Lesion recognition by XPC, TFIIH and XPA in DNA excision repair.

Nucleotide excision repair removes DNA lesions caused by ultraviolet light, cisplatin-like compounds and bulky adducts1. After initial recognition by XPC in global genome repair or a stalled RNA polymerase in transcription-coupled repair, damaged DNA is transferred to the seven-subunit TFIIH core complex (Core7) for verification and dual incisions by the XPF and XPG nucleases2. Structures capturing lesion recognition by the yeast XPC homologue Rad4 and TFIIH in transcription initiation or DNA repair have been separately reported3-7. How two different lesion recognition pathways converge and how the XPB and XPD helicases of Core7 move the DNA lesion for verification are unclear. Here we report on structures revealing DNA lesion recognition by human XPC and DNA lesion hand-off from XPC to Core7 and XPA. XPA, which binds between XPB and XPD, kinks the DNA duplex and shifts XPC and the DNA lesion by nearly a helical turn relative to Core7. The DNA lesion is thus positioned outside of Core7, as would occur with RNA polymerase. XPB and XPD, which track the lesion-containing strand but translocate DNA in opposite directions, push and pull the lesion-containing strand into XPD for verification.

The molecular basis of CRL4DDB2/CSA ubiquitin ligase architecture, targeting, and activation.

The DDB1-CUL4-RBX1 (CRL4) ubiquitin ligase family regulates a diverse set of cellular pathways through dedicated substrate receptors (DCAFs). The DCAF DDB2 detects UV-induced pyrimidine dimers in the genome and facilitates nucleotide excision repair. We provide the molecular basis for DDB2 receptor-mediated cyclobutane pyrimidine dimer recognition in chromatin. The structures of the fully assembled DDB1-DDB2-CUL4A/B-RBX1 (CRL4(DDB2)) ligases reveal that the mobility of the ligase arm creates a defined ubiquitination zone around the damage, which precludes direct ligase activation by DNA lesions. Instead, the COP9 signalosome (CSN) mediates the CRL4(DDB2) inhibition in a CSN5 independent, nonenzymatic, fashion. In turn, CSN inhibition is relieved upon DNA damage binding to the DDB2 module within CSN-CRL4(DDB2). The Cockayne syndrome A DCAF complex crystal structure shows that CRL4(DCAF(WD40)) ligases share common architectural features. Our data support a general mechanism of ligase activation, which is induced by CSN displacement from CRL4(DCAF) on substrate binding to the DCAF.

A formamidopyrimidine derivative from the deoxyguanosine adduct produced by food contaminant acrylamide induces DNA replication block and mutagenesis.

Acrylamide, a common food contaminant, is metabolically activated to glycidamide, which reacts with DNA at the N7 position of dG, forming N7-(2-carbamoyl-2-hydroxyethyl)-dG (GA7dG). Owing to its chemical lability, the mutagenic potency of GA7dG has not yet been clarified. We found that GA7dG undergoes ring-opening hydrolysis to form N6-(2-deoxy-d-erythro-pentofuranosyl)-2,6-diamino-3,4-dihydro-4-oxo-5-[N-(2-carbamoyl-2-hydroxyethyl)formamido]pyrimidine (GA-FAPy-dG), even at neutral pH. Therefore, we aimed to examine the effects of GA-FAPy-dG on the efficiency and fidelity of DNA replication using an oligonucleotide carrying GA-FAPy-9-(2-deoxy-2-fluoro-ß-d-arabinofuranosyl)guanine (dfG), a 2'-fluorine substituted analog of GA-FAPy-dG. GA-FAPy-dfG inhibited primer extension by both human replicative DNA polymerase ε and the translesion DNA synthesis polymerases (Polη, Polι, Polκ, and Polζ) and reduced the replication efficiency by less than half in human cells, with single base substitution at the site of GA-FAPy-dfG. Unlike other formamidopyrimidine derivatives, the most abundant mutation was G:C > A:T transition, which was decreased in Polκ- or REV1-KO cells. Molecular modeling suggested that a 2-carbamoyl-2-hydroxyethyl group at the N5 position of GA-FAPy-dfG can form an additional H-bond with thymidine, thereby contributing to the mutation. Collectively, our results provide further insight into the mechanisms underlying the mutagenic effects of acrylamide.

The intrinsically disordered N-terminal region of mouse DNA polymerase alpha mediates its interaction with POT1a/b at telomeres.

Mouse telomerase and the DNA polymerase alpha-primase complex elongate the leading and lagging strands of telomeres, respectively. To elucidate the molecular mechanism of lagging strand synthesis, we investigated the interaction between DNA polymerase alpha and two paralogs of the mouse POT1 telomere-binding protein (POT1a and POT1b). Yeast two-hybrid analysis and a glutathione S-transferase pull-down assay indicated that the C-terminal region of POT1a/b binds to the intrinsically disordered N-terminal region of p180, the catalytic subunit of mouse DNA polymerase alpha. Subcellular distribution analyses showed that although POT1a, POT1b, and TPP1 were localized to the cytoplasm, POT1a-TPP1 and POT1b-TPP1 coexpressed with TIN2 localized to the nucleus in a TIN2 dose-dependent manner. Coimmunoprecipitation and cell cycle synchronization experiments indicated that POT1b-TPP1-TIN2 was more strongly associated with p180 than POT1a-TPP1-TIN2, and this complex accumulated during the S phase. Fluorescence in situ hybridization and proximity ligation assays showed that POT1a and POT1b interacted with p180 and TIN2 on telomeric chromatin. Based on the present study and a previous study, we propose a model in which POT1a/b-TPP1-TIN2 translocates into the nucleus in a TIN2 dose-dependent manner to target the telomere, where POT1a/b interacts with DNA polymerase alpha for recruitment at the telomere for lagging strand synthesis.

Novel function of HATs and HDACs in homologous recombination through acetylation of human RAD52 at double-strand break sites.

The p300 and CBP histone acetyltransferases are recruited to DNA double-strand break (DSB) sites where they induce histone acetylation, thereby influencing the chromatin structure and DNA repair process. Whether p300/CBP at DSB sites also acetylate non-histone proteins, and how their acetylation affects DSB repair, remain unknown. Here we show that p300/CBP acetylate RAD52, a human homologous recombination (HR) DNA repair protein, at DSB sites. Using in vitro acetylated RAD52, we identified 13 potential acetylation sites in RAD52 by a mass spectrometry analysis. An immunofluorescence microscopy analysis revealed that RAD52 acetylation at DSBs sites is counteracted by SIRT2- and SIRT3-mediated deacetylation, and that non-acetylated RAD52 initially accumulates at DSB sites, but dissociates prematurely from them. In the absence of RAD52 acetylation, RAD51, which plays a central role in HR, also dissociates prematurely from DSB sites, and hence HR is impaired. Furthermore, inhibition of ataxia telangiectasia mutated (ATM) protein by siRNA or inhibitor treatment demonstrated that the acetylation of RAD52 at DSB sites is dependent on the ATM protein kinase activity, through the formation of RAD52, p300/CBP, SIRT2, and SIRT3 foci at DSB sites. Our findings clarify the importance of RAD52 acetylation in HR and its underlying mechanism.

Polη, a Y-family translesion synthesis polymerase, promotes cellular tolerance of Myc-induced replication stress.

Growth of precancerous and cancer cells relies on their tolerance of oncogene-induced replication stress (RS). Translesion synthesis (TLS) plays an essential role in the cellular tolerance of various types of RS and bypasses replication barriers by employing specialized polymerases. However, limited information is available about the role of TLS polymerases in oncogene-induced RS. Here, we report that Polη, a Y-family TLS polymerase, promotes cellular tolerance of Myc-induced RS. Polη was recruited to Myc-induced RS sites, and Polη depletion enhanced the Myc-induced slowing and stalling of replication forks and the subsequent generation of double-strand breaks (DSBs). Overexpression of a catalytically dead Polη also promoted Myc-induced DSB formation. In the absence of Polη, Myc-induced DSB formation depended on MUS81-EME2 (the S-phase-specific endonuclease complex), and concomitant depletion of MUS81-EME2 and Polη enhanced RS and cell death in a synergistic manner. Collectively, these results indicate that Polη facilitates fork progression during Myc-induced RS, thereby helping cells tolerate the resultant deleterious effects. Additionally, the present study highlights the possibility of a synthetic sickness or lethality between Polη and MUS81-EME2 in cells experiencing Myc-induced RS.

NBS1 recruits RAD18 via a RAD6-like domain and regulates Pol η-dependent translesion DNA synthesis.

Translesion DNA synthesis, a process orchestrated by monoubiquitinated PCNA, is critical for DNA damage tolerance. While the ubiquitin-conjugating enzyme RAD6 and ubiquitin ligase RAD18 are known to monoubiquitinate PCNA, how they are regulated by DNA damage is not fully understood. We show that NBS1 (mutated in Nijmegen breakage syndrome) binds to RAD18 after UV irradiation and mediates the recruitment of RAD18 to sites of DNA damage. Disruption of NBS1 abolished RAD18-dependent PCNA ubiquitination and Polη focus formation, leading to elevated UV sensitivity and mutation. Unexpectedly, the RAD18-interacting domain of NBS1, which was mapped to its C terminus, shares structural and functional similarity with the RAD18-interacting domain of RAD6. These domains of NBS1 and RAD6 allow the two proteins to interact with RAD18 homodimers simultaneously and are crucial for Polη-dependent UV tolerance. Thus, in addition to chromosomal break repair, NBS1 plays a key role in translesion DNA synthesis.

The Mcm2-7-interacting domain of human mini-chromosome maintenance 10 (Mcm10) protein is important for stable chromatin association and origin firing.

The protein mini-chromosome maintenance 10 (Mcm10) was originally identified as an essential yeast protein in the maintenance of mini-chromosome plasmids. Subsequently, Mcm10 has been shown to be required for both initiation and elongation during chromosomal DNA replication. However, it is not fully understood how the multiple functions of Mcm10 are coordinated or how Mcm10 interacts with other factors at replication forks. Here, we identified and characterized the Mcm2-7-interacting domain in human Mcm10. The interaction with Mcm2-7 required the Mcm10 domain that contained amino acids 530-655, which overlapped with the domain required for the stable retention of Mcm10 on chromatin. Expression of truncated Mcm10 in HeLa cells depleted of endogenous Mcm10 via siRNA revealed that the Mcm10 conserved domain (amino acids 200-482) is essential for DNA replication, whereas both the conserved and the Mcm2-7-binding domains were required for its full activity. Mcm10 depletion reduced the initiation frequency of DNA replication and interfered with chromatin loading of replication protein A, DNA polymerase (Pol) α, and proliferating cell nuclear antigen, whereas the chromatin loading of Cdc45 and Pol ϵ was unaffected. These results suggest that human Mcm10 is bound to chromatin through the interaction with Mcm2-7 and is primarily involved in the initiation of DNA replication after loading of Cdc45 and Pol ϵ.

The molecular chaperone Hsp90 regulates accumulation of DNA polymerase eta at replication stalling sites in UV-irradiated cells.

DNA polymerase eta (Pol eta) is a member of the mammalian Y family polymerases and performs error-free translesion synthesis across UV-damaged DNA. For this function, Pol eta accumulates in nuclear foci at replication stalling sites via its interaction with monoubiquitinated PCNA. However, little is known about the posttranslational control mechanisms of Pol eta, which regulate its accumulation in replication foci. Here, we report that the molecular chaperone Hsp90 promotes UV irradiation-induced nuclear focus formation of Pol eta through control of its stability and binding to monoubiquitinated PCNA. Our data indicate that Hsp90 facilitates the folding of Pol eta into an active form in which PCNA- and ubiquitin-binding regions are functional. Furthermore, Hsp90 inhibition potentiates UV-induced cytotoxicity and mutagenesis in a Pol eta-dependent manner. Our studies identify Hsp90 as an essential regulator of Pol eta-mediated translesion synthesis.

Two-step recognition of DNA damage for mammalian nucleotide excision repair: Directional binding of the XPC complex and DNA strand scanning.

For mammalian nucleotide excision repair (NER), DNA lesions are recognized in at least two steps involving detection of unpaired bases by the XPC protein complex and the subsequent verification of injured bases. Although lesion verification is important to ensure high damage discrimination and the accuracy of the repair system, it has been unclear how this is accomplished. Here, we show that damage verification involves scanning of a DNA strand from the site where XPC is initially bound. Translocation by the NER machinery exhibits a 5'-to-3' directionality, strongly suggesting involvement of the XPD helicase, a component of TFIIH. Furthermore, the initial orientation of XPC binding is crucial in that only one DNA strand is selected to search for the presence of lesions. Our results dissect the intricate molecular mechanism of NER and provide insights into a strategy for mammalian cells to survey large genomes to detect DNA damage.

Different types of interaction between PCNA and PIP boxes contribute to distinct cellular functions of Y-family DNA polymerases.

Translesion DNA synthesis (TLS) by the Y-family DNA polymerases Polη, Polι and Polκ, mediated via interaction with proliferating cell nuclear antigen (PCNA), is a crucial pathway that protects human cells against DNA damage. We report that Polη has three PCNA-interacting protein (PIP) boxes (PIP1, 2, 3) that contribute differentially to two distinct functions, stimulation of DNA synthesis and promotion of PCNA ubiquitination. The latter function is strongly associated with formation of nuclear Polη foci, which co-localize with PCNA. We also show that Polκ has two functionally distinct PIP boxes, like Polη, whereas Polι has a single PIP box involved in stimulation of DNA synthesis. All three polymerases were additionally stimulated by mono-ubiquitinated PCNA in vitro. The three PIP boxes and a ubiquitin-binding zinc-finger of Polη exert redundant and additive effects in vivo via distinct molecular mechanisms. These findings provide an integrated picture of the orchestration of TLS polymerases.

Functional regulation of the DNA damage-recognition factor DDB2 by ubiquitination and interaction with xeroderma pigmentosum group C protein.

In mammalian nucleotide excision repair, the DDB1-DDB2 complex recognizes UV-induced DNA photolesions and facilitates recruitment of the XPC complex. Upon binding to damaged DNA, the Cullin 4 ubiquitin ligase associated with DDB1-DDB2 is activated and ubiquitinates DDB2 and XPC. The structurally disordered N-terminal tail of DDB2 contains seven lysines identified as major sites for ubiquitination that target the protein for proteasomal degradation; however, the precise biological functions of these modifications remained unknown. By exogenous expression of mutant DDB2 proteins in normal human fibroblasts, here we show that the N-terminal tail of DDB2 is involved in regulation of cellular responses to UV. By striking contrast with behaviors of exogenous DDB2, the endogenous DDB2 protein was stabilized even after UV irradiation as a function of the XPC expression level. Furthermore, XPC competitively suppressed ubiquitination of DDB2 in vitro, and this effect was significantly promoted by centrin-2, which augments the DNA damage-recognition activity of XPC. Based on these findings, we propose that in cells exposed to UV, DDB2 is protected by XPC from ubiquitination and degradation in a stochastic manner; thus XPC allows DDB2 to initiate multiple rounds of repair events, thereby contributing to the persistence of cellular DNA repair capacity.

Association of the winged helix motif of the TFIIEα subunit of TFIIE with either the TFIIEß subunit or TFIIB distinguishes its functions in transcription.

In eukaryotes, the general transcription factor TFIIE consists of two subunits, α and ß, and plays essential roles in transcription. Structure-function studies indicate that TFIIE has three-winged helix (WH) motifs, with one in TFIIEα and two in TFIIEß. Recent studies suggested that, by binding to the clamp region of RNA polymerase II, TFIIEα-WH promotes the conformational change that transforms the promoter-bound inactive preinitiation complex to the active complex. Here, to elucidate its roles in transcription, functional analyses of point-mutated human TFIIEα-WH proteins were carried out. In vitro transcription analyses identified two classes of mutants. One class was defective in transcription initiation, and the other was defective in the transition from initiation to elongation. Analyses of the binding of this motif to other general transcription factors showed that the former class was defective in binding to the basic helix-loop-helix motif of TFIIEß and the latter class was defective in binding to the N-terminal cyclin homology region of TFIIB. Furthermore, TFIIEα-WH bound to the TFIIH XPB subunit at a third distinct region. Therefore, these results provide further insights into the mechanisms underlying RNA polymerase II activation at the initial stages of transcription.

Structure and mechanism of human DNA polymerase eta.

The variant form of the human syndrome xeroderma pigmentosum (XPV) is caused by a deficiency in DNA polymerase eta (Poleta), a DNA polymerase that enables replication through ultraviolet-induced pyrimidine dimers. Here we report high-resolution crystal structures of human Poleta at four consecutive steps during DNA synthesis through cis-syn cyclobutane thymine dimers. Poleta acts like a 'molecular splint' to stabilize damaged DNA in a normal B-form conformation. An enlarged active site accommodates the thymine dimer with excellent stereochemistry for two-metal ion catalysis. Two residues conserved among Poleta orthologues form specific hydrogen bonds with the lesion and the incoming nucleotide to assist translesion synthesis. On the basis of the structures, eight Poleta missense mutations causing XPV can be rationalized as undermining the molecular splint or perturbing the active-site alignment. The structures also provide an insight into the role of Poleta in replicating through D loop and DNA fragile sites.

A cyclobutane thymine-N4-methylcytosine dimer is resistant to hydrolysis but strongly blocks DNA synthesis.

Exposure of DNA to ultraviolet light produces harmful crosslinks between adjacent pyrimidine bases, to form cyclobutane pyrimidine dimers (CPDs) and pyrimidine(6-4)pyrimidone photoproducts. The CPD is frequently formed, and its repair mechanisms have been exclusively studied by using a CPD formed at a TT site. On the other hand, biochemical analyses using CPDs formed within cytosine-containing sequence contexts are practically difficult, because saturated cytosine easily undergoes hydrolytic deamination. Here, we found that N-alkylation of the exocyclic amino group of 2'-deoxycytidine prevents hydrolysis in CPD formation, and an N-methylated cytosine-containing CPD was stable enough to be derivatized into its phosphoramidite building block and incorporated into oligonucleotides. Kinetic studies of the CPD-containing oligonucleotide indicated that its lifetime under physiological conditions is relatively long (∼ 7 days). In biochemical analyses using human DNA polymerase η, incorporation of TMP opposite the N-methylcytosine moiety of the CPD was clearly detected, in addition to dGMP incorporation, and the incorrect TMP incorporation blocked DNA synthesis. The thermodynamic parameters confirmed the formation of this unusual base pair.

Mechanism of somatic hypermutation at the WA motif by human DNA polymerase η.

Somatic hypermutation is programmed base substitutions in the variable regions of Ig genes for high-affinity antibody generation. Two motifs, RGYW and WA (R, purine; Y, pyrimidine; W, A or T), have been found to be somatic hypermutation hotspots. Overwhelming evidence suggests that DNA polymerase η (Pol η) is responsible for converting the WA motif to WG by misincorporating dGTP opposite the templating T. To elucidate the molecular mechanism, crystal structures and kinetics of human Pol η substituting dGTP for dATP in four sequence contexts, TA, AA, GA, and CA, have been determined and compared. The T:dGTP wobble base pair is stabilized by Gln-38 and Arg-61, two uniquely conserved residues among Pol η. Weak base paring of the W (T:A or A:T) at the primer end and their distinct interactions with Pol η lead to misincorporation of G in the WA motif. Between two WA motifs, our kinetic and structural data indicate that A-to-G mutation occurs more readily in the TA context than AA. Finally, Pol η can extend the T:G mispair efficiently to complete the mutagenesis.

Sphingosine, a modulator of human translesion DNA polymerase activity.

Translesion (TLS) DNA polymerases are specialized, error-prone enzymes that synthesize DNA across bulky, replication-stalling DNA adducts. In so doing, they facilitate the progression of DNA synthesis and promote cell proliferation. To potentiate the effect of cancer chemotherapeutic regimens, we sought to identify inhibitors of TLS DNA polymerases. We screened five libraries of ∼ 3000 small molecules, including one comprising ∼ 600 nucleoside analogs, for their effect on primer extension activity of DNA polymerase η (Pol η). We serendipitously identified sphingosine, a lipid-signaling molecule that robustly stimulates the activity of Pol η by ∼ 100-fold at low micromolar concentrations but inhibits it at higher concentrations. This effect is specific to the Y-family DNA polymerases, Pols η, κ, and ι. The addition of a single phosphate group on sphingosine completely abrogates this effect. Likewise, the inclusion of other sphingolipids, including ceramide and sphingomyelin to extension reactions does not elicit this response. Sphingosine increases the rate of correct and incorrect nucleotide incorporation while having no effect on polymerase processivity. Endogenous Pol η activity is modulated similarly as the recombinant enzyme. Importantly, sphingosine-treated cells exhibit increased lesion bypass activity, and sphingosine tethered to membrane lipids mimics the effects of free sphingosine. Our studies have uncovered sphingosine as a modulator of TLS DNA polymerase activity; this property of sphingosine may be associated with its known role as a signaling molecule in regulating cell proliferation in response to cellular stress.

Transcription cofactor PC4 plays essential roles in collaboration with the small subunit of general transcription factor TFIIE.

In eukaryotes, positive cofactor 4 (PC4) stimulates activator-dependent transcription by facilitating transcription initiation and the transition from initiation to elongation. It also forms homodimers and binds to single-stranded DNA and various transcriptional activators, including the general transcription factor TFIIH. In this study, we further investigated PC4 from Homo sapiens and the nematode Caenorhabditis elegans (hPC4 and cePC4, respectively). hPC4 strongly stimulated transcription on a linearized template, whereas it alleviated transcription on a supercoiled template. Transcriptional stimulation by PC4 was also alleviated by increasing the amount of TFIID. GST pull-down studies with general transcription factors indicated that both hPC4 and cePC4 bind strongly to TFIIB, TFIIEß, TFIIFα, TFIIFß and TFIIH XPB subunits and weakly to TBP and TFIIH p62. However, only hPC4 bound to CDK7. The effect of each PC4 on transcription was studied in combination with TFIIEß. hPC4 stimulated both basal and activated transcription, whereas cePC4 primarily stimulated activated transcription, especially in the presence of TFIIEß from C. elegans. Finally, hPC4 bound to the C-terminal region of hTFIIEß adjacent to the basic region. These results indicate that PC4 plays essential roles in the transition step from transcription initiation to elongation by binding to melted DNA in collaboration with TFIIEß.

Structure-function analysis of the EF-hand protein centrin-2 for its intracellular localization and nucleotide excision repair.

Centrin-2 is an evolutionarily conserved, calmodulin-related protein, which is involved in multiple cellular functions including centrosome regulation and nucleotide excision repair (NER) of DNA. Particularly to exert the latter function, complex formation with the XPC protein, the pivotal NER damage recognition factor, is crucial. Here, we show that the C-terminal half of centrin-2, containing two calcium-binding EF-hand motifs, is necessary and sufficient for both its localization to the centrosome and interaction with XPC. In XPC-deficient cells, nuclear localization of overexpressed centrin-2 largely depends on co-overexpression of XPC, and mutational analyses of the C-terminal domain suggest that XPC and the major binding partner in the centrosome share a common binding surface on the centrin-2 molecule. On the other hand, the N-terminal domain of centrin-2 also contains two EF-hand motifs but shows only low-binding affinity for calcium ions. Although the N-terminal domain is dispensable for enhancement of the DNA damage recognition activity of XPC, it contributes to augmenting rather weak physical interaction between XPC and XPA, another key factor involved in NER. These results suggest that centrin-2 may have evolved to bridge two protein factors, one with high affinity and the other with low affinity, thereby allowing delicate regulation of various biological processes.

Structural basis of human DNA polymerase η-mediated chemoresistance to cisplatin.

Cisplatin (cis-diamminedichloroplatinum) and related compounds cause DNA damage and are widely used as anticancer agents. Chemoresistance to cisplatin treatment is due in part to translesion synthesis by human DNA polymerase η (hPol η). Here, we report crystal structures of hPol η complexed with intrastrand cisplatin-1,2-cross-linked DNA, representing four consecutive steps in translesion synthesis. In contrast to the generally enlarged and nondiscriminating active site of Y-family polymerases like Dpo4, Pol η is specialized for efficient bypass of UV-cross-linked pyrimidine dimers. Human Pol η differs from the yeast homolog in its binding of DNA template. To incorporate deoxycytidine opposite cisplatin-cross-linked guanines, hPol η undergoes a specific backbone rearrangement to accommodate the larger base dimer and minimizes the DNA distortion around the lesion. Our structural analyses show why Pol η is inefficient at extending primers after cisplatin lesions, which necessitates a second translesion DNA polymerase to complete bypass in vivo. A hydrophobic pocket near the primer-binding site in human Pol η is identified as a potential drug target for inhibiting translesion synthesis and, thereby, reducing chemoresistance.