Human DNA polymerase eta activity and translocation is regulated by phosphorylation

Human DNA polymerase eta (pol eta) can replicate across UV-induced pyrimidine dimers, and defects in the gene encoding pol eta result in a syndrome called xeroderma pigmentosum variant (XP-V). XP-V patients are prone to the development of cancer in sun-exposed areas, and cells derived from XP-V patients demonstrate increased sensitivity to UV radiation and a higher mutation rate compared with wild-type cells. pol eta has been shown to replicate across a wide spectrum of DNA lesions introduced by environmental or chemotherapeutic agents, or during nucleotide starvation, suggesting that the biological roles for pol eta are not limited to repair of UV-damaged DNA. The high error rate of pol eta requires that its intracellular activity be tightly regulated. Here, we show that the phosphorylation of pol eta increased after UV irradiation, and that treatment with caffeine, siRNA against ATR, or an inhibitor of PKC (calphostin C), reduced the accumulation of pol eta at stalled replication forks after UV irradiation or treatment with cisplatin and gemcitabine. Site-specific mutagenesis (S587A and T617A) of pol eta at two putative PKC phosphorylation sites located in the protein-protein interaction domain prevented nuclear foci formation induced by UV irradiation or treatment with gemcitabine/cisplatin. In addition, XP-V cell lines stably expressing either the S587A or T617A mutant form of pol eta were more sensitive to UV radiation and gemcitabine/cisplatin than control cells expressing wild-type pol eta. These results suggest that phosphorylation is one mechanism by which the cellular activity of pol eta is regulated.     

Essential function of Chk1 can be uncoupled from DNA damage checkpoint and replication control

Chk1 is widely known as a DNA damage checkpoint signaling protein. Unlike many other checkpoint proteins, Chk1 also plays an essential but poorly defined role in the proliferation of unperturbed cells. Activation of Chk1 after DNA damage is known to require the phosphorylation of several C-terminal residues, including the highly conserved S317 and S345 sites. To evaluate the respective roles of these individual sites and assess their contribution to the functions of Chk1, we used a gene targeting approach to introduce point mutations into the endogenous human CHK1 locus. We report that the essential and nonessential functions of Chk1 are regulated through distinct phosphorylation events and can be genetically uncoupled. The DNA damage response function of Chk1 was nonessential. Targeted mutation of S317 abrogated G₂/M checkpoint activation, prevented subsequent phosphorylation of Chk1, impaired efficient progression of DNA replication forks, and increased fork stalling, but did not impact viability. Thus, the nonessential DNA damage response function of Chk1 could be unambiguously linked to its role in DNA replication control. In contrast, a CHK1 allele with mutated S345 did not support viability, indicating an essential role for this residue during the unperturbed cell cycle. A distinct, physiologic mode of S345 phosphorylation, initiated at the centrosome during unperturbed mitosis was independent of codon 317 status and mechanistically distinct from the ordered and sequential phosphorylation of serine residues on Chk1 induced by DNA damage. Our findings suggest an essential regulatory role for Chk1 phosphorylation during mitotic progression.     

Tipin is required for stalled replication forks to resume DNA replication after removal of aphidicolin in Xenopus egg extracts

Tipin and its interacting partner Tim1 (Timeless) form a complex at replication forks that plays an important role in the DNA damage checkpoint response. Here we identify Xenopus laevis Tipin as a substrate for cyclin E/cyclin-dependent kinases 2 that is phosphorylated in interphase and undergoes further phosphorylation upon entry into mitosis. During unperturbed DNA replication, the Tipin/Tim1 complex is bound to chromatin, and we were able to detect interactions between Tipin and the MCM helicase. Depletion of Tipin from Xenopus extracts did not significantly impair normal replication but substantially blocked the ability of stalled replication forks to recover after removal of a block imposed by aphidicolin. Tipin-depleted extracts also showed defects in the activation of Chk1 in response to aphidicolin, probably because of a failure to load the checkpoint mediator protein Claspin onto chromatin.     

Chk1 promotes replication fork progression by controlling replication initiation

DNA replication starts at initiation sites termed replication origins. Metazoan cells contain many more potential origins than are activated (fired) during each S phase. Origin activation is controlled by the ATR checkpoint kinase and its downstream effector kinase Chk1, which suppresses origin firing in response to replication blocks and during normal S phase by inhibiting the cyclin-dependent kinase Cdk2. In addition to increased origin activation, cells deficient in Chk1 activity display reduced rates of replication fork progression. Here we investigate the causal relationship between increased origin firing and reduced replication fork progression. We use the Cdk inhibitor roscovitine or RNAi depletion of Cdc7 to inhibit origin firing in Chk1-inhibited or RNAi-depleted cells. We report that Cdk inhibition and depletion of Cdc7 can alleviate the slow replication fork speeds in Chk1-deficient cells. Our data suggest that increased replication initiation leads to slow replication fork progression and that Chk1 promotes replication fork progression during normal S phase by controlling replication origin activity.     

Roles of DNA polymerases V and II in SOS-induced error-prone and error-free repair in Escherichia coli

DNA polymerase V, composed of a heterotrimer of the DNA damage-inducible UmuC and UmuD(2)(') proteins, working in conjunction with RecA, single-stranded DNA (ssDNA)-binding protein (SSB), beta sliding clamp, and gamma clamp loading complex, are responsible for most SOS lesion-targeted mutations in Escherichia coli, by catalyzing translesion synthesis (TLS). DNA polymerase II, the product of the damage-inducible polB (dinA ) gene plays a pivotal role in replication-restart, a process that bypasses DNA damage in an error-free manner. Replication-restart takes place almost immediately after the DNA is damaged (approximately 2 min post-UV irradiation), whereas TLS occurs after pol V is induced approximately 50 min later. We discuss recent data for pol V-catalyzed TLS and pol II-catalyzed replication-restart. Specific roles during TLS for pol V and each of its accessory factors have been recently determined. Although the precise molecular mechanism of pol II-dependent replication-restart remains to be elucidated, it has recently been shown to operate in conjunction with RecFOR and PriA proteins.     

A phenotype for enigmatic DNA polymerase II: A pivotal role for pol II in replication restart in UV-irradiated Escherichia coli

DNA synthesis in Escherichia coli is inhibited transiently after UV irradiation. Induced replisome reactivation or "replication restart" occurs shortly thereafter, allowing cells to complete replication of damaged genomes. At the present time, the molecular mechanism underlying replication restart is not understood. DNA polymerase II (pol II), encoded by the dinA (polB) gene, is induced as part of the global SOS response to DNA damage. Here we show that pol II plays a pivotal role in resuming DNA replication in cells exposed to UV irradiation. There is a 50-min delay in replication restart in mutant cells lacking pol II. Although replication restart appears normal in DeltaumuDC strains containing pol II, the restart process is delayed for >90 min in cells lacking both pol II and UmuD'(2)C. Because of the presence of pol II, a transient replication-restart burst is observed in a "quick-stop" temperature-sensitive pol III mutant (dnaE486) at nonpermissive temperature. However, complete recovery of DNA synthesis requires the concerted action of both pol II and pol III. Our data demonstrate that pol II and UmuD'(2)C act in independent pathways of replication restart, thereby providing a phenotype for pol II in the repair of UV-damaged DNA.     

Chromatin assembly factor 1 is essential and couples chromatin assembly to DNA replication in vivo

De novo chromatin assembly maintains histone density on the daughter strands in the wake of the replication fork. The heterotrimer chromatin assembly factor 1 (CAF-1) couples DNA replication to histone deposition in vitro, but is not essential for yeast cell proliferation. Depletion of CAF-1 in human cell lines demonstrated that CAF-1 was required for efficient progression through S-phase. Cells lacking CAF-1 accumulated in early and mid S-phase and replicated DNA slowly. The checkpoint kinase Chk1, but not Chk2, was phosphorylated in response to CAF-1 depletion, consistent with a DNA replication defect. CAF-1-depleted cell extracts completely lacked DNA replication-coupled chromatin assembly activity, suggesting that CAF-1 is required for efficient S-phase progression in human cells. These results indicate that, in contrast to yeast, human CAF-1 is necessary for coupling chromatin assembly with DNA replication.     

Human Claspin works with BRCA1 to both positively and negatively regulate cell proliferation

Claspin is a homolog of Mrc1, a checkpoint protein required for the DNA replication checkpoint in yeast. In Xenopus, phosphorylated Claspin binds to xChk1 and regulates xChk1 activation in response to replication stress. In this study, we have shown that the human homolog of Claspin is required for resistance to multiple forms of genotoxic stress including UV, IR, and hydroxyurea. Phosphorylation of Claspin was found to depend on the ataxia telangiectasia mutated-Rad3 related (ATR) pathway. DNA damage induces the formation of a complex between Claspin and BRCA1, a second regulator of Chk1 activation. Claspin was found to control BRCA1 phosphorylation on serine 1524, a site whose phosphorylation is controlled by the ATR pathway. These results are consistent with a model in which ATR regulates Claspin phosphorylation in response to DNA damage and replication stress resulting in recruitment and phosphorylation of BRCA1. BRCA1 and Claspin then function to activate the tumor suppressor Chk1. Unexpectedly, we found that Claspin has a second, positive role in control of the cell cycle as Claspin overexpression increased cell proliferation. These results suggest that Claspin has properties of both a tumor suppressor and an oncogene.     

From the Cover: Reconstitution of RPA-covered single-stranded DNA-activated ATR-Chk1 signaling

ATR kinase is a critical upstream regulator of the checkpoint response to various forms of DNA damage. Previous studies have shown that ATR is recruited via its binding partner ATR-interacting protein (ATRIP) to replication protein A (RPA)-covered single-stranded DNA (RPA-ssDNA) generated at sites of DNA damage where ATR is then activated by TopBP1 to phosphorylate downstream targets including the Chk1 signal transducing kinase. However, this critical feature of the human ATR-initiated DNA damage checkpoint signaling has not been demonstrated in a defined system. Here we describe an in vitro checkpoint system in which RPA-ssDNA and TopBP1 are essential for phosphorylation of Chk1 by the purified ATR-ATRIP complex. Checkpoint defective RPA mutants fail to activate ATR kinase in this system, supporting the conclusion that this system is a faithful representation of the in vivo reaction. Interestingly, we find that an alternative form of RPA (aRPA), which does not support DNA replication, can substitute for the checkpoint function of RPA in vitro, thus revealing a potential role for aRPA in the activation of ATR kinase. We also find that TopBP1 is recruited to RPA-ssDNA in a manner dependent on ATRIP and that the N terminus of TopBP1 is required for efficient recruitment and activation of ATR kinase.     

Werner syndrome protein interacts functionally with translesion DNA polymerases

Werner syndrome (WS) is characterized by premature onset of age-associated disorders and predisposition to cancer. The WS protein, WRN, encodes 3' --> 5' DNA helicase and 3' --> 5' DNA exonuclease activities, and is implicated in the maintenance of genomic stability. Translesion (TLS) DNA polymerases (Pols) insert nucleotides opposite replication-blocking DNA lesions and presumably prevent replication fork stalling/collapse. Here, we present in vitro and in vivo data that demonstrate functional interaction between WRN and the TLS Pols, Poleta, Polkappa, and Poliota. In vitro, WRN stimulates the extension activity of TLS Pols on lesion-free and lesion-containing DNA templates, and alleviates pausing at stalling lesions. Stimulation is mediated through an increase in the apparent V(max) of the polymerization reaction. Notably, by accelerating the rate of nucleotide incorporation, WRN increases mutagenesis by Poleta. In vivo, WRN and Poleta colocalize at replication-dependent foci in response to UVC irradiation. The functional interaction between WRN and TLS Pols may promote replication fork progression, at the expense of increased mutagenesis, and obviate the need to resolve stalled/collapsed forks by processes involving chromosomal rearrangements.     

Rad51 recombinase prevents Mre11 nuclease-dependent degradation and excessive PrimPol-mediated elongation of nascent DNA after UV irradiation

After UV irradiation, DNA polymerases specialized in translesion DNA synthesis (TLS) aid DNA replication. However, it is unclear whether other mechanisms also facilitate the elongation of UV-damaged DNA. We wondered if Rad51 recombinase (Rad51), a factor that escorts replication forks, aids replication across UV lesions. We found that depletion of Rad51 impairs S-phase progression and increases cell death after UV irradiation. Interestingly, Rad51 and the TLS polymerase polη modulate the elongation of nascent DNA in different ways, suggesting that DNA elongation after UV irradiation does not exclusively rely on TLS events. In particular, Rad51 protects the DNA synthesized immediately before UV irradiation from degradation and avoids excessive elongation of nascent DNA after UV irradiation. In Rad51-depleted samples, the degradation of DNA was limited to the first minutes after UV irradiation and required the exonuclease activity of the double strand break repair nuclease (Mre11). The persistent dysregulation of nascent DNA elongation after Rad51 knockdown required Mre11, but not its exonuclease activity, and PrimPol, a DNA polymerase with primase activity. By showing a crucial contribution of Rad51 to the synthesis of nascent DNA, our results reveal an unanticipated complexity in the regulation of DNA elongation across UV-damaged templates.The DNA-binding protein Rad51 is a central component of homologous recombination repair (HRR). HRR repairs double-strand breaks (DSBs) in an error-free way and processes one-ended DSBs to reactivate collapsed replication forks (1). During HRR, DSBs are processed by the 3′-to-5′ exonuclease activity of the double strand break repair nuclease (Mre11) to generate protruding 3′ ssDNA at DSBs. The ssDNA is then coated with Rad51, a factor that catalyzes homology search and strand invasion. The loading and stabilization of Rad51/ssDNA complexes are supported by multiple mediators, such as the tumor suppressor BRCA2 (breast cancer 2) (1). Moreover, Rad51 promotes XPF1- and Exo1-mediated DSB formation after gemcitabine-induced irreversible ribonucleotide reductase inhibition, thus promoting cell death (2). The signals that redirect Rad51 into a DSB formation pathway rather than DSB repair are not yet known.The functions of Rad51 are not limited to the processing/generation of DSBs. Over the past few years, it has become evident that Rad51 escorts ongoing replication forks regardless of the presence of DSBs (3–5). Specifically, Rad51 protects persistently stalled replication forks from Mre11-mediated nucleolytic degradation and facilitates replication fork restart when the replication-halting agent hydroxyurea (HU) or aphidicolin (APH) is removed (6–19). Such novel functions of Rad51 require many HRR factors, including BCRA2, FANCD2 (Fanconi Anemia Complementation group protein D2), CtIP, BRCA1, and the WRN helicase, but are independent of HRR effectors, such as Rad54 (6, 7). Rad51-dependent fork-restart and fork-protection are distinct mechanisms, because proteins like the BLM helicase promote the former, but not the latter, process after HU treatment (6, 20). Mre11-mediated nucleolytic degradation of nascent DNA in BCRA2- and FANCD2-depleted, HU-treated cells was suggested to take place at the unprotected ends of reversed forks, which may mimic DSBs (6–8). Conversely, two recent reports suggest that Rad51 prevents pathological Mre11-dependent nucleolytic degradation of nonreversed stalled forks and promotes controlled DNA2-dependent exonucleolytic processing of reversed forks (15, 21).DSB-independent Rad51 functions were revealed by the use of agents that cause a significant degree (more than 40%) of replication fork stalling, such as HU, camptothecin (CPT), and mitomycin C (MMC) (15). Much less is known about the participation of Rad51 in the replication across DNA lesions that do not persistently halt replication forks and only cause a moderate reduction in the replication speed (e.g., UV-induced DNA lesions) (22). Multiple mechanisms aid DNA replication after UV irradiation. First, strongly distorting UV lesions are effectively removed by nucleotide excision repair (NER). Second, mildly distorting lesions, which are less efficiently removed by NER, can be used as replication templates in translesion DNA synthesis (TLS) events (23). TLS avoids fork stalling by loading specialized DNA polymerases that use damaged DNA as replication templates (24).It is currently unclear whether non-TLS events aid DNA replication across UV-damaged DNA in mammalian systems. Importantly, Rad51 is recruited to DNA after UV irradiation (4, 21, 25). HRR factors contribute to the repair of infrequent DSBs generated by high UV doses (80 J/m²) in NER-deficient backgrounds (26). Interestingly, however, cancer cells depleted from Rad51 were sensitive to much lower UV doses (1–5 J/m²) (26), thus suggesting DSB-independent functions of Rad51 in the cellular response to UV light. More recently, Rad51 was shown to maintain continuous DNA replication after treatment with methyl-methane sulfonate (MMS), a DNA-damaging agent that induces bulky lesions similar to the lesions caused by UV radiation (4). Whether Rad51 prevents nucleolytic degradation of nascent DNA in response to UV irradiation has not yet been explored. Remarkably, fork reversal takes place frequently after UV irradiation, similar to the case with HU, MMC, and CPT (21).We therefore set out to investigate the contribution of Rad51 to DNA replication across UV lesions. Using the DNA stretching technique (27), we uncovered two DSB-independent roles of Rad51 in the replication of UV-damaged DNA. First, Rad51 protected the nascent DNA from rapid and time-limited Mre11-dependent exonucleolytic degradation. Second, Rad51 prevented excessive DNA elongation after UV irradiation. Such dysregulated elongation of DNA was orchestrated by Mre11, but not by its exonuclease activity, and a DNA polymerase with primase activity, PrimPol (primase polymerase). Our results therefore suggest that Rad51 depletion increases repriming after UV irradiation. Intriguingly, both Rad51-mediated functions affected the accumulation of DNA damage response (DDR) markers at later time points, but only the excessive fork elongation in Rad51-depleted cells was associated with cell death. Finally, we demonstrate that the TLS DNA polymerase polη and Rad51 are both required to achieve optimal elongation of nascent UV-damaged DNA.     

Damage-induced DNA replication stalling relies on MAPK-activated protein kinase 2 activity

DNA damage can obstruct replication forks, resulting in replicative stress. By siRNA screening, we identified kinases involved in the accumulation of phosphohistone 2AX (γH2AX) upon UV irradiation-induced replication stress. Surprisingly, the strongest reduction of phosphohistone 2AX followed knockdown of the MAP kinase-activated protein kinase 2 (MK2), a kinase currently implicated in p38 stress signaling and G2 arrest. Depletion or inhibition of MK2 also protected cells from DNA damage-induced cell death, and mice deficient for MK2 displayed decreased apoptosis in the skin upon UV irradiation. Moreover, MK2 activity was required for damage response, accumulation of ssDNA, and decreased survival when cells were treated with the nucleoside analogue gemcitabine or when the checkpoint kinase Chk1 was antagonized. By using DNA fiber assays, we found that MK2 inhibition or knockdown rescued DNA replication impaired by gemcitabine or by Chk1 inhibition. This rescue strictly depended on translesion DNA polymerases. In conclusion, instead of being an unavoidable consequence of DNA damage, alterations of replication speed and origin firing depend on MK2-mediated signaling.Replicative stress is a consequence of nonperfect DNA replication, resulting in DNA damage response (DDR) signaling. In contrast to the DDR induced by double strand breaks, our current understanding of replicative stress is still far from complete. However, replicative stress constitutes a limiting factor in cancer cell proliferation (1) and a major mechanism of chemotherapy, and thus merits detailed understanding.Exogenous damage can enhance replicative stress. UV irradiation forms cross-links between DNA bases at any stage of the cell cycle, but damage is strongly enhanced when the cell tries to use such DNA as a template for replication. Nucleoside analogues, such as gemcitabine or cytarabine, perturb replication by being incorporated into nascent DNA strands, and/or by inducing an imbalance of nucleoside pools. Hence, a deeper understanding of how nucleoside analogues help to eliminate cancer cells can only be achieved through knowledge of how these cells respond to replicative stress.One way to avoid replicative stress consists in the avoidance of replication itself. Along this line, nongenotoxic activation of p53 induces G1 or G2 arrest that leads to profound resistance toward gemcitabine and UV irradiation (2, 3). This prompted us to ask more generally whether replicative stress represents merely a function of DNA damage before or during S phase, or whether it also depends on the activity of cellular signaling pathways. Indeed, the factors Chk1 and Wee1 are required to avoid replicative stress, and their knockdown induces a severe DDR (4, 5). We were now asking whether some factors can also act in a reverse fashion, provoking a more profound DDR and possibly cell death in response to misincorporations and other conditions that lead to replicative stress. Such mediators of detrimental outcome would contribute to the radiation sensitivity and chemosensitivity of cells.In this study, we have performed a siRNA screen, interrogating all known human kinases as to their contribution to the cellular response upon UV. We have identified MAP kinase-activated protein kinase 2 (MK2) as a major mediator of this response. MK2 suppresses replication fork progression and conversely enhances the firing frequency of new replication origins in the presence of replicative stress. It dampens translesion synthesis (TLS)-dependent and ongoing replication while promoting stalling of the replication fork.     

Protection from UV-induced skin carcinogenesis by genetic inhibition of the ataxia telangiectasia and Rad3-related (ATR) kinase

Multiple human epidemiologic studies link caffeinated (but not decaffeinated) beverage intake with significant decreases in several types of cancer, including highly prevalent UV-associated skin carcinomas. The mechanism by which caffeine protects against skin cancer is unknown. Ataxia telangiectasia and Rad3-related (ATR) is a replication checkpoint kinase activated by DNA stresses and is one of several targets of caffeine. Suppression of ATR, or its downstream target checkpoint kinase 1 (Chk1), selectively sensitizes DNA-damaged and malignant cells to apoptosis. Agents that target this pathway are currently in clinical trials. Conversely, inhibition of other DNA damage response pathways, such as ataxia telangiectasia mutated (ATM) and BRCA1, promotes cancer. To determine the effect of replication checkpoint inhibition on carcinogenesis, we generated transgenic mice with diminished ATR function in skin and crossed them into a UV-sensitive background, Xpc(-/-). Unlike caffeine, this genetic approach was selective and had no effect on ATM activation. These transgenic mice were viable and showed no histological abnormalities in skin. Primary keratinocytes from these mice had diminished UV-induced Chk1 phosphorylation and twofold augmentation of apoptosis after UV exposure (P = 0.006). With chronic UV treatment, transgenic mice remained tumor-free for significantly longer (P = 0.003) and had 69% fewer tumors at the end of observation of the full cohort (P = 0.019), compared with littermate controls with the same genetic background. This study suggests that inhibition of replication checkpoint function can suppress skin carcinogenesis and supports ATR inhibition as the relevant mechanism for the protective effect of caffeinated beverage intake in human epidemiologic studies.     

Intra-G1 arrest in response to UV irradiation in fission yeast

G1 is a crucial phase of cell growth because the decision to begin another mitotic cycle is made during this period. Occurrence of DNA damage in G1 poses a particular challenge, because replication of damaged DNA can be deleterious and because no sister chromatid is present to provide a template for recombinational repair. We therefore have studied the response of Schizosaccharomyces pombe cells to UV irradiation in early G1 phase. We find that irradiation results in delayed progression through G1, as manifested most critically in the delayed formation of the pre-replication complex. This delay does not have the molecular hallmarks of known checkpoint responses: it is independent of the checkpoint proteins Rad3, Cds1, and Chk1 and does not elicit inhibitory phosphorylation of Cdc2. Irradiated cells eventually progress into S phase and arrest in early S by a rad3- and cds1-dependent mechanism, most likely the intra-S checkpoint. Caffeine alleviates both the intra-G1- and intra-S-phase delays. We suggest that intra-G1 delay may be widely conserved and discuss significance and possible mechanisms.     

Requirement of proliferating cell nuclear antigen in RAD6-dependent postreplicational DNA repair.

The proliferating cell nuclear antigen (PCNA) acts as a processivity factor for replicative DNA polymerases and is essential for DNA replication. In vitro studies have suggested a role for PCNA-in the repair synthesis step of nucleotide excision repair, and PCNA interacts with the cyclin-dependent kinase inhibitor p21. However, because of the lack of genetic evidence, it is not clear which of the DNA repair processes are in fact affected by PCNA in vivo. Here, we describe a PCNA mutation, pol30-46, that confers ultraviolet (UV) sensitivity but has no effect on growth or cell cycle progression, and the mutant pcna interacts normally with DNA polymerase delta and epsilon. Genetic studies indicate that the pol30-46 mutation is specifically defective in RAD6-dependent postreplicational repair of UV damaged DNA, and this mutation impairs the error-free mode of bypass repair. These results implicate a role for PCNA as an intermediary between DNA replication and postreplicational DNA repair.     

RPA phosphorylation facilitates mitotic exit in response to mitotic DNA damage

Human replication protein A (RPA) becomes phosphorylated on the RPA2 subunit by cyclin B-Cdc2 during mitosis, although the functional role of this modification is unclear. We find that this modification stimulates RPA2 to become hyperphosphorylated in response to mitotic DNA damage caused by bleomycin treatment. Cells in which endogenous RPA2 was replaced by a mutant subunit lacking both Cdc2 sites had a significant defect in mitotic release into a 2N G₁ phase after exposure to bleomycin. An increased percentage of these mutant cells also was positive initially for cyclin B expression and BubR1 chromatin staining, indicative of an extended spindle assembly checkpoint. The mutant cells that experienced mitotic DNA damage also underwent apoptosis at higher levels than cells expressing the WT subunit. Even so, we did not find the mutation had any dramatic effects on the level of DNA repair in mitosis. Cells lacking ATM (a checkpoint factor and RPA2 kinase) also were severely defective in mitotic exit and were unable to support RPA hyperphosphorylation after mitotic DNA damage. Although checkpoint 1 effector kinase (Chk1) had a more complex role, inhibition of Chk1 activity with UCN-01 also reduced mitotic exit. Chk1 activation and mitotic RPA hyperphosphorylation were found to be independent events. Our results demonstrate that mitotic RPA hyperphosphorylation facilitates release of cells from a damaged mitosis into a 2N G₁ phase, thereby increasing cell viability.     

A DinB variant reveals diverse physiological consequences of incomplete TLS extension by a Y-family DNA polymerase

The only Y-family DNA polymerase conserved among all domains of life, DinB and its mammalian ortholog pol κ, catalyzes proficient bypass of damaged DNA in translesion synthesis (TLS). Y-family DNA polymerases, including DinB, have been implicated in diverse biological phenomena ranging from adaptive mutagenesis in bacteria to several human cancers. Complete TLS requires dNTP insertion opposite a replication blocking lesion and subsequent extension with several dNTP additions. Here we report remarkably proficient TLS extension by DinB from Escherichia coli. We also describe a TLS DNA polymerase variant generated by mutation of an evolutionarily conserved tyrosine (Y79). This mutant DinB protein is capable of catalyzing dNTP insertion opposite a replication-blocking lesion, but cannot complete TLS, stalling three nucleotides after an N²-dG adduct. Strikingly, expression of this variant transforms a bacteriostatic DNA damaging agent into a bactericidal drug, resulting in profound toxicity even in a dinB⁺ background. We find that this phenomenon is not exclusively due to a futile cycle of abortive TLS followed by exonucleolytic reversal. Rather, gene products with roles in cell death and metal homeostasis modulate the toxicity of DinB(Y79L) expression. Together, these results indicate that DinB is specialized to perform remarkably proficient insertion and extension on damaged DNA, and also expose unexpected connections between TLS and cell fate.     

Ataxia telangiectasia-mutated-Rad3-related DNA damage checkpoint signaling pathway triggered by hepatitis B virus infection

AIM： To explore whether acute cellular DNA damage response is induced upon hepatitis B virus （HBV） infection and the effects of the HBV infection. METHODS： We incubated HL7702 hepatocytes with HBV-positive serum, mimicking a natural HBV infection process. We used immunoblotting to evaluate protein expression levels in HBV-infected cells or in non-infected cells; immunofluorescence to show ATR foci ands Chkl phosphorylation foci formation; flow cytometry to analyze the cell cycle and apoptosis; ultraviolet （UV） radiation and ionizing radiation （IR）-treated cells to mimic DNA damage; and Trypan blue staining to count the viable cells. RESULTS： We found that HBV infection induced an increased steady state of ATR protein and increased phosphorylation of multiple downstream targets including Chkl, p53 and H2AX. In contrast to ATR and its target, the phosphorylated form of ATM at Ser-1981 and its downstream substrate Chk2 phosphorylation at Thr-68 did not visibly increase upon infection. However, the level of Mre11 and p21 were reduced beginning at 0.5 h aEer HBV-positive serum addition. Also, HBV infection led to transient cell cycle arrest in the S and the G2 phases without accompanying increasedapoptosis. Research on cell survival changes upon radiation following HBV infection showed that survival of UV-treated host cells was greatly increased by HBV infection, owing to the reduced apoptosis. Meanwhile, survival of IR-treated host cells was reduced by HBV infection. CONCLUSION： HBV infection activates ATR DNA damage response to replication stress and abrogates the checkpoint signaling controlled by DNA damage response.