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Ceramide synthase 6 (CERS6) promotes lung cancer metastasis by stimulating cancer cell migration. To examine the underlying mechanisms, we performed luciferase analysis of the CERS6 promoter region and identified the Y‐box as a cis‐acting element. As a parallel analysis of database records for 149 non–small‐cell lung cancer (NSCLC) cancer patients, we screened for trans‐acting factors with an expression level showing a correlation with CERS6 expression. Among the candidates noted, silencing of either CCAAT enhancer‐binding protein γ (CEBPγ) or Y‐box binding protein 1 (YBX1) reduced the CERS6 expression level. Following knockdown, CEBPγ and YBX1 were found to be independently associated with reductions in ceramide‐dependent lamellipodia formation as well as migration activity, while only CEBPγ may have induced CERS6 expression through specific binding to the Y‐box. The mRNA expression levels of CERS6, CEBPγ, and YBX1 were positively correlated with adenocarcinoma invasiveness. YBX1 expression was observed in all 20 examined clinical lung cancer specimens, while 6 of those showed a staining pattern similar to that of CERS6. The present findings suggest promotion of lung cancer migration by possible involvement of the transcription factors CEBPγ and YBX1., Schematic illustration showing CEBPγ functions for cancer cell migration.

Background Disease progression in castrate-resistant prostate cancer (PCa) is most commonly driven by the reactivation of androgen receptor (AR) signaling and involves AR splice variants including ARV7. Materials and methods We used the ARV7-positive PCa cell line, 22Rv1, to study the relationship of the PCa marker α-methylacyl-CoA racemase (AMACR), AR, and ARV7 in PCa. Results Docetaxel addition but not AMACR inhibition decreased the proliferation of 22Rv1 cells. The combination of AMACR inhibition and docetaxel treatment resulted in a maximum reduction of cell proliferation. The Western blotting analysis revealed that both AR and ARV7 expression were significantly decreased with the use of charcoal-stripped serum following AMACR inhibition and docetaxel treatment. AMACR inhibition and docetaxel treatment in the charcoal-stripped serum condition reduced the proliferation of 22Rv1, possibly via the downregulation of the heat shock protein 27. Conclusion Using cell proliferation and Western blot analysis, we demonstrated that AMACR inhibition and docetaxel treatment, under androgen deprivation conditions, significantly reduced the proliferation of ARV7 positive cancer cells and decreased the levels of AR and ARV7 expression, possibly via downregulation of heat shock protein 27.

Sphingolipids constitute a class of bio‐reactive molecules that transmit signals and exhibit a variety of physical properties in various cell types, though their functions in cancer pathogenesis have yet to be elucidated. Analyses of gene expression profiles of clinical specimens and a panel of cell lines revealed that the ceramide synthase gene CERS6 was overexpressed in non–small‐cell lung cancer (NSCLC) tissues, while elevated expression was shown to be associated with poor prognosis and lymph node metastasis. NSCLC profile and in vitro luciferase analysis results suggested that CERS6 overexpression is promoted, at least in part, by reduced miR‐101 expression. Under a reduced CERS6 expression condition, the ceramide profile became altered, which was determined to be associated with decreased cell migration and invasion activities in vitro. Furthermore, CERS6 knockdown suppressed RAC1‐positive lamellipodia/ruffling formation and attenuated lung metastasis efficiency in mice, while forced expression of CERS6 resulted in an opposite phenotype in examined cell lines. Based on these findings, we consider that ceramide synthesis by CERS6 has important roles in lung cancer migration and metastasis.

Photons, such as X- or γ-rays, induce DNA damage (distributed throughout the nucleus) as a result of low-density energy deposition. In contrast, particle irradiation with high linear energy transfer (LET) deposits high-density energy along the particle track. High-LET heavy-ion irradiation generates a greater number and more complex critical chromosomal aberrations, such as dicentrics and translocations, compared with X-ray or γ irradiation. In addition, the formation of >1000 bp deletions, which is rarely observed after X-ray irradiation, has been identified following high-LET heavy-ion irradiation. Previously, these chromosomal aberrations have been thought to be the result of misrepair of complex DNA lesions, defined as DNA damage through DNA double-strand breaks (DSBs) and single-strand breaks as well as base damage within 1–2 helical turns (

Programmed cell death-1 (PD-1) and its ligand (programmed death-ligand 1, PD-L1) are key factors that regulate a cytotoxic immune reaction. Anti-PD-1 therapy provides significant clinical benefits for patients with cancer, even those with advanced-stage cancer. We have recently demonstrated that DNA damage signaling from DNA double-strand breaks (DSBs) promotes PD-L1 upregulation in cancer cells. In the present study, we aimed to investigate PD-L1 expression in primary normal human dermal fibroblasts (NHDFs) in response to DSBs. We demonstrated that PD-L1 expression in NHDFs is not upregulated after ionizing radiation (IR). In addition, interferon (IFN) regulatory factor 1 (IRF1) and signal transducer and activator of transcription 1 (STAT1) phosphorylation do not respond in NHDFs after IR. In contrast, IFNγ treatment upregulates PD-L1 and IRF1 expressions and STAT1 phosphorylation. The nonresponsiveness was also observed after treatment with other DNA-damaging agents, such as camptothecin and etoposide. Treatment with a histone deacetylase inhibitor (HDACi), which causes chromatin relaxation and restores gene silencing, upregulates PD-L1 without exogenous DNA damage; however, IR-dependent upregulation is not observed in NHDFs treated with HDACi. Taken together, our data suggest that DNA-damage signaling is insufficient for upregulating PD-L1 in NHDFs.

DNA double-strand breaks (DSBs) induced by ionising radiation are considered the major cause of genotoxic mutations and cell death. While DSBs are dispersed throughout chromatin after X-rays or γ-irradiation, multiple types of DNA damage including DSBs, single-strand breaks and base damage can be generated within 1–2 helical DNA turns, defined as a complex DNA lesion, after high Linear Energy Transfer (LET) particle irradiation. In addition to the formation of complex DNA lesions, recent evidence suggests that multiple DSBs can be closely generated along the tracks of high LET particle irradiation. Herein, by using three dimensional (3D)-structured illumination microscopy, we identified the formation of 3D widespread γH2AX foci after high LET carbon-ion irradiation. The large γH2AX foci in G2-phase cells encompassed multiple foci of replication protein A (RPA), a marker of DSBs undergoing resection during homologous recombination. Furthermore, we demonstrated by 3D analysis that the distance between two individual RPA foci within γH2AX foci was approximately 700 nm. Together, our findings suggest that high LET heavy-ion particles induce clustered DSB formation on a scale of approximately 1 μm3. These closely localised DSBs are considered to be a risk for the formation of chromosomal rearrangement after heavy-ion irradiation.

Immunotherapy is expected to be promising as a next generation cancer therapy. Immunoreceptors are often activated constitutively in cancer cells, however, such levels of ligand expression are not effectively recognized by the native immune system due to tumor microenvironmental adaptation. Studies have demonstrated that natural-killer group 2, member D (NKG2D), a major activating immunoreceptor, responds to DNA damage. The upregulation of major histocompatibility complex class I-related chain A and B (MICA/B) (members of NKG2D ligands) expression after DNA damage is associated with NK cell-mediated killing of cancer cells. However, the regulation of DNA damage-induced MICA/B expression has not been fully elucidated in the context of the types of cancer cell lines. In the present study, we found that MICA/B expression varied between cancer cell lines after DNA damage. Screening in terms of chromatin remodeling identified that inhibitors related to chromatin relaxation via post-translational modification on histone H3K9, i.e. HDAC, Suv39 or G9a inhibition, restored DNA damage-dependent MICA/B expression in insensitive cells. In addition, we revealed that the restored MICA/B expression was dependent on ATR as well as E2F1, a transcription factor. We further revealed that low-dose treatment of an HDAC inhibitor was sufficient to restore MICA/B expression in insensitive cells. Finally, we demonstrated that HDAC inhibition restored DNA damage-dependent cytotoxic NK activity against insensitive cells. Thus, the present study revealed that DNA damage-dependent MICA/B expression in insensitive cancer cells can be restored by chromatin relaxation via the HDAC/Suv39/G9a pathway. Collectively, manipulation of chromatin status by therapeutic cancer drugs may potentiate the antitumor effect by enhancing immune activation following radiotherapy and DNA damage-associated chemotherapy.

Chromosomal translocations arise from misrejoining of DNA double strand breaks (DSBs) between loci located on two chromosomes. One current model suggests that spatial proximity of potential chromosomal translocation partners influences translocation probability. Ionizing radiation (IR) is a potent inducer of translocations. Accumulating evidence demonstrates that particle irradiation more frequently causes translocations compared with X-ray irradiation. This observation has led to the hypothesis that the high frequency of translocations after particle irradiation may be due to the formation of DSBs at chromosome boundaries along the particle track, because such DSBs can be misrejoined between distinct chromosomes. In this study, we simultaneously visualized the site of IR-induced DSBs and chromosome position by combining Immunofluorescence and fluorescence in situ hybridization. Importantly, the frequency of γH2AX foci at the chromosome boundary of chromosome 1 after carbon-ion irradiation was >4-fold higher than that after X-ray irradiation. This observation is consistent with the idea that particle irradiation generates DSBs at the boundaries of two chromosomes along the track. Further, we showed that resolution of γH2AX foci at chromosome boundaries is prevented by inhibition of DNA-PKcs activity, indicating that the DSB repair is NHEJ-dependent. Finally, we found that γH2AX foci at chromosome boundaries after carbon-ion irradiation contain DSBs undergoing DNA-end resection, which promotes repair utilizing microhomology mediated end-joining during translocation. Taken together, our study suggests that the frequency of DSB formation at chromosome boundaries is associated with the incidence of chromosomal translocations, supporting the notion that the spatial proximity between breaks is an important factor in translocation formation. © 2016 Wiley Periodicals, Inc.

Monoubiquitination of proliferating cell nuclear antigen (PCNA) is a critical regulator of post replication repair (PRR). The depletion of BAF180, a unique subunit of the PBAF chromatin remodeling complex in human cells results in reduced PCNA ubiquitination leading to less efficient fork progression following DNA damage, but little is known about the mechanism. Here, we report that the expression of exogenous BAF180 in cells promotes PCNA ubiquitination during S-phase after UV irradiation and it persists for many hours. No correlation was observed between the protein level of ubiquitin-specific protease 1 (USP1) and ubiquitinated PCNA in BAF180 expressing cells. Analysis of cells expressing BAF180 deletion mutants showed that the bromo-adjacent homology (BAH) domains are responsible for this effect. Surprisingly, a deletion construct encoding only the BAH domain region is able to increase the level of ubiquitinated PCNA, even though it is unable to be assembled into the PBAF complex. These results suggest that the ATPase-dependent chromatin remodeling activity of PBAF is not necessary, but instead the BAH domains are sufficient to promote PCNA ubiquitination.

Summary: BRCA1 promotes homologous recombination (HR) by activating DNA-end resection. By contrast, 53BP1 forms a barrier that inhibits DNA-end resection. Here, we show that BRCA1 promotes DNA-end resection by relieving the 53BP1-dependent barrier. We show that 53BP1 is phosphorylated by ATM in S/G2 phase, promoting RIF1 recruitment, which inhibits resection. 53BP1 is promptly dephosphorylated and RIF1 released, despite remaining unrepaired DNA double-strand breaks (DSBs). When resection is impaired by CtIP/MRE11 endonuclease inhibition, 53BP1 phosphorylation and RIF1 are sustained due to ongoing ATM signaling. BRCA1 depletion also sustains 53BP1 phosphorylation and RIF1 recruitment. We identify the phosphatase PP4C as having a major role in 53BP1 dephosphorylation and RIF1 release. BRCA1 or PP4C depletion impairs 53BP1 repositioning, EXO1 recruitment, and HR progression. 53BP1 or RIF1 depletion restores resection, RAD51 loading, and HR in PP4C-depleted cells. Our findings suggest that BRCA1 promotes PP4C-dependent 53BP1 dephosphorylation and RIF1 release, directing repair toward HR. : Following induction of DNA double-strand break, a pro-end-joining environment is created in G2 by transient 53BP1 phosphorylation and RIF1 recruitment. Here, Isono et al. show that, if timely repair does not ensue, BRCA1 promotes 53BP1 dephosphorylation and RIF1 release, favoring repair by homologous recombination. Keywords: ATM, DNA-end resection, BRCA1, 53BP1, RIF1, PP4C, NHEJ, HR

After exposure to DNA-damaging agents that block the progress of the replication fork, monoubiquitination of proliferating cell nuclear antigen (PCNA) mediates the switch from replicative to translesion synthesis DNA polymerases. We show that in human cells, PCNA is monoubiquitinated in response to methyl methanesulfonate and mitomycin C, as well as UV light, albeit with different kinetics, but not in response to bleomycin or camptothecin. Cyclobutane pyrimidine dimers are responsible for most of the PCNA ubiquitination events after UV-irradiation. Failure to ubiquitinate PCNA results in substantial sensitivity to UV and methyl methanesulfonate, but not to camptothecin or bleomycin. PCNA ubiquitination depends on Replication Protein A (RPA), but is independent of ATR-mediated checkpoint activation. After UV-irradiation, there is a temporal correlation between the disappearance of the deubiquitinating enzyme USP1 and the presence of PCNA ubiquitination, but this correlation was not found after chemical mutagen treatment. By using cells expressing photolyases, we are able to remove the UV lesions, and we show that PCNA ubiquitination persists for many hours after the damage has been removed. We present a model of translesion synthesis behind the replication fork to explain the persistence of ubiquitinated PCNA.

In eukaryotic cells, replication past damaged sites in DNA is regulated by the ubiquitination of proliferating cell nuclear antigen (PCNA). Little is known about how this process is affected by chromatin structure. There are two isoforms of the Remodels the Structure of Chromatin (RSC) remodelling complex in yeast. We show that deletion of RSC2 results in a dramatic reduction in the level of PCNA ubiquitination after DNA-damaging treatments, whereas no such effect was observed after deletion of RSC1. Similarly, depletion of the BAF180 component of the corresponding PBAF (Polybromo BRG1 (Brahma-Related Gene 1) Associated Factor) complex in human cells led to a similar reduction in PCNA ubiquitination. Remarkably, we found that depletion of BAF180 resulted after UV-irradiation, in a reduction not only of ubiquitinated PCNA but also of chromatin-associated unmodified PCNA and Rad18 (the E3 ligase that ubiquitinates PCNA). This was accompanied by a modest decrease in fork progression. We propose a model to account for these findings that postulates an involvement of PBAF in repriming of replication downstream from replication forks blocked at sites of DNA damage. In support of this model, chromatin immunoprecipitation data show that the RSC complex in yeast is present in the vicinity of the replication forks, and by extrapolation, this is also likely to be the case for the PBAF complex in human cells.

We demonstrate that firefly luciferase is a good reporter in Escherichia coli for transcription dynamics in response to the environment. E. coli strains, carrying a fusion of the promoter of the ycgZ gene and the coding region of the luciferase gene, showed transient bioluminescence on receiving blue light. This response was compromised in mutants lacking known regulators in manners consistent with each regulator’s function. We also show that relA , a gene encoding a (p)ppGpp synthetase, affects ycgZ dynamics when nullified. Moreover, two unstable luciferase variants showed improved response dynamics and should be useful to study quick changes of gene expression.

Translesion DNA synthesis (TLS) involves PCNA mono-ubiquitination and TLS DNA polymerases (pols). Recent evidence has shown that the mono-ubiquitination is induced not only by DNA damage but also by other factors that induce stalling of the DNA replication fork. We studied the effect of spontaneous DNA replication errors on PCNA mono-ubiquitination and TLS induction. In the pol1L868F strain, which expressed an error-prone pol alpha, PCNA was spontaneously mono-ubiquitinated. Pol alpha L868F had a rate-limiting step at the extension from mismatched primer termini. Electron microscopic observation showed the accumulation of a single-stranded region at the DNA replication fork in yeast cells. For pol alpha errors, pol zeta participated in a generation of +1 frameshifts. Furthermore, in the pol1L868F strain, UV-induced mutations were lower than in the wild-type and a pol delta mutant strain (pol3-5DV), and deletion of the RAD30 gene (pol eta) suppressed this defect. These data suggest that nucleotide misincorporation by pol alpha induces exposure of single-stranded DNA, PCNA mono-ubiquitination and activates TLS pols.

Eukaryotic replication begins at origins and on the lagging strand with RNA-primed DNA synthesis of a few nucleotides by polymerase alpha, which lacks proofreading activity. A polymerase switch then allows chain elongation by proofreading-proficient pol delta and pol epsilon. Pol delta and pol epsilon are essential, but their roles in replication are not yet completely defined . Here, we investigate their roles by using yeast pol alpha with a Leu868Met substitution . L868M pol alpha copies DNA in vitro with normal activity and processivity but with reduced fidelity. In vivo, the pol1-L868M allele confers a mutator phenotype. This mutator phenotype is strongly increased upon inactivation of the 3' exonuclease of pol delta but not that of pol epsilon. Several nonexclusive explanations are considered, including the hypothesis that the 3' exonuclease of pol delta proofreads errors generated by pol alpha during initiation of Okazaki fragments. Given that eukaryotes encode specialized, proofreading-deficient polymerases with even lower fidelity than pol alpha, such intermolecular proofreading could be relevant to several DNA transactions that control genome stability.

We isolated active mutants in Saccharomyces cerevisiae DNA polymerase alpha that were associated with a defect in error discrimination. Among them, L868F DNA polymerase alpha has a spontaneous error frequency of 3 in 100 nucleotides and 570-fold lower replication fidelity than wild-type (WT) polymerase alpha. In vivo, mutant DNA polymerases confer a mutator phenotype and are synergistic with msh2 or msh6, suggesting that DNA polymerase alpha-dependent replication errors are recognized and repaired by mismatch repair. In vitro, L868F DNA polymerase alpha catalyzes efficient bypass of a cis-syn cyclobutane pyrimidine dimer, extending the 3' T 26000-fold more efficiently than the WT. Phe34 is equivalent to residue Leu868 in translesion DNA polymerase eta, and the F34L mutant of S. cerevisiae DNA polymerase eta has reduced translesion DNA synthesis activity in vitro. These data suggest that high-fidelity DNA synthesis by DNA polymerase alpha is required for genomic stability in yeast. The data also suggest that the phenylalanine and leucine residues in translesion and replicative DNA polymerases, respectively, might have played a role in the functional evolution of these enzyme classes.

Structural differences between class A and B DNA polymerases suggest that the motif B region, a wall of the catalytic pocket, may have evolved differentially in the two polymerase families. This study examines the function of the motif B residues in Saccharomyces cerevisiae DNA polymerase alpha (pol alpha). Effects of the mutations were determined by biochemical analysis and genetic complementation of a yeast strain carrying a temperature-sensitive pol alpha mutant. Many conserved residues were viable with a variety of substitutions. Among them, mutations at Asn-948 or Tyr-951 conferred up to 8-fold higher colony formation frequency in a URA3 forward mutation assay, and 79-fold higher trp1 reversion frequency was observed for Y951P in yeast. Purified Y951P was as accurate as wild type in DNA synthesis but approximately 6-fold less processive and 22-fold less active in vitro. Therefore, Y951P may increase the frequency of mutant colony formation because of its low level of DNA polymerase activity in yeast. Mutations at Lys-944 or Gly-952 were not viable, which is consistent with the observation that mutants with substitutions at Gly-952 have strongly reduced catalytic activity in vitro. Gly-952 may provide a space for the nascent base pair and thus may play an essential function in S. cerevisiae DNA pol alpha. These results suggest that class B DNA polymerases have a unique structure in the catalytic pocket, which is distinct from the corresponding region in class A DNA polymerases.

Gly-952 is a conserved residue in Saccharomyces cerevisiae DNA polymerase alpha (pol alpha) that is strictly required for catalytic activity and for genetic complementation of a pol alpha-deficient yeast strain. This study analyzes the role of Gly-952 by characterizing the biochemical properties of Gly-952 mutants. Analysis of the nucleotide incorporation specificity of pol alpha G952A showed that this mutant incorporates nucleotides with extraordinarily low fidelity. In a steady-state kinetic assay to measure nucleotide misincorporation, pol alpha G952A incorporated incorrect nucleotides more efficiently than correct nucleotides opposite template C, G, and T. The fidelity of the G952A mutant polymerase was highest at template A, where the ratio of incorporation of dCMP to dTMP was as high as 0.37. Correct nucleotide insertion was 500- to 3500-fold lower for G952A than for wild type pol alpha, with up to 22-fold increase in pyrimidine misincorporation. The Km for G952A pol alpha bound to mismatched termini T:T, T:C, C:A, and A:C was 71- to 460-fold lower than to a matched terminus. Furthermore, pol alpha G952A preferentially incorporated pyrimidine instead of dAMP opposite an abasic site, cis-syn cyclobutane di-thymine, or (6-4) di-thymine photoproduct. These data demonstrate that Gly-952 is a critical residue for catalytic efficiency and error prevention in S. cerevisiae pol alpha.

Chromosome rearrangement is clinically and physiologically important because it can produce oncogenic fusion genes. Chromosome rearrangement requires DNA double-strand breaks (DSBs) at two genomic locations and misrejoining between the DSBs. Before DSB misrejoining, two DSB-containing chromatin regions move and pair with each other; however, the molecular mechanism underlying this process is largely unknown. We performed a spatiotemporal analysis of ionizing radiation-induced foci of p53-binding protein 1 (53BP1), a marker for DSB-containing chromatin. We found that some 53BP1 foci were paired, indicating that the two damaged chromatin regions neighboured one another. We searched for factors regulating the foci pairing and found that the number of paired foci increased when Ku80, DNA-PKcs, or ATM was absent. In contrast, 53BP1 depletion reduced the number of paired foci and dicentric chromosomes - an interchromosomal rearrangement. Foci were paired more frequently in heterochromatin than in euchromatin in control cells. Additionally, the reduced foci pairing in 53BP1-depleted cells was rescued by concomitant depletion of a heterochromatin building factor such as Kruppel-associated box-associated protein 1 or chromodomain helicase DNA-binding protein 3. These findings indicate that pairing between DSB-containing chromatin regions was suppressed by Ku80, DNA-PKcs, and ATM, and this pairing was promoted by 53BP1 through chromatin relaxation., Scientific Reports, 7, 41812; 2017

Clones for DNA topoisomerase IIalpha and beta (topo-IIalpha and beta) were isolated from a cDNA expression library of chicken MSB-1 cells by immunoscreening. The deduced sequences of chicken topo-IIalpha and beta were about 80% identical for the N-terminal ATPase domain and the central core domain but only 37% for the C-terminal domain. Polyclonal antibodies were raised against C-terminal polypeptides specific to topo-IIalpha and beta. Indirect immunofluorescence with these antibodies to chicken embryonic fibroblasts demonstrated that topo-IIalpha was distributed in discrete intranuclear spots, which coincided with sites of DNA replication as indicated by incorporation of 5-bromo-2'-deoxyuridine, whereas topo-IIbeta was distributed rather uniformly within a nucleus. Examination of intranuclear distribution patterns of chimeric constructs between topo-IIalpha and beta suggested that a sequence region (residues 1280-1294) in the C-terminal domain of topo-IIalpha was effective in co-localization with sites of DNA replication. This region consists of a QTxhxF motif (x, any residue; h, hydrophobic residue) followed by a KR-rich sequence, which resembles those found in several proteins known to associate with proliferating cell nuclear antigen (PCNA) or targeted to the replication factory. An in vitro pull-down assay with glutathione-S-transferase-PCNA and (His)6-tagged truncated forms of topo-IIalpha demonstrated that polypeptides containing the above region (residues 1158-1553 or 1158-1294) bound to PCNA in vitro.

Nucleotide excision repair (NER) is the most versatile DNA repair system that deals with the major UV photoproducts in DNA, as well as many other DNA adducts. The early steps of NER are well understood, whereas the later steps of repair synthesis and ligation are not. In particular, which polymerases are definitely involved in repair synthesis and how they are recruited to the damaged sites has not yet been established. We report that, in human fibroblasts, approximately half of the repair synthesis requires both pol kappa and pol delta, and both polymerases can be recovered in the same repair complexes. Pol kappa is recruited to repair sites by ubiquitinated PCNA and XRCC1 and pol delta by the classical replication factor complex RFC1-RFC, together with a polymerase accessory factor, p66, and unmodified PCNA. The remaining repair synthesis is dependent on pol epsilon, recruitment of which is dependent on the alternative clamp loader CTF18-RFC.

DNA polymerase eta is a Y family polymerase involved in translesion synthesis (TLS). Its action is initiated by simultaneous interaction between the PIP box in pol eta and PCNA and between the UBZ in pol eta and monoubiquitin attached to PCNA. Whereas monoubiquitination of PCNA is required for its interaction with pol eta during TLS, we now show that monoubiquitination of pol eta inhibits this interaction, preventing its functions in undamaged cells. Identification of monoubiquitination sites within pol eta nuclear localization signal (NLS) led to the discovery that pol eta NLS directly contacts PCNA, forming an extended pol eta-PCNA interaction surface. We name this the PCNA-interacting region (PIR) and show that its monoubiquitination is downregulated by various DNA-damaging agents. We propose that this mechanism ensures optimal availability of nonubiquitinated, TLS-competent pol eta after DNA damage. Our work shows how monoubiquitination can either positively or negatively regulate the assembly of a protein complex, depending on which substrates are targeted by ubiquitin.

Recent studies have revealed that the base selection step of DNA polymerases (pol) plays a role in prevention of DNA replication errors. We investigated whether base selection is required for the DNA replication fidelity of pol alpha and genomic stability in human cells. We introduced an Leu864 to Phe substitution (L864F) into human pol alpha and performed an in vitro LacZ alpha forward mutation assay. Our results showed that the overall mutation rate was increased by 180-fold as compared to that of the wild-type. Furthermore, steady state kinetics analyses consistently showed that L864F pol alpha had a decreased discrimination ability between correct and incorrect nucleotide incorporation, as well as between matched and mismatched primer termini. L864F pol alpha also exhibited increased translesion activity over the abasic, etheno-A, O(4)-methyl-T, and O(6)-methyl-G sites. In addition, our steady state kinetics analyses supported the finding of increased translesion activity of L864F pol alpha over O(6)-methyl-G. We also established stable clones transfected with pola1L864F utilizing the human cancer cell line HCT116. Using the HPRT gene as a reporter, the spontaneous mutation rate of pola1L864F cells was determined to be 2.4-fold greater than that of wild-type cells. Mutation assays were also carried out using cells transiently transfected with the wild-type or pola1L864F, and increased mutant frequencies were observed in pola1L864F cells under both spontaneous and methyl methanesulfonate-induced conditions. Together, our results indicate that the base selection step in human pol alpha functions to prevent DNA replication errors and maintain genomic integrity in HCT116 cells.

Following exposure of human cells to DNA damaging agents that block the progress of the replication fork, mono-ubiquitination of PCNA mediates the switch from replicative DNA polymerases to polymerases specialised for translesion synthesis. We have shown that this modification of PCNA is necessary for the survival of cells after UV-irradiation and methyl methanesulfonate, that it is independent of cell cycle checkpoint activation, and that it persists after UV damage has been removed. In this Extra-view, we compare the regulation and biological significance of PCNA ubiquitination following treatments with UV light and the replication inhibitor hydroxyurea. We show that ubiquitination persists after removal of the replication block in both cases. With UV however, the persistence of ubiquitinated PCNA correlates with disappearance of the PCNA deubiquitinating enzyme USP1, whereas this is not the case for HU. Prevention of PCNA ubiquitination sensitises the cells to killing by both UV and HU.

Proteasome inhibitors are novel antitumor agents against multiple myeloma and other malignancies. Despite the increasing clinical application, the molecular basis of their antitumor effect has been poorly understood due to the involvement of the ubiquitin-proteasome pathway in multiple cellular metabolisms. Here, we show that treatment of cells with proteasome inhibitors has no significant effect on nonhomologous end joining but suppresses homologous recombination (HR), which plays a key role in DNA double-strand break (DSB) repair. In this study, we treat human cells with proteasome inhibitors and show that the inhibition of the proteasome reduces the efficiency of HR-dependent repair of an artificial HR substrate. We further show that inhibition of the proteasome interferes with the activation of Rad51, a key factor for HR, although it does not affect the activation of ATM, γH2AX, or Mre11. These data show that the proteasome-mediated destruction is required for the promotion of HR at an early step. We suggest that the defect in HR-mediated DNA repair caused by proteasome inhibitors contributes to antitumor effect, as HR plays an essential role in cellular proliferation. Moreover, because HR plays key roles in the repair of DSBs caused by chemotherapeutic agents such as cisplatin and by radiotherapy, proteasome inhibitors may enhance the efficacy of these treatments through the suppression of HR-mediated DNA repair pathways. [Cancer Res 2007;67(18):8536–43]

Replicative DNA polymerases are blocked at DNA lesions. Synthesis past DNA damage requires the replacement of the replicative polymerase by one of a group of specialised translesion synthesis (TLS) polymerases, most of which belong to the Y-family. Each of these has different substrate specificities for different types of damage. In eukaryotes mono-ubiquitination of PCNA plays a crucial role in the switch from replicative to TLS polymerases at stalled forks. All the Y-family polymerases have ubiquitin binding sites that increase their binding affinity for ubiquitinated PCNA at the sites of stalled forks.

Xeroderma pigmentosum (XP) is an autosomal recessive genetic disorder. Afflicted patients show extreme sun-sensitivity and skin cancer predisposition. XP is in most cases associated with deficient nucleotide excision repair (NER), which is the process responsible for removing photolesions from DNA. Measuring NER activity by nucleotide incorporation into repair patches, termed 'unscheduled DNA synthesis (UDS)', is one of the most commonly used assays for XP-diagnosis and NER research. We have established a rapid and accurate procedure for measuring UDS by replacement of thymidine with 5-ethynyl-2'-deoxyuridine (EdU). EdU incorporated into repair patches can be directly conjugated to fluorescent azide derivatives, thereby obviating the need for either radiolabeled thymidine or denaturation and antibody detection of incorporated bromodeoxyuridine (BrdU). We demonstrate that the EdU incorporation assay is compatible with conventional techniques such as immunofluorescent staining and labeling of cells with micro-latex beads. Importantly, we can complete the entire UDS assay within half a day from preparation of the assay coverslips; this technique may prove useful as a method for XP diagnosis., Nucleic Acids Research 37(4), e31; 2009