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1.
Mouse Rev1 protein interacts with multiple DNA polymerases involved in translesion DNA synthesis 总被引:9,自引:0,他引:9
Guo C Fischhaber PL Luk-Paszyc MJ Masuda Y Zhou J Kamiya K Kisker C Friedberg EC 《The EMBO journal》2003,22(24):6621-6630
Pol kappa and Rev1 are members of the Y family of DNA polymerases involved in tolerance to DNA damage by replicative bypass [translesion DNA synthesis (TLS)]. We demonstrate that mouse Rev1 protein physically associates with Pol kappa. We show too that Rev1 interacts independently with Rev7 (a subunit of a TLS polymerase, Pol zeta) and with two other Y-family polymerases, Pol iota and Pol eta. Mouse Pol kappa, Rev7, Pol iota and Pol eta each bind to the same approximately 100 amino acid C-terminal region of Rev1. Furthermore, Rev7 competes directly with Pol kappa for binding to the Rev1 C-terminus. Notwithstanding the physical interaction between Rev1 and Pol kappa, the DNA polymerase activity of each measured by primer extension in vitro is unaffected by the complex, either when extending normal primer-termini, when bypassing a single thymine glycol lesion, or when extending certain mismatched primer termini. Our observations suggest that Rev1 plays a role(s) in mediating protein-protein interactions among DNA polymerases required for TLS. The precise function(s) of these interactions during TLS remains to be determined. 相似文献
2.
Rev1p in yeast is essential for the translesion of abasic sites and 6-4 photoproducts. It plays a role as a translesion polymerase, but also supports translesion catalyzed by other polymerases. The protein has two domains, BRCT and Y-family polymerase. A point mutation in the BRCT domain is known to abolish the second function. In the present research, we have studied the effects of deletion of the BRCT domain and a point mutation at the two amino acids in the putative polymerase active center. We have introduced an abasic site, its tetrahydrofuran analog, and a 6-4 thymine-thymine photoproduct using the oligonucleotide transformation assay. Translesion efficiencies were estimated from the transforming activities of the oligonucleotides with a lesion, and the mutation spectra were analyzed by DNA sequencing of the transformants. Results showed that the lack of the BRCT domain reduced translesion efficiencies, but that substantial translesion synthesis took place. The mutation spectra of the lesions were not greatly affected. Therefore, the BRCT domain may be important, but dispensable for translesion synthesis. In contrast, the polymerase mutation, rev1AA, has only small effects on the translesion efficiencies, but the mutation spectra were greatly affected; the incorporation of dCMP opposite the lesions was specifically lost. This clearly shows that the polymerase domain is responsible for the dCMP incorporation. The effect of Poleta was also analyzed. From all the results DNA polymerases other than these two translesion polymerases, too, seem to initiate the translesion synthesis. 相似文献
3.
Rev1 is a eukaryotic DNA polymerase of the Y family involved in translesion synthesis (TLS), a major damage tolerance pathway that allows DNA replication at damaged templates. Uniquely amongst the Y family polymerases, the N-terminal part of Rev1, dubbed the BRCA1 C-terminal homology (BRCT) region, includes a BRCT domain. While most BRCT domains mediate protein-protein interactions, Rev1 contains a predicted α-helix N-terminal to the BRCT domain and in human Replication Factor C (RFC) such a BRCT region endows the protein with DNA binding capacity. Here, we studied the DNA binding properties of yeast and mouse Rev1. Our results show that the BRCT region of Rev1 specifically binds to a 5' phosphorylated, recessed, primer-template junction. This DNA binding depends on the extra α-helix, N-terminal to the BRCT domain. Surprisingly, a stretch of 20 amino acids N-terminal to the predicted α-helix is also critical for high-affinity DNA binding. In addition to 5' primer-template junction binding, Rev1 efficiently binds to a recessed 3' primer-template junction. These dual DNA binding characteristics are discussed in view of the proposed recruitment of Rev1 by 5' primer-template junctions, downstream of stalled replication forks. 相似文献
4.
《Cell cycle (Georgetown, Tex.)》2013,12(4):729-735
The encounter of replication forks with DNA lesions may lead to fork arrest and/or the formation of single-stranded gaps. A major strategy to cope with these replication irregularities is translesion DNA replication (TLS), in which specialized error-prone DNA polymerases bypass the blocking lesions. Recent studies suggest that TLS across a particular DNA lesion may involve as many as four different TLS polymerases, acting in two-polymerase reactions in which insertion by a particular polymerase is followed by extension by another polymerase. Insertion determines the accuracy and mutagenic specificity of the TLS reaction, and is carried out by one of several polymerases such as polη, polκ or polι. In contrast, extension is carried out primarily by polζ. In cells from XPV patients, which are deficient in TLS across cyclobutane pyrimidine dimers (CPD) due to a deficiency in polη, TLS is carried out by at least two backup reactions each involving two polymerases: One reaction involves polκ and polζ, and the other polι and polζ. These mechanisms may also assist polη in normal cells under an excessive amount of UV lesions. 相似文献
5.
The BRCT domain of mammalian Pes1 is crucial for nucleolar localization and rRNA processing 总被引:1,自引:0,他引:1
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Hölzel M Grimm T Rohrmoser M Malamoussi A Harasim T Gruber-Eber A Kremmer E Eick D 《Nucleic acids research》2007,35(3):789-800
The nucleolar protein Pes1 interacts with Bop1 and WDR12 in a stable complex (PeBoW-complex) and its expression is tightly associated with cell proliferation. The yeast homologue Nop7p (Yph1p) functions in both, rRNA processing and cell cycle progression. The presence of a BRCT-domain (BRCA1 C-terminal) within Pes1 is quite unique for an rRNA processing factor, as this domain is normally found in factors involved in DNA-damage or repair pathways. Thus, the function of the BRCT-domain in Pes1 remains elusive. We established a conditional siRNA-based knock-down-knock-in system and analysed a panel of Pes1 truncation mutants for their functionality in ribosome synthesis in the absence of endogenous Pes1. Deletion of the BRCT-domain or single point mutations of highly conserved residues caused diffuse nucleoplasmic distribution and failure to replace endogenous Pes1 in rRNA processing. Further, the BRCT-mutants of Pes1 were less stable and not incorporated into the PeBoW-complex. Hence, the integrity of the BRCT-domain of Pes1 is crucial for nucleolar localization and its function in rRNA processing. 相似文献
6.
The DNA single-strand break repair (SSBR) protein XRCC1 is required for genetic stability and for embryonic viability. XRCC1 possesses two BRCA1 carboxyl-terminal (BRCT) protein interaction domains, denoted BRCT I and II. BRCT II is required for SSBR during G(1) but is dispensable for this process during S/G(2) and consequently for cell survival following DNA alkylation. Little is known about BRCT I, but this domain has attracted considerable interest because it is the site of a genetic polymorphism that epidemiological studies have associated with altered cancer risk. We report that the BRCT I domain comprises the evolutionarily conserved core of XRCC1 and that this domain is required for efficient SSBR during both G(1) and S/G(2) cell cycle phases and for cell survival following treatment with methyl methanesulfonate. However, the naturally occurring human polymorphism in BRCT I supported XRCC1-dependent SSBR and cell survival after DNA alkylation equally well. We conclude that while the BRCT I domain is critical for XRCC1 to maintain genetic integrity and cell survival, the polymorphism does not impact significantly on this function and therefore is unlikely to impact significantly on susceptibility to cancer. 相似文献
7.
Translesion synthesis (TLS) in Saccharomyces cerevisiae requires at least Rev1p and polymerase zeta (Pol zeta), a complex of the Rev3 polymerase and its accessory factor Rev7p. Although their precise role(s) are poorly characterized, in vitro studies suggest that each protein contributes to TLS in a manner dependent on the particular lesion and surrounding DNA sequence. In the present study, strand segregation analysis is used to attempt to identify the role(s) of the Rev1 and Rev7 proteins during TLS. This assay uses double-stranded plasmids containing a genetic marker opposite to a replication blocking lesion (N-2-acetylaminofluorene; AAF) to measure TLS quantitatively and qualitatively in vivo. The AAF adduct is localized within a repetitive sequence in a manner that allows the formation of misaligned primer-template replication intermediates. Elongation from a misaligned intermediate fixes a frameshift mutation (slipped TLS), while extension of the correctly aligned lesion terminus yields error-free (non-slipped) TLS. The results indicate that there is a strong requirement for Rev7p during Pol zeta-mediated TLS measured in vivo. Furthermore, Rev1p is needed only for non-slipped TLS; slipped TLS remains efficient in its absence, revealing a previously uncharacterized Rev1p activity similar to Escherichia coli UmuDC function. Specifically, this activity is required for elongation from a correctly aligned lesion terminus. 相似文献
8.
PCNA ubiquitination is important, but not essential for translesion DNA synthesis in mammalian cells
Hendel A Krijger PH Diamant N Goren Z Langerak P Kim J Reissner T Lee KY Geacintov NE Carell T Myung K Tateishi S D'Andrea A Jacobs H Livneh Z 《PLoS genetics》2011,7(9):e1002262
Translesion DNA synthesis (TLS) is a DNA damage tolerance mechanism in which specialized low-fidelity DNA polymerases bypass replication-blocking lesions, and it is usually associated with mutagenesis. In Saccharomyces cerevisiae a key event in TLS is the monoubiquitination of PCNA, which enables recruitment of the specialized polymerases to the damaged site through their ubiquitin-binding domain. In mammals, however, there is a debate on the requirement for ubiquitinated PCNA (PCNA-Ub) in TLS. We show that UV-induced Rpa foci, indicative of single-stranded DNA (ssDNA) regions caused by UV, accumulate faster and disappear more slowly in Pcna(K164R/K164R) cells, which are resistant to PCNA ubiquitination, compared to Pcna(+/+) cells, consistent with a TLS defect. Direct analysis of TLS in these cells, using gapped plasmids with site-specific lesions, showed that TLS is strongly reduced across UV lesions and the cisplatin-induced intrastrand GG crosslink. A similar effect was obtained in cells lacking Rad18, the E3 ubiquitin ligase which monoubiquitinates PCNA. Consistently, cells lacking Usp1, the enzyme that de-ubiquitinates PCNA exhibited increased TLS across a UV lesion and the cisplatin adduct. In contrast, cells lacking the Rad5-homologs Shprh and Hltf, which polyubiquitinate PCNA, exhibited normal TLS. Knocking down the expression of the TLS genes Rev3L, PolH, or Rev1 in Pcna(K164R/K164R) mouse embryo fibroblasts caused each an increased sensitivity to UV radiation, indicating the existence of TLS pathways that are independent of PCNA-Ub. Taken together these results indicate that PCNA-Ub is required for maximal TLS. However, TLS polymerases can be recruited to damaged DNA also in the absence of PCNA-Ub, and perform TLS, albeit at a significantly lower efficiency and altered mutagenic specificity. 相似文献
9.
Irene Gallina Ivo A. Hendriks Saskia Hoffmann Nicolai B. Larsen Joachim Johansen Camilla S. Colding-Christensen Lisa Schubert Selene Sellés-Baiget Zita Fábián Ulrike Kühbacher Alan O. Gao Markus Räschle Simon Rasmussen Michael L. Nielsen Niels Mailand Julien P. Duxin 《Molecular cell》2021,81(3):442-458.e9
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10.
Murakumo Y 《Mutation research》2002,510(1-2):37-44
Translesion DNA synthesis (TLS) is an important damage tolerance system which rescues cells from severe injuries caused by DNA damage. Specialized low fidelity DNA polymerases in this system synthesize DNA past lesions on the template DNA strand, that replicative DNA polymerases are usually unable to pass through. However, in compensation for cell survival, most polymerases in this system are potentially mutagenic and sometimes introduce mutations in the next generation. In yeast Saccharomyces cerevisiae (S. cerevisiae), DNA polymerase ζ, which consists of Rev3 and Rev7 proteins, and Rev1 are known to be involved in most damage-induced and spontaneous mutations. The human homologs of S. cerevisiae REV1, REV3, and REV7 were identified, and it is revealed that the human REV proteins have similar functions to their yeast counterparts, however, a large part of the mechanisms of mutagenesis employing REV proteins are still unclear. Recently, the new findings about REV proteins were reported, which showed that REV7 interacts not only with REV3 but also with REV1 in human and that REV7 is involved in cell cycle control in Xenopus. These findings give us a new point of view for further investigation about REV proteins. Recent studies of REV proteins are summarized and several points are discussed. 相似文献
11.
12.
Friedberg EC 《Molecular cell》2006,22(2):150-152
Deubiquitination of monoubiquitinated proteins is effected by a large number of deubiquitinating enzymes (DUBs) that function as cysteine proteases in eukaryotic cells. Huang et al. (2006) report the regulation of monoubiquitination of PCNA by autocleavage of a DUB called USP1 in cells exposed to UV radiation. 相似文献
13.
A cell cycle-specific requirement for the XRCC1 BRCT II domain during mammalian DNA strand break repair 总被引:1,自引:0,他引:1
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Taylor RM Moore DJ Whitehouse J Johnson P Caldecott KW 《Molecular and cellular biology》2000,20(2):735-740
XRCC1 protein is essential for viability in mammals and is required for efficient DNA single-strand break repair and genetic stability following DNA base damage. We report here that XRCC1-dependent strand break repair in G(1) phase of the cell cycle is abolished by mutations created within the XRCC1 BRCT domain that interact with DNA ligase III. In contrast, XRCC1-dependent DNA strand break repair in S phase is largely unaffected by these mutations. These data describe a cell cycle-specific role for a BRCT domain, and we conclude that the XRCC1-DNA ligase III complex is required for DNA strand break repair in G(1) phase of the cell cycle but is dispensable for this process in S phase. The S-phase DNA repair process can remove both strand breaks induced in S phase and those that persist from G(1) and can in part compensate for lack of repair in G(1). This process correlates with the appearance of XRCC1 nuclear foci that colocalize with Rad51 and may thus function in concert with homologous recombination. 相似文献
14.
The Rev1-Polζ pathway is believed to be the major mechanism of translesion DNA synthesis and base damage-induced mutagenesis in eukaryotes. While it is widely believed that Rev1 plays a non-catalytic function in translesion synthesis, the role of its dCMP transferase activity remains uncertain. To determine the relevance of its catalytic function in translesion synthesis, we separated the Rev1 dCMP transferase activity from its non-catalytic function in yeast. This was achieved by mutating two conserved amino acid residues in the catalytic domain of Rev1, i.e. D467A/E468A, where its catalytic function was abolished but its non-catalytic function remained intact. In this mutant strain, whereas translesion synthesis and mutagenesis of UV radiation were fully functional, those of a site-specific 1,N6-ethenoadenine were severely deficient. Specifically, the predominant A→G mutations resulting from C insertion opposite the lesion were abolished. Therefore, translesion synthesis and mutagenesis of 1,N6-ethenoadenine require the catalytic function of the Rev1 dCMP transferase, in contrast to those of UV lesions, which only require the non-catalytic function of Rev1. These results show that the catalytic function of the Rev1 dCMP transferase is required in a lesion-specific manner for translesion synthesis and base damage-induced mutagenesis. 相似文献
15.
Yang IY Hashimoto K de Wind N Blair IA Moriya M 《The Journal of biological chemistry》2009,284(1):191-198
Translesion DNA synthesis (TLS) of damaged DNA templates is catalyzed by specialized DNA polymerases. To probe the cellular TLS mechanism, a host-vector system consisting of mouse fibroblasts and a replicating plasmid bearing a single DNA adduct was developed. This system was used to explore the TLS mechanism of a heptanone-etheno-dC (H-epsilondC) adduct, an endogenous lesion produced by lipid peroxidation. In wild-type cells, H-epsilondC almost exclusively directed incorporation of dT and dA. Whereas knockout of the Y family TLS polymerase genes, Polh, Polk, or Poli, did not qualitatively affect these TLS events, inactivation of the Rev3 gene coding for a subunit of polymerase zeta or of the Rev1 gene abolished TLS associated with dA, but not dT, insertion. The analysis of results of the cellular studies and in vitro TLS studies using purified polymerases has revealed that the insertion of dA and dT was catalyzed by different polymerases in cells. While insertion of dT can be catalyzed by polymerase eta, kappa, and iota, insertion of dA is catalyzed by an unidentified polymerase that cannot catalyze extension from the resulting dA terminus. Therefore, the extension from this terminus requires the activity of polymerase zeta-REV1. These results provide new insight into how cells use different TLS pathways to overcome a synthesis block. 相似文献
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17.
Ward I Kim JE Minn K Chini CC Mer G Chen J 《The Journal of biological chemistry》2006,281(50):38472-38477
53BP1 plays an important role in cellular response to DNA damage. It is thought to be the mammalian homologue of budding yeast Rad9 and/or fission yeast Crb2. Rad9/Crb2 are bona fide checkpoint proteins whose activation requires their corresponding C-terminal tandem BRCT (BRCA1 C-terminal) motifs, which mediate their oligomerization and phosphorylation at multiple sites following DNA damage. Here we show that the function of human 53BP1 similarly depends on its oligomerization and phosphorylation at multiple sites but in a BRCT domain-independent manner. Moreover, unlike its proposed yeast counterparts, human 53BP1 only has limited checkpoint functions but rather acts as an adaptor in the repair of DNA double strand breaks. This difference in function may reflect the higher complexity of the DNA damage response network in metazoa including the evolution of other BRCT domain-containing proteins that may have functions redundant or overlapping with those of 53BP1. 相似文献
18.
In their seminal publication describing the structure of the DNA double helix , Watson and Crick wrote what may be one of the greatest understatements in the scientific literature, namely that "It has not escaped our notice that the specific pairing we have postulated immediately suggests a possible copying mechanism for the genetic material." Half a century later, we more fully appreciate what a huge challenge it is to replicate six billion nucleotides with the accuracy needed to stably maintain the human genome over many generations. This challenge is perhaps greater than was realized 50 years ago, because subsequent studies have revealed that the genome can be destabilized not only by environmental stresses that generate a large number and variety of potentially cytotoxic and mutagenic lesions in DNA but also by various sequence motifs of normal DNA that present challenges to replication. Towards a better understanding of the many determinants of genome stability, this chapter reviews the fidelity with which undamaged and damaged DNA is copied, with a focus on the eukaryotic B- and Y-family DNA polymerases, and considers how this fidelity is achieved. 相似文献
19.
Dulic A Bates PA Zhang X Martin SR Freemont PS Lindahl T Barnes DE 《Biochemistry》2001,40(20):5906-5913
Proteins involved in DNA repair, or its coordination with DNA replication and mitosis through cell cycle checkpoints, are vital in the concerted cellular response to DNA damage that maintains the integrity of the genome. The "BRCT" domain (BRCA1 carboxy terminal) was noted as a putative protein-protein interaction motif in the breast cancer suppressor gene, BRCA1, and subsequently identified in over 50 proteins involved in DNA repair, recombination, or cell cycle control. The heterodimer of the DNA repair proteins, XRCC1 and DNA ligase III, was the first example of a functional interaction via BRCT modules. The only three-dimensional crystal structure of a BRCT domain was solved for this region of XRCC1. Key amino acid residues mediating the interaction with DNA ligase III were identified here by targeted mutagenesis of the XRCC1 BRCT domain. The consequences of these mutations on protein folding were assessed. A structural model of the DNA ligase III BRCT domain was constructed and similarly tested by mutation of corresponding residues required for the interaction with XRCC1. These data identify the XRCC1-DNA ligase III heterodimer interface and provide the first demonstration of the surface contacts coordinating a functional BRCT-BRCT protein interaction. 相似文献
20.
A single domain in human DNA polymerase iota mediates interaction with PCNA: implications for translesion DNA synthesis
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Haracska L Acharya N Unk I Johnson RE Hurwitz J Prakash L Prakash S 《Molecular and cellular biology》2005,25(3):1183-1190
DNA polymerases (Pols) of the Y family rescue stalled replication forks by promoting replication through DNA lesions. Humans have four Y family Pols, eta, iota, kappa, and Rev1, of which Pols eta, iota, and kappa have been shown to physically interact with proliferating cell nuclear antigen (PCNA) and be functionally stimulated by it. However, in sharp contrast to the large increase in processivity that PCNA binding imparts to the replicative Pol, Poldelta, the processivity of Y family Pols is not enhanced upon PCNA binding. Instead, PCNA binding improves the efficiency of nucleotide incorporation via a reduction in the apparent K(m) for the nucleotide. Here we show that Poliota interacts with PCNA via only one of its conserved PCNA binding motifs, regardless of whether PCNA is bound to DNA or not. The mode of PCNA binding by Poliota is quite unlike that in Poldelta, where multisite interactions with PCNA provide for a very tight binding of the replicating Pol with PCNA. We discuss the implications of these observations for the accuracy of DNA synthesis during translesion synthesis and for the process of Pol exchange at the lesion site. 相似文献