand by replicative B-family polymerases. Misincorporations shift the balance toward the exonuclease site, and the balance tips back in favor of DNA synthesis when the incorrect nucleotides have been removed. Most B-family DNA polymerases have an extended β-hairpin loop that appears to be important for switching from the exonuclease site to the polymerase site, a process that affects fidelity of the DNA polymerase. Here, we show that DNA polymerase ε can switch between the polymerase site and exonuclease site in a processive manner despite the absence of an extended β-hairpin loop. K967 and R988 are two conserved amino acids in the palm and thumb domain that interact with bases on the primer strand in the minor groove at positions n−2 and n−4/n−5, respectively. DNA polymerase ε depends on both K967 and R988 to stabilize the 3′-terminus of the DNA within the polymerase site and on R988 to processively switch between the exonuclease and polymerase sites. Based on a structural alignment with DNA polymerase δ, we propose that arginines corresponding to R988 might have a similar function in other B-family polymerases. 相似文献
7.
8.
Akari Yoshimura Yume Kobayashi Shusuke Tada Masayuki Seki Takemi Enomoto 《Biochemical and biophysical research communications》2014
WRNIP1 (WRN-interacting protein 1) was first identified as a factor that interacts with WRN, the protein that is defective in Werner syndrome (WS). WRNIP1 associates with DNA polymerase η (Polη), but the biological significance of this interaction remains unknown. In this study, we analyzed the functional interaction between WRNIP1 and Polη by generating knockouts of both genes in DT40 chicken cells. Disruption of WRNIP1 in Polη-disrupted (POLH−/−) cells suppressed the phenotypes associated with the loss of Polη: sensitivity to ultraviolet light (UV), delayed repair of cyclobutane pyrimidine dimers (CPD), elevated frequency of mutation, elevated levels of UV-induced sister chromatid exchange (SCE), and reduced rate of fork progression after UV irradiation. These results suggest that WRNIP1 functions upstream of Polη in the response to UV irradiation. 相似文献
9.
《Biochimica et Biophysica Acta (BBA) - Nucleic Acids and Protein Synthesis》1981,652(2):324-333
The amount of DNA synthesis in vitro with the ultraviolet-irradiated poly-(dT) · oligo(rA) template initiators catalysed by DNA polymerase α (Masaki, S. and Yoshida, S., Biochim. Biophys. Acta 521, 74–88) decreased with the dose of ultraviolet-irradiation. The ultraviolet irradiation to the template, however, did not affect the rate of incorporation of incorrect deoxynycleotides into the newly synthesized poly(dA). The addition of terminal deoxynucleotidyl transferase to this system enhanced the DNA synthesis to a level which is comparable to that of the control and it concomitantly increased the incorporation of the mismatched deoxynucleotide into the newly synthesized poly(dA) strands. On the other hand, with an unirradiated template initiator, the misincorporation was only slightly enhanced by the addition of terminal deoxynucleotidyl transferase. The sizes of newly synthesized DNA measured by the sedimentation velocities were found to be smaller with the ultraviolet-irradiated templates but they increased to the control level with the addition of terminal deoxynucleotidyl transferase to the systems. These results suggest that terminal deoxynucleotidyl transferase can help DNA polymerase α to ‘bypass’ thymine dimers in vitro by the formation of mismatched regions at the positions opposite to pyrimidine dimers on the template. 相似文献
10.
Translesion synthesis (TLS), the process by which DNA polymerases replicate through DNA lesions, is the source of most DNA damage-induced mutations. Sometimes TLS is carried out by replicative polymerases that have evolved to synthesize DNA on non-damaged templates. Most of the time, however, TLS is carried out by specialized translesion polymerases that have evolved to synthesize DNA on damaged templates. TLS requires the mono-ubiquitylation of the replication accessory factor proliferating cell nuclear antigen (PCNA). PCNA and ubiquitin-modified PCNA (UbPCNA) stimulate TLS by replicative and translesion polymerases. Two mutant forms of PCNA, one with an E113G substitution and one with a G178S substitution, support normal cell growth but inhibit TLS thereby reducing mutagenesis in yeast. A re-examination of the structures of both mutant PCNA proteins revealed substantial disruptions of the subunit interface that forms the PCNA trimer. Both mutant proteins have reduced trimer stability with the G178S substitution causing a more severe defect. The mutant forms of PCNA and UbPCNA do not stimulate TLS of an abasic site by either replicative Pol δ or translesion Pol η. Normal replication by Pol η was also impacted, but normal replication by Pol δ was much less affected. These findings support a model in which reduced trimer stability causes these mutant PCNA proteins to occasionally undergo conformational changes that compromise their ability to stimulate TLS by both replicative and translesion polymerases. 相似文献
11.
Silverstein TD Jain R Johnson RE Prakash L Prakash S Aggarwal AK 《Structure (London, England : 1993)》2010,18(11):1463-1470
7,8-dihydro-8-oxoguanine (8-oxoG) adducts are formed frequently by the attack of oxygen-free radicals on DNA. They are among the most mutagenic lesions in cells because of their dual coding potential, where, in addition to normal base-pairing of 8-oxoG(anti) with dCTP, 8-oxoG in the syn conformation can base pair with dATP, causing G to T transversions. We provide here for the first time a structural basis for the error-free replication of 8-oxoG lesions by yeast DNA polymerase η (Polη). We show that the open active site cleft of Polη can accommodate an 8-oxoG lesion in the anti conformation with only minimal changes to the polymerase and the bound DNA: at both the insertion and post-insertion steps of lesion bypass. Importantly, the active site geometry remains the same as in the undamaged complex and provides a basis for the ability of Polη to prevent the mutagenic replication of 8-oxoG lesions in cells. 相似文献
12.
When cells are exposed to radiation serious lesions are introduced into the DNA including double strand breaks (DSBs), single strand breaks (SSBs), base modifications and clustered damage sites (a specific feature of ionizing radiation induced DNA damage). Radiation induced DNA damage has the potential to initiate events that can lead ultimately to mutations and the onset of cancer and therefore understanding the cellular responses to DNA lesions is of particular importance. Using γH2AX as a marker for DSB formation and RAD51 as a marker of homologous recombination (HR) which is recruited in the processing of frank DSBs or DSBs arising from stalled replication forks, we have investigated the contribution of SSBs and non-DSB DNA damage to the induction of DSBs in mammalian cells by ionizing radiation during the cell cycle. V79-4 cells and human HF19 fibroblast cells have been either irradiated with 0–20 Gy of γ radiation or, for comparison, treated with a low concentration of hydrogen peroxide, which is known to induce SSBs but not DSBs. Inhibition of the repair of oxidative DNA lesions by poly(ADP ribose) polymerase (PARP) inhibitor leads to an increase in radiation induced γH2AX and RAD51 foci which we propose is due to these lesions colliding with replication forks forming replication induced DSBs. It was confirmed that DSBs are not induced in G1 phase cells by treatment with hydrogen peroxide but treatment does lead to DSB induction, specifically in S phase cells. We therefore suggest that radiation induced SSBs and non-DSB DNA damage contribute to the formation of replication induced DSBs, detected as RAD51 foci. 相似文献
13.
Krijger PH van den Berk PC Wit N Langerak P Jansen JG Reynaud CA de Wind N Jacobs H 《DNA Repair》2011,10(10):1051-1059
The generation of high affinity antibodies in B cells critically depends on translesion synthesis (TLS) polymerases that introduce mutations into immunoglobulin genes during somatic hypermutation (SHM). The majority of mutations at A/T base pairs during SHM require ubiquitination of PCNA at lysine 164 (PCNA-Ub), which activates TLS polymerases. By comparing the mutation spectra in B cells of WT, TLS polymerase η (Polη)-deficient, PCNA(K164R)-mutant, and PCNA(K164R);Polη double-mutant mice, we now find that most PCNA-Ub-independent A/T mutagenesis during SHM is mediated by Polη. In addition, upon exposure to various DNA damaging agents, PCNA(K164R) mutant cells display strongly impaired recruitment of TLS polymerases, reduced daughter strand maturation and hypersensitivity. Interestingly, compared to the single mutants, PCNA(K164R);Polη double-mutant cells are dramatically delayed in S phase progression and far more prone to cell death following UV exposure. Taken together, these data support the existence of PCNA ubiquitination-dependent and -independent activation pathways of Polη during SHM and DNA damage tolerance. 相似文献
14.
Igor B. Rogozin Alexander Goncearenco Artem G. Lada Subhajyoti De Vyacheslav Yurchenko German Nudelman 《Cell cycle (Georgetown, Tex.)》2018,17(3):348-355
DNA polymerase (pol) η is a specialized error-prone polymerase with at least two quite different and contrasting cellular roles: to mitigate the genetic consequences of solar UV irradiation, and promote somatic hypermutation in the variable regions of immunoglobulin genes. Misregulation and mistargeting of pol η can compromise genome integrity. We explored whether the mutational signature of pol η could be found in datasets of human somatic mutations derived from normal and cancer cells. A substantial excess of single and tandem somatic mutations within known pol η mutable motifs was noted in skin cancer as well as in many other types of human cancer, suggesting that somatic mutations in A:T bases generated by DNA polymerase η are a common feature of tumorigenesis. Another peculiarity of pol ηmutational signatures, mutations in YCG motifs, led us to speculate that error-prone DNA synthesis opposite methylated CpG dinucleotides by misregulated pol η in tumors might constitute an additional mechanism of cytosine demethylation in this hypermutable dinucleotide. 相似文献
15.
Aguilera A 《Trends in genetics : TIG》2001,17(6):318-321
The central step of homologous recombination is the DNA strand exchange reaction catalyzed by bacterial RecA or eukaryotic Rad51. Besides Rad51-mediated synthesis-dependent strand annealing (SDSA), DNA ends can promote replication in Escherichia coli (recombination-dependent replication, RDR) and yeast (break-induced replication, BIR). However, what causes a DNA end to be repaired via SDSA or via BIR/RDR? I propose that Rad51/RecA--DNA plectonemic joints act as barriers to DNA replication and that BIR/RDR is only possible when the DNA polymerase that synthesizes DNA from the invading 3' end does not encounter RecA/Rad51--DNA joints in its path. 相似文献
16.
Humans are exposed to N-nitroso compounds (NOCs) both endogenously and exogenously from a number of environmental sources, and NOCs are both mutagenic and carcinogenic. After metabolic activation, some NOCs can induce carboxymethylation of nucleobases through a diazoacetate intermediate, which could give rise to p53 mutations similar to those seen in human gastrointestinal cancers. It was previously found that the growth of polymerase η-deficient human cells was inhibited by treatment with azaserine, a DNA carboxymethylation agent, suggesting the importance of this polymerase in bypassing the azaserine-induced carboxymethylated DNA lesions. In this study, we examined how carboxymethylated DNA lesions, which included N(6)-carboxymethyl-2'-deoxyadenosine (N(6)-CMdA), N(4)-carboxymethyl-2'-deoxycytidine (N(4)-CMdC), N3-carboxymethylthymidine (N3-CMdT), and O(4)-carboxymethylthymidine (O(4)-CMdT), perturbed the efficiency and fidelity of DNA replication mediated by Saccharomyces cerevisiae polymerase η (pol η). Our results from steady-state kinetic assay showed that pol η could readily bypass and extend past N(6)-CMdA and incorporated the correct nucleotides opposite the lesion and its neighboring 5'-nucleoside with high efficiency. By contrast, the polymerase could bypass N(4)-CMdC inefficiently, with substantial misincorporation of dCMP followed by dAMP, though pol η could extend past the lesion with high fidelity and efficiency when dGMP was incorporated opposite the lesion. On the other hand, yeast pol η experienced great difficulty in bypassing O(4)-CMdT and N3-CMdT, and the polymerase inserted preferentially the incorrect dGMP opposite these two DNA lesions; the extension step, nevertheless, occurred with high fidelity and efficiency when the correct dAMP was opposite the lesion, as opposed to the preferentially incorporated incorrect dGMP. These results suggest that these lesions may contribute significantly to diazoacetate-induced mutations and those in the p53 gene observed in human gastrointestinal tumors. 相似文献
17.
DNA polymerase η (polη) belongs to the Y-family of DNA polymerases and facilitates translesion synthesis past UV damage. We show that, after UV irradiation, polη becomes phosphorylated at Ser601 by the ataxia-telangiectasia mutated and Rad3-related (ATR) kinase. DNA damage-induced phosphorylation of polη depends on its physical interaction with Rad18 but is independent of PCNA monoubiquitination. It requires the ubiquitin-binding domain of polη but not its PCNA-interacting motif. ATR-dependent phosphorylation of polη is necessary to restore normal survival and postreplication repair after ultraviolet irradiation in xeroderma pigmentosum variant fibroblasts, and is involved in the checkpoint response to UV damage. Taken together, our results provide evidence for a link between DNA damage-induced checkpoint activation and translesion synthesis in mammalian cells. 相似文献
18.
Ummat A Silverstein TD Jain R Buku A Johnson RE Prakash L Prakash S Aggarwal AK 《Journal of molecular biology》2012,415(4):627-634
Pre-steady-state kinetic studies on Y-family DNA polymerase η (Polη) have suggested that the polymerase undergoes a rate-limiting conformational change step before the phosphoryl transfer of the incoming nucleotide to the primer terminus. However, the nature of this rate-limiting conformational change step has been unclear, due in part to the lack of structural information on the Polη binary complex. We present here for the first time a crystal structure of human Polη (hPolη) in binary complex with its DNA substrate. We show that the hPolη domains move only slightly on dNTP binding and that the polymerase by and large is pre-aligned for dNTP binding and catalysis. We also show that there is no major reorientation of the DNA from a nonproductive to a productive configuration and that the active site is devoid of metals in the absence of dNTP. Together, these observations lead us to suggest that the rate-limiting conformational change step in the Polη replication cycle likely corresponds to a rate-limiting entry of catalytic metals in the active site. 相似文献
19.
Rie Kanao Fumio Hanaoka Chikahide Masutani 《Biochemical and biophysical research communications》2009,389(1):40-1141
Human DNA polymerase η (Polη) is the gene product underlying xeroderma pigmentosum variant, and plays principal roles in translesion DNA synthesis. Here, we identified human MLH1, an essential component of mismatch repair (MMR), as a Polη-interacting protein. The middle area residues, which include the little finger domain, of Polη are important for the interaction with MLH1. Polη also interacts with the MLH1/PMS2 heterodimer (MutLα). Co-immunoprecipitation analyses revealed that MutLα, and also MSH2 and MSH6, components of the MutSα heterodimer, form complexes with Polη in human cells. Although MutSα had been reported to interact with C-terminal residues of Polη, MutLα and MutSα co-precipitated with C-terminally truncated Polη, suggesting that MutSα can interact with Polη through MutLα. MMR proteins were more abundant in the Polη complex on the chromatin of S phase-synchronized cells than of asynchronous cells, suggesting that the interaction between Polη and MLH1 is involved in DNA replication. 相似文献
20.
TopBP1 and the Rad9–Rad1–Hus1 (9-1-1) complex activate the ataxia telangiectasia mutated and Rad3-related (ATR) protein kinase at stalled replication forks. ATR is recruited to stalled forks through its binding partner, ATR-interacting protein (ATRIP); however, it is unclear how TopBP1 and 9-1-1 are recruited so that they may join ATR–ATRIP and initiate signaling. In this study, we use Xenopus laevis egg extracts to determine the requirements for 9-1-1 loading. We show that TopBP1 is required for the recruitment of both 9-1-1 and DNA polymerase (pol)-α to sites of replication stress. Furthermore, we show that pol-α is also directly required for Rad9 loading. Our study identifies an assembly pathway, which is controlled by TopBP1 and includes pol-α, that mediates the loading of the 9-1-1 complex onto stalled replication forks. These findings clarify early events in the assembly of checkpoint signaling complexes on DNA and identify TopBP1 as a critical sensor of replication stress. 相似文献