Collapsed DNA Replication Forks Research Articles

SLFN11 inhibits checkpoint maintenance and homologous recombination repair.

High expression levels of SLFN11 correlate with the sensitivity of human cancer cells to DNA-damaging agents. However, little is known about the underlying mechanism. Here, we show that SLFN11 interacts directly with RPA1 and is recruited to sites of DNA damage in an RPA1-dependent manner. Furthermore, we establish that SLFN11 inhibits checkpoint maintenance and homologous recombination repair by promoting the destabilization of the RPA-ssDNA complex, thereby sensitizing cancer cell lines expressing high endogenous levels of SLFN11 to DNA-damaging agents. Finally, we demonstrate that the RPA1-binding ability of SLFN11 is required for its function in the DNA damage response. Our findings not only provide novel insight into the molecular mechanisms underlying the drug sensitivity of cancer cell lines expressing SLFN11 at high levels, but also suggest that SLFN11 expression can serve as a biomarker to predict responses to DNA-damaging therapeutic agents.

Distinct mechanisms of cell-kill by triapine and its terminally dimethylated derivative Dp44mT due to a loss or gain of activity of their copper(II) complexes

Triapine, currently being evaluated as an antitumor agent in phase II clinical trials, and its terminally dimethylated derivative Dp44mT share the α-pyridyl thiosemicarbazone backbone that functions as ligands for transition metal ions. Yet, Dp44mT is approximately 100-fold more potent than triapine in cytotoxicity assays. The aims of this study were to elucidate the mechanisms underlying their potency disparity and to determine their kinetics of cell-kill in culture to aid in the formulation of their clinical dosing schedules. The addition of Cu2+ inactivated triapine in a 1:1 stoichiometric fashion, while it potentiated the cytotoxicity of Dp44mT. Clonogenic assays after finite-time drug-exposure revealed that triapine produced cell-kill in two phases, one completed within 20min that caused limited cell-kill, and the other occurring after 16h of exposure that produced extensive cell-kill. The ribonucleotide reductase inhibitor triapine at 0.4μM caused immediate complete arrest of DNA synthesis, whereas Dp44mT at this concentration did not appreciably inhibit DNA synthesis. The inhibition of DNA synthesis by triapine was reversible upon its removal from the medium. Cell death after 16h exposure to triapine paralleled the appearance of phospho-(γ)H2AX, a marker of DNA double-strand breaks induced by collapse of DNA replication forks after prolonged replication arrest. In contrast to triapine, Dp44mT produced robust cell-kill within 1h in a concentration-dependent manner. The short-term action of both agents was prevented by thiols, indicative of the involvement of reactive oxygen species. The time dependency in the production of cell-kill by triapine should be considered in treatment regimens.

Putative antirecombinase Srs2 DNA helicase promotes noncrossover homologous recombination avoiding loss of heterozygosity

DNA damage alone or DNA replication fork arrest at damaged sites may induce DNA double-strand breaks and initiate homologous recombination. This event can result in a crossover with a homologous chromosome, causing loss of heterozygosity along the chromosome. It is known that Srs2 acts as an antirecombinase at the replication fork: it is recruited by the SUMO (a small ubiquitin-related modifier)-conjugated DNA-polymerase sliding clamp (PCNA) and interferes with Rad51/Rad52-mediated homologous recombination. Here, we report that Srs2 promotes another type of homologous recombination that produces noncrossover products only, in collaboration with PCNA and Rad51. Srs2 proteins lacking the Rad51-binding domain, PCNA-SUMO-binding motifs, or ATP hydrolysis-dependent DNA helicase activity reduce this noncrossover recombination. However, the removal of either the Rad51-binding domain or the PCNA-binding motif strongly increases crossovers. Srs2 gene mutations are epistatic to mutations in the PCNA modification-related genes encoding PCNA, Siz1 (a SUMO ligase) and Rad6 (a ubiquitin-conjugating protein). Knocking out RAD51 blocked this recombination but enhanced nonhomologous end-joining. We hypothesize that, during DNA double-strand break repair, Srs2 mediates collaboration between the Rad51 nucleofilament and PCNA-SUMO and directs the heteroduplex intermediate to DNA synthesis in a moving bubble. This Rad51/Rad52/Srs2/PCNA-mediated noncrossover pathway avoids both interchromosomal crossover and imprecise end-joining, two potential paths leading to loss of heterozygosity, and contributes to genome maintenance and human health.

Abstract 4482: Pemetrexed treatment results in DNA replication fork instability and double strand breaks formation in UNG-/- human cancer cells.

Abstract Misincorporation of genomic uracil and formation of DNA double strand breaks are known consequences of exposure to TS inhibitors such as 5-fluorouracil, and pemetrexed. Uracil DNA glycosylase catalyzes the excision of genomic uracil and initiates DNA base excision repair (BER). Thus, a relationship between antifolate cytotoxicity and UNG expression and activity has been hypothesized. However, a precise mechanism linking antifolate-induced formation of DSBs to genomic uracil accumulation and UNG-initiated BER has not been described. Herein, we report that despite equivalent proliferation indices, DLD1 UNG-/- cells are more sensitive to pemetrexed mediated intra S-phase arrest, DNA double strand break formation and apoptosis compared to UNG+/+ cells. Using data from western blots in chromatin extracts, PCNA staining of cells in S-phase, and pulse-chase labeling of replicating cells with CldU and IdU, we surmise that the accumulation of uracil in pemetrexed-treated UNG-/- cells is associated with significant replication fork instability. In addition, UNG-/- cells have reduced capacity to recover from pemetrexed-mediated DNA damage, as indicated by the persistence of S-phase arrest and gamma-H2AX foci. This defect in recovery was not explained by double strand break repair capacity, which was equivalent in UNG+/+ and UNG-/- cells. Using γ-H2AX ChIP sequencing, we observed a 5-fold increase in the number of γ-H2AX binding sites in UNG-/- cells compared to UNG+/+ cells treated at IC50 levels of pemetrexed. This analysis evinced distinct patterns of γ-H2AX binding in UNG+/+ and UNG-/- cells. Double strand breaks (γ-H2AX) were more significantly associated with transcription start sites and putative origins of replication in UNG-/- cells compared to UNG+/+ cells. Based on these data we conclude that uracil accumulation, and thus UNG activity, during pemetrexed exposure directs both the quantity and the location of double strand breaks. These findings support uracil mediated S-phase arrest and DNA replication fork collapse as the mechanism of double strand break formation and cell death in pemetrexed treated UNG-/- cells. Citation Format: Lachelle D. Weeks, Gabriel Zentner, Peter Scacheri, Stanton L. Gerson. Pemetrexed treatment results in DNA replication fork instability and double strand breaks formation in UNG-/- human cancer cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4482. doi:10.1158/1538-7445.AM2013-4482

Discordance between phosphorylation and recruitment of 53BP1 in response to DNA double-strand breaks

During the DNA damage response (DDR), chromatin modifications contribute to localization of 53BP1 to sites of DNA double-strand breaks (DSBs). 53BP1 is phosphorylated during the DDR, but it is unclear whether phosphorylation is directly coupled to chromatin binding. In this study, we used human diploid fibroblasts and HCT116 tumor cells to study 53BP1 phosphorylation at Serine-25 and Serine-1778 during endogenous and exogenous DSBs (DNA replication and whole-cell or sub-nuclear microbeam irradiation, respectively). In non-stressed conditions, endogenous DSBs in S-phase cells led to accumulation of 53BP1 and γH2AX into discrete nuclear foci. Only the frank collapse of DNA replication forks following hydroxyurea treatment initiated 53BP1Ser25 and 53BP1Ser1778 phosphorylation. In response to exogenous DSBs, 53BP1Ser25 and 53BP1Ser1778 phosphoforms localized to sites of initial DSBs in a cell cycle-independent manner. 53BP1 phosphoforms also localized to late residual foci and associated with PML-NBs during IR-induced senescence. Using isogenic cell lines and small-molecule inhibitors, we observed that DDR-induced 53BP1 phosphorylation was dependent on ATM and DNA-PKcs kinase activity but independent of MRE11 sensing or RNF168 chromatin remodeling. However, loss of RNF168 blocked recruitment of phosphorylated 53BP1 to sites of DNA damage. Our results uncouple 53BP1 phosphorylation from DSB localization and support parallel pathways for 53BP1 biology during the DDR. As relative 53BP1 expression may be a biomarker of DNA repair capacity in solid tumors, the tracking of 53BP1 phosphoforms in situ may give unique information regarding different cancer phenotypes or response to cancer treatment.

Abstract B27: The PARP inhibitor rucaparib radiosensitizes prostate cancer cells, most effectively those that are PTEN-deficient and are expressing ETS gene fusion proteins, which inhibit NHEJ DNA repair

Abstract Exposure to genotoxic agents, such as irradiation produces DNA damage and its toxicity is augmented when the DNA repair is impaired. Poly ADP ribose polymerase (PARP) inhibitors, such as rucaparib (CO-338; formerly known as AG014699 and PF-01367338) have been also found to be most effective in cells deficient in DNA repair. Radiation dose and scheduling experiments were performed in the prostate cancer cell line C4-2 in conditions mimicking brachytherapy with low dose radiation (Ir-192) as compared to external beam radiotherapy (EBRT). These studies have established the effectiveness of rucaparib to radiosensitize prostate cancer cells when the two clinical modalities are used: EBRT or brachytherapy, with a clear benefit of lower doses administered over a longer period of time, that caused DNA damage due to DNA replication fork collapse when rucaparib was present. Clonogenic survival assay were then performed on a panel of prostate cancer cell lines (LNCaP, C4-2, PC3, DU145, MDaPc2B, CWR22rv1, and VCaP) following radiation and rucaparib, used either alone or together. The combination index (CI) calculated for these various combinations revealed synergistic interactions, which was strongest for LNCaP cells. In addition to apoptosis, senescence was an important response, particularly in PTEN-negative tumors, as it was not observed in Du145 cells that have a wild-type PTEN allele. Absence of PTEN therefore allows activation of senescence, which contributes to decreased clonogenic survival. Increased radiosensitivity in presence of rucaparib was associated with persistent DNA breaks as determined by gamma-H2AX and p53BP1 foci. Interestingly, VCaP cells showed such foci constitutively, indicative of persistent DNA damage that was not present if the TMPRSS2/ERG gene fusion was depleted by siRNA. This response correlated with the ability of the gene fusion protein to bind DNA-PKcs on the chromatin and, as a result, inhibited its kinase activity. DNA-PKcs functional deficiency caused by TMPRSS2/ERG destabilized critical nonhomologous end-joining (NHEJ) components, such as Ku70 and XRCC4. When the TMPRSS2/ERG gene fusion protein was depleted by siRNA the DNA-PKcs kinase activity was restored and the DNA damage response (constitutive or induced) diminished, as measured by gamma-H2AX and p53BP1 foci. Therefore, the presence of the TMPRSS2/ERG fusion gene protein, by inhibiting NHEJ DNA repair, sensitized prostate cancer cells to TREATMENT WITH radiation and/or rucaparib. These results support the clinical testing of rucaparib in combination with radiotherapy. Late stage metastatic tumors, which harbor a gene fusion and are defective in PTEN could benefit most from a combination therapy using brachytherapy and rucaparib in a neoadjuvant setting. Citation Format: Payel Chatterjee, Gaurav Choudhary, Warren D. Heston, Eric A. Klein, Alex Almasan. The PARP inhibitor rucaparib radiosensitizes prostate cancer cells, most effectively those that are PTEN-deficient and are expressing ETS gene fusion proteins, which inhibit NHEJ DNA repair [abstract]. In: Proceedings of the AACR Special Conference on Advances in Prostate Cancer Research; 2012 Feb 6-9; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2012;72(4 Suppl):Abstract nr B27.

Polyploid cells rewire DNA damage response networks to overcome replication stress-induced barriers for tumour progression.

Mutations in genes involved in DNA replication such as FEN1, can cause single-stranded DNA breaks (SSBs) and subsequent collapse of DNA replication forks leading to DNA replication stresses. Persistent replication stresses normally induce p53-mediated senescence or apoptosis to prevent tumor progression. It is unclear how some mutant cells can overcome persistent replication stresses and bypass the p53-mediated pathways to develop malignancy. Here we show that formation of polyploidy, which is often observed in human cancers, leads to overexpression of BRCA1, p19arf and other DNA repair genes in FEN1 mutant cells. This overexpression triggers SSB repair and non-homologous end joining pathways to increase DNA repair activity, but at the cost of frequent chromosomal translocations. Meanwhile, DNA methylation silences p53 target genes, to bypass the p53-mediated senescence and apoptosis. These molecular changes rewire DNA damage response and repair gene networks in polyploid tumor cells, enabling them to escape replication stress-induced senescence barriers.

ATR-p53 Restricts Homologous Recombination in Response to Replicative Stress but Does Not Limit DNA Interstrand Crosslink Repair in Lung Cancer Cells

Homologous recombination (HR) is required for the restart of collapsed DNA replication forks and error-free repair of DNA double-strand breaks (DSB). However, unscheduled or hyperactive HR may lead to genomic instability and promote cancer development. The cellular factors that restrict HR processes in mammalian cells are only beginning to be elucidated. The tumor suppressor p53 has been implicated in the suppression of HR though it has remained unclear why p53, as the guardian of the genome, would impair an error-free repair process. Here, we show for the first time that p53 downregulates foci formation of the RAD51 recombinase in response to replicative stress in H1299 lung cancer cells in a manner that is independent of its role as a transcription factor. We find that this downregulation of HR is not only completely dependent on the binding site of p53 with replication protein A but also the ATR/ATM serine 15 phosphorylation site. Genetic analysis suggests that ATR but not ATM kinase modulates p53's function in HR. The suppression of HR by p53 can be bypassed under experimental conditions that cause DSB either directly or indirectly, in line with p53's role as a guardian of the genome. As a result, transactivation-inactive p53 does not compromise the resistance of H1299 cells to the interstrand crosslinking agent mitomycin C. Altogether, our data support a model in which p53 plays an anti-recombinogenic role in the ATR-dependent mammalian replication checkpoint but does not impair a cell's ability to use HR for the removal of DSB induced by cytotoxic agents.

Rad18-mediated Translesion Synthesis of Bulky DNA Adducts Is Coupled to Activation of the Fanconi Anemia DNA Repair Pathway

Fanconi anemia (FA) is a cancer susceptibility syndrome characterized by sensitivity to DNA-damaging agents. The FA proteins (FANCs) are implicated in DNA repair, although the precise mechanisms by which FANCs process DNA lesions are not fully understood. An epistatic relationship between the FA pathway and translesion synthesis (TLS, a post-replication DNA repair mechanism) has been suggested, but the basis for cross-talk between the FA and TLS pathways is poorly understood. We show here that ectopic overexpression of the E3 ubiquitin ligase Rad18 (a central regulator of TLS) induces DNA damage-independent mono-ubiquitination of proliferating cell nuclear antigen (PCNA) (a known Rad18 substrate) and FANCD2. Conversely, DNA damage-induced mono-ubiquitination of both PCNA and FANCD2 is attenuated in Rad18-deficient cells, demonstrating that Rad18 contributes to activation of the FA pathway. WT Rad18 but not an E3 ubiquitin ligase-deficient Rad18 C28F mutant fully complements both PCNA ubiquitination and FANCD2 activation in Rad18-depleted cells. Rad18-induced mono-ubiquitination of FANCD2 is not observed in FA core complex-deficient cells, demonstrating that Rad18 E3 ligase activity alone is insufficient for FANCD2 ubiquitylation. Instead, Rad18 promotes FA core complex-dependent FANCD2 ubiquitination in a manner that is secondary to PCNA mono-ubiquitination. Taken together, these results demonstrate a novel Rad18-dependent mechanism that couples activation of the FA pathway with TLS.

The role of RPA2 phosphorylation in homologous recombination in response to replication arrest

Failure to reactivate stalled or collapsed DNA replication forks is a potential source of genomic instability. Homologous recombination (HR) is a major mechanism for repairing the DNA damage resulting from replication arrest. The single-strand DNA (ssDNA)-binding protein, replication protein A (RPA), plays a major role in multiple processes of DNA metabolism. However, the role of RPA2 hyperphosphorylation, which occurs in response to DNA damage, had been unclear. Here, we show that hyperphosphorylated RPA2 associates with ssDNA and recombinase protein Rad51 in response to replication arrest by hydroxyurea (HU) treatment. In addition, RPA2 hyperphosphorylation is critical for Rad51 recruitment and HR-mediated repair following HU. However, RPA2 hyperphosphorylation is not essential for both ionizing radiation (IR)-induced Rad51 foci formation and I-Sce-I endonuclease-stimulated HR. Moreover, we show that expression of a phosphorylation-deficient mutant of RPA2 leads to increased chromosomal aberrations following HU treatment but not after exposure to IR. Finally, we demonstrate that loss of RPA2 hyperphosphorylation results in a loss of viability when cells are confronted with replication stress whereas cells expressing hyperphosphorylation-defective RPA2 or wild-type RPA2 have a similar sensitivity to IR. Thus, our data suggest that RPA2 hyperphosphorylation plays a critical role in maintenance of genomic stability and cell survival after a DNA replication block via promotion of HR.

Mammalian TIMELESS Is Required for ATM-dependent CHK2 Activation and G2/M Checkpoint Control

Timeless (Tim), a core circadian clock gene in Drosophila, is retained in mammals but has no apparent mammalian circadian clock function. Mammalian TIM is essential for ATR-dependent Chk1 activation and S-phase arrest. We report that TIM is likewise essential for ATM-dependent Chk2-mediated signaling of doxorubicin-induced DNA double strand breaks. TIM depletion attenuates doxorubicin-induced G(2)/M cell cycle arrest and sensitizes cancer cells to doxorubicin-induced cytotoxicity. TIM is, thereby, a potential novel anticancer drug target whose inhibition may enhance the therapeutic cytotoxicity of agents that activate DNA damage pathways as part of their mechanism.

Effect of Single-Strand Break on Holliday Junction Migration Dynamics: A Single-Molecule Fluorescence Study

Single-strand breaks (SSBs) are the most common DNA lesions in living cells. SSBs occur three orders of magnitude more frequently than double-strand breaks. Unrepaired SSBs lead to blockage or collapse of DNA replication forks, possibly causing formation of double-strand breaks. Holliday junctions (HJ) play central role in various DNA functions including repair of lesions, replication, homologous and site-specific recombination. Branch migration, either spontaneous or protein-mediated, is among widely employed mechanisms in these functions. Therefore, elucidation of the SSB effect on spontaneous branch migration of HJs is a problem of great importance. To accomplish this task, we employed single-molecule FRET approach developed before, allowing us to follow spontaneous branch migration of one HJ at a single base pair level in real time. One SSB was incorporated in the middle of the homologous region of mobile HJ with the donor and acceptor dyes placed on its opposite arms. The data showed that branch migration does not stop at these lesions or reflects from them. Our previous results showed that branch migration is a step-wise process and one step can cover entire homology region. The analysis of the time trajectories showed that such long steps are not prevented by SSBs. Step size distributions for HJs containing SSB were obtained and such characteristics of these distributions as the mean hop size and the distributions width remain virtually the same as in the designs without a break. Lifetimes of folded states were found to be very similar to that in control, just slightly increasing around the SSB in the AT-rich design. We also studied an immobile HJ with a SSB at the junction. Its arms remain in antiparallel orientation, very similarly to our previous studies.Supported by the NSF grant PHY-0615590 (YLL).

RecA family proteins in archaea: RadA and its cousins

Recombinases of the RecA family are essential for homologous recombination and underpin genome stability, by promoting the repair of double-stranded DNA breaks and the rescue of collapsed DNA replication forks. Until now, our understanding of homologous recombination has relied on studies of bacterial and eukaryotic model organisms. Archaea provide new opportunities to study how recombination operates in a lineage distinct from bacteria and eukaryotes. In the present paper, we focus on RadA, the archaeal RecA family recombinase, and its homologues in archaea and other domains. On the basis of phylogenetic analysis, we propose that a family of archaeal proteins with a single RecA domain, which are currently annotated as KaiC, be renamed aRadC.

An Oncogene-Induced DNA Damage Model for Cancer Development

Of all types of DNA damage, DNA double-strand breaks (DSBs) pose the greatest challenge to cells. One might have, therefore, anticipated that a sizable number of DNA DSBs would be incompatible with cell proliferation. Yet recent experimental findings suggest that, in both precancerous lesions and cancers, activated oncogenes induce stalling and collapse of DNA replication forks, which in turn leads to formation of DNA DSBs. This continuous formation of DNA DSBs may contribute to the genomic instability that characterizes the vast majority of human cancers. In addition, in precancerous lesions, these DNA DSBs activate p53, which, by inducing apoptosis or senescence, raises a barrier to tumor progression. Breach of this barrier by various mechanisms, most notably by p53 mutations, that impair the DNA damage response pathway allows cancers to develop. Thus, oncogene-induced DNA damage may explain two key features of cancer: genomic instability and the high frequency of p53 mutations.

Essential and distinct roles of the F-box and helicase domains of Fbh1 in DNA damage repair

BackgroundDNA double-strand breaks (DSBs) are induced by exogenous insults such as ionizing radiation and chemical exposure, and they can also arise as a consequence of stalled or collapsed DNA replication forks. Failure to repair DSBs can lead to genomic instability or cell death and cancer in higher eukaryotes. The Schizosaccharomyces pombe fbh1 gene encodes an F-box DNA helicase previously described to play a role in the Rhp51 (an orthologue of S. cerevisiae RAD51)-dependent recombinational repair of DSBs. Fbh1 fused to GFP localizes to discrete nuclear foci following DNA damage.ResultsTo determine the functional roles of the highly conserved F-box and helicase domains, we have characterized fbh1 mutants carrying specific mutations in these domains. We show that the F-box mutation fbh1-fb disturbs the nuclear localization of Fbh1, conferring an fbh1 null-like phenotype. Moreover, nuclear foci do not form in fbh1-fb cells with DNA damage even if Fbh1-fb is targeted to the nucleus by fusion to a nuclear localization signal sequence. In contrast, the helicase mutation fbh1-hl causes the accumulation of Fbh1 foci irrespective of the presence of DNA damage and confers damage sensitivity greater than that conferred by the null allele. Additional mutation of the F-box alleviates the hypermorphic phenotype of the fbh1-hl mutant.ConclusionThese results suggest that the F-box and DNA helicase domains play indispensable but distinct roles in Fbh1 function. Assembly of the SCFFbh1 complex is required for both the nuclear localization and DNA damage-induced focus formation of Fbh1 and is therefore prerequisite for the Fbh1 recombination function.

BCCIP regulates homologous recombination by distinct domains and suppresses spontaneous DNA damage

Homologous recombination (HR) is critical for maintaining genome stability through precise repair of DNA double-strand breaks (DSBs) and restarting stalled or collapsed DNA replication forks. HR is regulated by many proteins through distinct mechanisms. Some proteins have direct enzymatic roles in HR reactions, while others act as accessory factors that regulate HR enzymatic activity or coordinate HR with other cellular processes such as the cell cycle. The breast cancer susceptibility gene BRCA2 encodes a critical accessory protein that interacts with the RAD51 recombinase and this interaction fluctuates during the cell cycle. We previously showed that a BRCA2- and p21-interacting protein, BCCIP, regulates BRCA2 and RAD51 nuclear focus formation, DSB-induced HR and cell cycle progression. However, it has not been clear whether BCCIP acts exclusively through BRCA2 to regulate HR and whether BCCIP also regulates the alternative DSB repair pathway, non-homologous end joining. In this study, we found that BCCIP fragments that interact with BRCA2 or with p21 each inhibit DSB repair by HR. We further show that transient down-regulation of BCCIP in human cells does not affect non-specific integration of transfected DNA, but significantly inhibits homology-directed gene targeting. Furthermore, human HT1080 cells with constitutive down-regulation of BCCIP display increased levels of spontaneous single-stranded DNA (ssDNA) and DSBs. These data indicate that multiple BCCIP domains are important for HR regulation, that BCCIP is unlikely to regulate non-homologous end joining, and that BCCIP plays a critical role in resolving spontaneous DNA damage.

A Hand-Off Mechanism for Primosome Assembly in Replication Restart

Collapsed DNA replication forks must be reactivated through origin-independent reloading of the replication machinery (replisome) to ensure complete duplication of cellular genomes. In E. coli, the PriA-dependent pathway is the major replication restart mechanism and requires primosome proteins PriA, PriB, and DnaT for replisome reloading. However, the molecular mechanisms that regulate origin-independent replisome loading are not fully understood. Here, we demonstrate that assembly of primosome protein complexes represents a key regulatory mechanism, as inherently weak PriA-PriB and PriB-DnaT interactions are strongly stimulated by single-stranded DNA. Furthermore, the binding site on PriB for single-stranded DNA partially overlaps the binding sites for PriA and DnaT, suggesting a dynamic primosome assembly process in which single-stranded DNA is handed off from one primosome protein to another as a repaired replication fork is reactivated. This model helps explain how origin-independent initiation of DNA replication is restricted to repaired replication forks, preventing overreplication of the genome.

The Endonuclease Hje Catalyses Rapid, Multiple Turnover Resolution of Holliday junctions

Holliday junction-resolving enzymes are ubiquitous, structure-specific endonucleases that resolve four-way DNA junctions by the introduction of paired nicks in opposing strands, and are required for homologous recombination, double-strand break repair, recombination-dependent restart of stalled or collapsed DNA replication forks, and phage DNA processing. Here, we present the first steady-state kinetic characterisation of a junction-resolving enzyme; the Hje endonuclease from Sulfolobus solfataricus. We demonstrate that substrate turnover by Hje is sequence-independent and limited largely by the rate of cleavage of the phosphodiester bonds of the bound Holliday junction substrate, rather than substrate association or product dissociation. Reaction rates under multiple turnover conditions compare favourably with type II restriction enzymes. These properties, coupled with a high level of specificity for four-way junctions over all other DNA substrates, make Hje a suitable enzyme for applications requiring the detection and cleavage of Holliday junctions in vitro.

Involvement of Poly(ADP-ribose) Polymerase-1 and XRCC1/DNA Ligase III in an Alternative Route for DNA Double-strand Breaks Rejoining

The efficient repair of DNA double-strand breaks (DSBs) is critical for the maintenance of genomic integrity. In mammalian cells, the nonhomologous end-joining process that represents the predominant repair pathway relies on the DNA-dependent protein kinase (DNA-PK) and the XRCC4-DNA ligase IV complex. Nonetheless, several in vitro and in vivo results indicate that mammalian cells use more than a single end-joining mechanism. While searching for a DNA-PK-independent end-joining activity, we found that the pretreatment of DNA-PK-proficient and -deficient rodent cells with an inhibitor of the poly(ADP-ribose) polymerase-1 enzyme (PARP-1) led to increased cytotoxicity of the highly efficient DNA double-strand breaking compound calicheamicin gamma1. In addition, the repair kinetics of the DSBs induced by calicheamicin gamma1 was delayed both in PARP-1-proficient cells pretreated with the PARP-1 inhibitor and in PARP-1-deficient cells. In order to get new insights into the mechanism of an alternative route for DSBs repair, we have established a new synapsis and end-joining two-step assay in vitro, operating on DSBs with either nuclear protein extracts or recombinant proteins. We found an end-joining activity independent of the DNA-PK/XRCC4-ligase IV complex but that actually required a novel synapsis activity of PARP-1 and the ligation activity of the XRCC1-DNA ligase III complex, proteins otherwise involved in the base excision repair pathway. Taken together, these results strongly suggest that a PARP-1-dependent DSBs end-joining activity may exist in mammalian cells. We propose that this mechanism could act as an alternative route of DSBs repair that complements the DNA-PK/XRCC4/ligase IV-dependent nonhomologous end-joining.

Cellular machineries for chromosomal DNA repair.

Each day a cell must face a constant onslaught of DNA lesions. Although we tend to worry most about environmental sources of DNA damage (e.g., chemical agents, UV radiation, ionizing radiation), a human cell must repair over 10,000 DNA lesions per day to counteract endogenous sources of DNA damage (Lindahl 1993). DNA itself has a measurable half-life—spontaneous depurination can generate abasic sites in DNA strands at an estimated rate of 2,000–10,000 lesions per human cell per day (Lindahl 1993). Indeed, it has been proposed that much of the DNA repair machinery has evolved to contend with DNA damage generated by the byproducts of cellular metabolism—reactive oxygen species, endogenous alkylating agents, and DNA singleand doublestrand breaks resulting from collapsed DNA replication forks or from oxidative destruction of deoxyribose residues (Lindahl and Wood 1999; Lindahl 2000). Failure to repair such lesions can lead to a deleterious mutation rate, genomic instability, or cell death. In higher eukaryotes, the damage that occurs in genes responsible for DNA repair and/or cell cycle regulation can lead to threatening diseases such as cancer. The timely and efficient repair of eukaryotic DNA damage is further complicated by the realization that DNA lesions must be detected and repaired in the context of highly condensed, 100–400-nm-thick chromatin fibers (Belmont and Bruce 1994). The nucleosome is the first level of DNA compaction in the nucleus and is formed by the wrapping of 147 bp of DNA around a histone octamer composed of two H2A–H2B dimers and a H3–H4 tetramer. Linear arrays of nucleosomes are then folded into more compact structures, stabilized by linker histones such as histone H1. These compact structures are well known to hinder nuclear processes like transcription, and several in vitro studies have demonstrated that the assembly of DNA lesions into chromatin greatly hinders their detection and subsequent repair (see below). Within cells, of course, the repair machinery has created the means to contend with chromosomal structures, and an enormous number of DNA lesions are faithfully repaired each cell cycle. Here we review recent studies that have begun to elucidate this cellular machinery that facilitates DNA repair within the context of chromatin.