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ABSTRACT: 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.
Nucleic Acids Research 01/2013; · 8.03 Impact Factor
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ABSTRACT: The E3 ubiquitin ligase Rad18 mediates tolerance of replication fork-stalling bulky DNA lesions, but whether Rad18 mediates tolerance of bulky DNA lesions acquired outside S-phase is unclear. Using synchronized cultures of primary human cells, we defined cell cycle stage-specific contributions of Rad18 to genome maintenance in response to ultraviolet C (UVC) and H(2)O(2)-induced DNA damage. UVC and H(2)O(2) treatments both induced Rad18-mediated proliferating cell nuclear antigen mono-ubiquitination during G(0), G(1) and S-phase. Rad18 was important for repressing H(2)O(2)-induced (but not ultraviolet-induced) double strand break (DSB) accumulation and ATM S1981 phosphorylation only during G(1), indicating a specific role for Rad18 in processing of oxidative DNA lesions outside S-phase. However, H(2)O(2)-induced DSB formation in Rad18-depleted G1 cells was not associated with increased genotoxin sensitivity, indicating that back-up DSB repair mechanisms compensate for Rad18 deficiency. Indeed, in DNA LigIV-deficient cells Rad18-depletion conferred H(2)O(2)-sensitivity, demonstrating functional redundancy between Rad18 and non-homologous end joining for tolerance of oxidative DNA damage acquired during G(1). In contrast with G(1)-synchronized cultures, S-phase cells were H(2)O(2)-sensitive following Rad18-depletion. We conclude that although Rad18 pathway activation by oxidative lesions is not restricted to S-phase, Rad18-mediated trans-lesion synthesis by Polη is dispensable for damage-tolerance in G(1) (because of back-up non-homologous end joining-mediated DSB repair), yet Rad18 is necessary for damage tolerance during S-phase.
Nucleic Acids Research 01/2013; · 8.03 Impact Factor
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ABSTRACT: 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.
Molecular biology of the cell 03/2012; 23(10):1943-54. · 5.98 Impact Factor
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ABSTRACT: 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.
Cell cycle (Georgetown, Tex.) 05/2011; 10(10):1625-38. · 5.36 Impact Factor
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ABSTRACT: Fanconi anemia (FA) is a rare genetic disorder characterized by bone marrow failure, congenital abnormalities, and an increased risk for cancer and leukemia. Components of the FA-BRCA pathway are thought to function in the repair of DNA interstrand cross-links. Central to this pathway is the monoubiquitylation and chromatin localization of 2 FA proteins, FA complementation group D2 (FANCD2) and FANCI. In the present study, we show that RAD18 binds FANCD2 and is required for efficient monoubiquitylation and chromatin localization of both FANCD2 and FANCI. Human RAD18-knockout cells display increased sensitivity to mitomycin C and a delay in FANCD2 foci formation compared with their wild-type counterparts. In addition, RAD18-knockout cells display a unique lack of FANCD2 and FANCI localization to chromatin in exponentially growing cells. FANCD2 ubiquitylation is normal in cells containing a ubiquitylation-resistant form of proliferating cell nuclear antigen, and chromatin loading of FA core complex proteins appears normal in RAD18-knockout cells. Mutation of the RING domain of RAD18 ablates the interaction with and chromatin loading of FANCD2. These data suggest a key role for the E3 ligase activity of RAD18 in the recruitment of FANCD2 and FANCI to chromatin and the events leading to their ubiquitylation during S phase.
Blood 02/2011; 117(19):5078-87. · 9.90 Impact Factor
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ABSTRACT: Following acquisition of DNA damage S-phase progression may potentially be affected via multiple mechanisms. For example DNA damage-activated signal transduction pathways negatively regulate the initiation of DNA synthesis at unfired origins of replication, a process termed the 'S-phase checkpoint' or the 'intra-S-phase checkpoint'. Additionally, many DNA lesions pose physical barriers to replication forks and therefore inhibit DNA synthesis directly by blocking the elongation of active replicons. Inhibition of DNA synthesis in response to DNA damage is commonly assayed by measuring incorporation of radiolabeled or halogenated nucleotides into bulk genomic DNA. However, these techniques do not distinguish between effects of DNA damage on initiation and elongation phases of DNA synthesis. The velocity sedimentation protocol described here allows investigators to determine the effects of DNA damage on initiation and elongation events. This technique involves labeling replicating DNA with (3)H-thymidine, then analyzing the size distribution of labeled ssDNAs based on their differential density sedimentation profiles after centrifugation through alkaline sucrose gradients. Determining the relative abundance and growth rates of small nascent ssDNAs provides an index of initiation and elongation events, respectively. Therefore, analysis of replication dynamics using velocity sedimentation provides a potentially valuable tool for assaying S-phase checkpoints as well as other aspects of DNA replication.
Methods in molecular biology (Clifton, N.J.) 01/2011; 782:159-70.
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ABSTRACT: It has long been appreciated that Cdc7 is an essential protein kinase that phosphorylates Mcm2-7 helicase subunits to promote initiation of DNA replication. In addition to its well-elucidated role in DNA replication, recent studies suggest that DDK is active in genotoxin-treated cells and may mediate aspects of the DNA damage response. However, specific role(s) of DDK and its effector targets in DNA damage signaling have not been defined. A recent study from our laboratories has identified the E3 ubiquitin ligase Rad18 as novel substrate of DDK in vitro and in human cells. Rad18 plays a central role in a post-replication DNA repair pathway termed 'Trans-Lesion Synthesis' (TLS) by promoting recruitment of DNA Polymerase eta (Polη) and other TLS polymerases to stalled replication forks. DDK-mediated Rad18 phosphorylation promotes Rad18-Polη complex formation and facilitates Rad18-dependent recruitment of Polη to stalled replication forks. The mechanisms that regulate Rad18-dependent TLS are incompletely understood. Our study provides the first demonstration of Rad18 regulation by direct phosphorylation and defines a novel mechanism for Rad18-dependent recruitment of TLS polymerases to stalled forks. This study also demonstrates a molecular basis for integration of TLS with S-phase progression via the essential Cdc7 kinase. These findings reveal unexpected mechanistic insights to the regulation of the TLS pathway and Polη recruitment.
Cell cycle (Georgetown, Tex.) 12/2010; 9(24):4818-23. · 5.36 Impact Factor
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ABSTRACT: The efficiency of metazoan origins of DNA replication is known to be enhanced by histone acetylation near origins. Although this correlates with increased MCM recruitment, the mechanism by which such acetylation regulates MCM loading is unknown. We show here that Cdt1 induces large-scale chromatin decondensation that is required for MCM recruitment. This process occurs in G₁, is suppressed by Geminin, and requires HBO1 HAT activity and histone H4 modifications. HDAC11, which binds Cdt1 and replication origins during S-phase, potently inhibits Cdt1-induced chromatin unfolding and re-replication, suppresses MCM loading and binds Cdt1 more efficiently in the presence of Geminin. We also demonstrate that chromatin at endogenous origins is more accessible in G₁ relative to S-phase. These results provide evidence that histone acetylation promotes MCM loading via enhanced chromatin accessibility. This process is regulated positively by Cdt1 and HBO1 in G₁ and repressed by Geminin-HDAC11 association with Cdt1 in S-phase, and represents a novel form of replication licensing control.
Cell cycle (Georgetown, Tex.) 11/2010; 9(21):4351-63. · 5.36 Impact Factor
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ABSTRACT: The E3 ubiquitin ligase Rad18 guides DNA Polymerase eta (Polη) to sites of replication fork stalling and mono-ubiquitinates proliferating cell nuclear antigen (PCNA) to facilitate binding of Y family trans-lesion synthesis (TLS) DNA polymerases during TLS. However, it is unclear exactly how Rad18 is regulated in response to DNA damage and how Rad18 activity is coordinated with progression through different phases of the cell cycle. Here we identify Rad18 as a novel substrate of the essential protein kinase Cdc7 (also termed Dbf4/Drf1-dependent Cdc7 kinase [DDK]). A serine cluster in the Polη-binding motif of Rad 18 is phosphorylated by DDK. Efficient association of Rad18 with Polη is dependent on DDK and is necessary for redistribution of Polη to sites of replication fork stalling. This is the first demonstration of Rad18 regulation by direct phosphorylation and provides a novel mechanism for integration of S phase progression with postreplication DNA repair to maintain genome stability.
The Journal of Cell Biology 11/2010; 191(5):953-66. · 10.26 Impact Factor
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ABSTRACT: Fanconi anemia (FA) is a cancer susceptibility syndrome characterized by sensitivity to DNA-damaging agents. The FA proteins (FANCs) are implicated in DNA repair, although the precise mechanisms by which FANCs process DNA lesions are not fully understood. An epistatic relationship between the FA pathway and translesion synthesis (TLS, a post-replication DNA repair mechanism) has been suggested, but the basis for cross-talk between the FA and TLS pathways is poorly understood. We show here that ectopic overexpression of the E3 ubiquitin ligase Rad18 (a central regulator of TLS) induces DNA damage-independent mono-ubiquitination of proliferating cell nuclear antigen (PCNA) (a known Rad18 substrate) and FANCD2. Conversely, DNA damage-induced mono-ubiquitination of both PCNA and FANCD2 is attenuated in Rad18-deficient cells, demonstrating that Rad18 contributes to activation of the FA pathway. WT Rad18 but not an E3 ubiquitin ligase-deficient Rad18 C28F mutant fully complements both PCNA ubiquitination and FANCD2 activation in Rad18-depleted cells. Rad18-induced mono-ubiquitination of FANCD2 is not observed in FA core complex-deficient cells, demonstrating that Rad18 E3 ligase activity alone is insufficient for FANCD2 ubiquitylation. Instead, Rad18 promotes FA core complex-dependent FANCD2 ubiquitination in a manner that is secondary to PCNA mono-ubiquitination. Taken together, these results demonstrate a novel Rad18-dependent mechanism that couples activation of the FA pathway with TLS.
Journal of Biological Chemistry 10/2010; 285(41):31525-36. · 4.77 Impact Factor
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ABSTRACT: Fanconi Anemia (FA) is a cancer-susceptibility syndrome characterized by cellular sensitivity to DNA inter-strand cross-link (ICL)-inducing agents. The Fanconia Anemia D2 (FANCD2) protein is implicated in repair of various forms of DNA damage including ICLs. Studies with replicating extracts from Xenopus eggs indicate a role for FANCD2 in processing and repair of DNA replication-associated double stranded breaks (DSB). We have investigated the role of FANCD2 in cell cycle progression of cultured human cells. Similar to Xenopus cell-free extracts, we show that chromatin association of FANCD2 in human cells is coupled to ongoing DNA replication. siRNA depletion experiments demonstrate that FANCD2 is necessary for efficient DNA synthesis. However, in contrast with Xenopus extracts, FANCD2-deficiency does not elicit a DNA damage response, and does not affect the elongation phase of DNA synthesis, suggesting that FANCD2 is dispensable for repair of replication-associated DNA damage. Using synchronized cultures of primary untransformed human dermal fibroblasts we demonstrate that FANCD2 is necessary for efficient initiation of DNA synthesis. Taken together, our results suggest a novel role for the FA pathway in regulation of DNA synthesis and cell cycle progression. Inefficient DNA replication may contribute to the genome instability and cancer-propensity of FA patients.
Cell cycle (Georgetown, Tex.) 06/2010; 9(12):2375-88. · 5.36 Impact Factor
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Junjie Wu,
Jun Qian,
Chun Li,
Letty Kwok,
Feng Cheng,
Peijun Liu,
Catalina Perdomo,
Darrell Kotton, Cyrus Vaziri,
Christina Anderlind,
Avrum Spira,
Wellington V Cardoso,
Jining Lü
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ABSTRACT: Reduced expression of miR-129 has been reported in multiple tumor cell lines and in primary tumors including medulloblastoma, undifferentiated gastric cancers, lung adenocarcinoma, endometrial cancer and colorectal carcinoma. There is also recent evidence of an anti-proliferative activity of miR-129 in tumor cell lines. Still, little is known about how miR-129 regulates cell proliferation. Here we found that lentiviral-mediated overexpression of miR-129 in mouse lung epithelial cells (E10 cells) results in significant G(1) phase arrest that eventually leads to cell death. miR-129 induce G(1) phase arrest in multiple human lung adenocarcinoma cell lines, suggesting miR-129 targeting of G(1)/S phase-specific regulators. Interestingly, we show that Cdk6, a kinase involved in G(1)-S transition, is a direct target of miR-129. We also found the downregulation of three other cell cycle-related novel targets of miR-129, including Erk1, Erk2 and protein kinase C epsilon (Prkce). We further show that among these targets, only Cdk6 is functionally relevant. Restoring expression of Cdk6, but not Prkce partially rescues the cell growth arrest and cell death phenotype that results from miR-129 overexpression. Together, our data indicate that miR-129 plays an important role in regulating cell proliferation by downregulation of Cdk6.
Cell cycle (Georgetown, Tex.) 05/2010; 9(9):1809-18. · 5.36 Impact Factor
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Cell cycle (Georgetown, Tex.) 08/2009; 8(14):2138. · 5.36 Impact Factor
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ABSTRACT: In cancer cells ablation of the GINS complex member Psf2 elicits chromosome mis-segregation yet the precise role of Psf2 in mitosis is unknown. We investigated the putative mitotic role of the GINS complex using synchronized cultures of untransformed Human Dermal Fibroblasts (HDF). Metaphase spreads from Psf1/Psf2-depleted HDF were normal and mitotic exit of Psf1/Psf2-depleted cells was only slightly delayed, suggesting no direct role for the GINS complex in mitosis of untransformed cells. Because the GINS complex is required for initiation and elongation events during DNA replication we hypothesized that the mitotic delay of Psf1/Psf2-deficient cells resulted indirectly from defective DNA synthesis during a prior S-phase. Therefore, we investigated the effects of Psf1/Psf2-depletion on DNA replication. Recruitment of Mcm7 to chromatin during G(1) was unaffected by Psf1/Psf2-ablation, indicating that replication licensing does not require GINS. However, chromatin-binding of Cdc45 and PCNA, onset of DNA synthesis and accumulation of G(2)/M markers were delayed in Psf1/Psf2-ablated cells. The cell cycle delay of Psf1/Psf2-depleted HDF was associated with several hallmarks of pre-malignancy including gammaH2AX, Thr 68-phosphorylated Chk2, and increased numbers of aberrant fragmented nuclei. Ectopic expression of catalytically-inactive Chk2 promoted S-phase and G(2)/M progression in Psf1/Psf2-depleted cells, as evidenced by modestly-increased rates of DNA synthesis and increased dephosphorylation of Cdc2. Therefore, S-phase progression of untransformed cells containing sub-optimal levels of Psf1/2 is associated with replication stress and acquisition of DNA damage. The ensuing Chk2-mediated DNA damage signaling likely contributes to maintenance of chromosomal integrity.
Cell cycle (Georgetown, Tex.) 06/2009; 8(10):1577-88. · 5.36 Impact Factor
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ABSTRACT: When a replicative DNA polymerase (Pol) is stalled by damaged DNA, a "polymerase switch" recruits specialized translesion synthesis (TLS) DNA polymerase(s) to sites of damage. Mammalian cells have several TLS DNA polymerases, including the four Y-family enzymes (Poleta, Poliota, Polkappa and REV1) that share multiple primary sequence motifs, but show preferential bypass of different DNA lesions. REV1 interacts with Poleta, Poliota, and Polkappa and therefore appears to play a central role during TLS in vivo. Here we have investigated the molecular basis for interactions between REV1 and Polkappa. We have identified novel REV1-interacting regions (RIRs) present in Polkappa, Poliota and Poleta. Within the RIRs, the presence of two consecutive phenylalanines (FF) is essential for REV1-binding. The consensus sequence for REV1-binding is denoted by x-x-x-F-F-y-y-y-y (x, no specific residue and y, no specific residue but not proline). Our results identify structural requirements that are necessary for FF-flanking residues to confer interactions with REV1. A Polkappa mutant lacking REV1-binding activity did not complement the genotoxin-sensitivity of Polk-null mouse embryonic fibroblast cells, thereby demonstrating that the REV1-interaction is essential for Polkappa function in vivo.
Genes to Cells 03/2009; 14(2):101-11. · 2.68 Impact Factor
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ABSTRACT: Defects in DNA replication are implicated as early and causal events in malignancy. However, the immediate effects of impaired DNA replication licensing on cell cycle progression of non-malignant human cells are unknown. Therefore, we have investigated the acute effects of Mcm7 ablation using synchronized cultures of untransformed Human Dermal Fibroblasts (HDF). Mcm7 ablation elicited a G(1) delay associated with impaired activation of CDK4 and CDK2 and reduced Rb phosphorylation. The cell cycle delay of Mcm7-ablated cells was not associated with a DNA damage response. However, levels of cyclin D1 mRNA were specifically reduced and binding of RNA Polymerase II to the CYCD1 promoter was decreased in Mcm7-depleted cells. Similar to Mcm7-deficiency, Mcm2- or Cdc6-depletion led to impaired cyclin D expression. Ectopic overexpression of Cdc6 in quiescent cells promoted cyclin D1 expression, CDK4 activation and G(1) progression. Therefore timely and efficient expression of cyclin D1 during G(1) phase requires replication licensing. Reconstitution of cyclin D1 expression was insufficient to correct the G(1) delay of Mcm7-depleted cells, indicating that additional cell cycle events during G(1) are dependent on replication licensing. However, ectopic expression of the HPV-E7 oncoprotein, and the resulting bypass of the requirement for cyclin D1-Rb signaling enabled Mcm7-depleted cells to enter S-phase. HPV-E7-induced S-phase entry of Mcm7-depleted cells led to a DNA damage response, a hallmark of pre-malignancy. Taken together, our results suggest the existence of a 'replication licensing restriction point' that couples pre-RC assembly with G(1) progression in normal cells to minimize replication stress, DNA damage and tumorigenesis.
Cell cycle (Georgetown, Tex.) 02/2009; 8(1):125-36. · 5.36 Impact Factor
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Frank Schembri,
Sriram Sridhar,
Catalina Perdomo,
Adam M Gustafson,
Xiaoling Zhang,
Ayla Ergun,
Jining Lu,
Gang Liu,
Xiaohui Zhang,
Jessica Bowers, Cyrus Vaziri,
Kristen Ott,
Kelly Sensinger,
James J Collins,
Jerome S Brody,
Robert Getts,
Marc E Lenburg,
Avrum Spira
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ABSTRACT: We have shown that smoking impacts bronchial airway gene expression and that heterogeneity in this response associates with smoking-related disease risk. In this study, we sought to determine whether microRNAs (miRNAs) play a role in regulating the airway gene expression response to smoking. We examined whole-genome miRNA and mRNA expression in bronchial airway epithelium from current and never smokers (n = 20) and found 28 miRNAs to be differentially expressed (P < 0.05) with the majority being down-regulated in smokers. We further identified a number of mRNAs whose expression level is highly inversely correlated with miRNA expression in vivo. Many of these mRNAs contain potential binding sites for the differentially expressed miRNAs in their 3'-untranslated region (UTR) and are themselves affected by smoking. We found that either increasing or decreasing the levels of mir-218 (a miRNA that is strongly affected by smoking) in both primary bronchial epithelial cells and H1299 cells was sufficient to cause a corresponding decrease or increase in the expression of predicted mir-218 mRNA targets, respectively. Further, mir-218 expression is reduced in primary bronchial epithelium exposed to cigarette smoke condensate (CSC), and alteration of mir-218 levels in these cells diminishes the induction of the predicted mir-218 target MAFG in response to CSC. These data indicate that mir-218 levels modulate the airway epithelial gene expression response to cigarette smoke and support a role for miRNAs in regulating host response to environmental toxins.
Proceedings of the National Academy of Sciences 01/2009; 106(7):2319-24. · 9.68 Impact Factor
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ABSTRACT: SPARC-Related Modular Calcium Binding Protein-2 (Smoc-2) is a broadly-expressed matricellular protein which contributes to mitogenesis via activation of Integrin-Linked Kinase (ILK). Here we show that expression of Smoc2 is repressed in cultured cells following treatment with Aryl-hydrocarbon receptor (Ahr) ligands including the ubiquitous environmental pollutants Benzo[a]pyrene (B[a]P) and 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD). The Smoc2 promoter contains two consensus putative Ahr-binding sites and Smoc2 promoter-driven reporter genes are repressed in response to B[a]P in an Ahr-dependent manner in cultured cells. Using organ culture experiments we show that TCDD also represses Smoc2 mRNA expression in testes from Ahr(+/+) but not Ahr(-/-) mice. Therefore, exposure to Ahr ligands is likely to affect Smoc2 expression in vivo. Taken together our results indicate that Smoc2 is a novel transcriptional target of activated Ahr. Perturbation of Smoc2 expression may mediate the adverse developmental effects of environmental aryl-hydrocarbon exposure.
Gene 01/2009; 433(1-2):72-80. · 2.34 Impact Factor
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ABSTRACT: All organisms have multiple DNA polymerases specialized for translesion DNA synthesis (TLS) on damaged DNA templates. Mammalian TLS DNA polymerases include Pol eta, Pol iota, Pol kappa, and Rev1 (all classified as "Y-family" members) and Pol zeta (a "B-family" member). Y-family DNA polymerases have highly structured catalytic domains; however, some of these proteins adopt different structures when bound to DNA (such as archaeal Dpo4 and human Pol kappa), while others maintain similar structures independently of DNA binding (such as archaeal Dbh and Saccharomyces cerevisiae Pol eta). DNA binding-induced structural conversions of TLS polymerases depend on flexible regions present within the catalytic domains. In contrast, noncatalytic regions of Y-family proteins, which contain multiple domains and motifs for interactions with other proteins, are predicted to be mostly unstructured, except for short regions corresponding to ubiquitin-binding domains. In this review we discuss how the organization of structured and unstructured regions in TLS polymerases is relevant to their regulation and function during lesion bypass.
Advances in protein chemistry and structural biology. 01/2009; 78:99-146.
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Junya Tomida,
Yuji Masuda,
Hidekazu Hiroaki,
Tomoko Ishikawa,
Ihnyoung Song,
Toshiki Tsurimoto,
Satoshi Tateishi,
Tadahiro Shiomi,
Yasuhiro Kamei,
Jinhyeong Kim,
Kenji Kamiya, Cyrus Vaziri,
Haruo Ohmori,
Takeshi Todo
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ABSTRACT: Many proteins involved in DNA replication and repair undergo post-translational modifications such as phosphorylation and ubiquitylation. Proliferating cell nuclear antigen (PCNA; a homotrimeric protein that encircles double-stranded DNA to function as a sliding clamp for DNA polymerases) is monoubiquitylated by the RAD6-RAD18 complex and further polyubiquitylated by the RAD5-MMS2-UBC13 complex in response to various DNA-damaging agents. PCNA mono- and polyubiquitylation activate an error-prone translesion synthesis pathway and an error-free pathway of damage avoidance, respectively. Here we show that replication factor C (RFC; a heteropentameric protein complex that loads PCNA onto DNA) was also ubiquitylated in a RAD18-dependent manner in cells treated with alkylating agents or H(2)O(2). A mutant form of RFC2 with a D228A substitution (corresponding to a yeast Rfc4 mutation that reduces an interaction with replication protein A (RPA), a single-stranded DNA-binding protein) was heavily ubiquitylated in cells even in the absence of DNA damage. Furthermore RFC2 was ubiquitylated by the RAD6-RAD18 complex in vitro, and its modification was inhibited in the presence of RPA. The inhibitory effect of RPA on RFC2 ubiquitylation was relatively specific because RAD6-RAD18-mediated ubiquitylation of PCNA was RPA-insensitive. Our findings suggest that RPA plays a regulatory role in DNA damage responses via repression of RFC2 ubiquitylation in human cells.
Journal of Biological Chemistry 05/2008; 283(14):9071-9. · 4.77 Impact Factor
