DNA Replication Arrest Research Articles

The minor groove-binding agent ELB-21 forms multiple interstrand and intrastrand covalent cross-links with duplex DNA and displays potent bactericidal activity against methicillin-resistant Staphylococcus aureus.

ObjectivesThe antistaphylococcal pyrrolobenzodiazepine dimer ELB-21 forms multiple adducts with duplex DNA through covalent interactions with appropriately spaced guanine residues; it is now known to form interstrand and intrastrand adducts with oligonucleotide sequences of variable length. We determined the DNA sequence preferences of ELB-21 in relation to its capacity to exert a bactericidal effect by damaging DNA.MethodsFormation of adducts by ELB-21 and 12- to 14-mer DNA duplexes was investigated using ion-pair reversed phase liquid chromatography and mass spectrometry. Drug-induced changes in gene expression were measured in prophage-free Staphylococcus aureus RN4220 by microarray analysis.ResultsELB-21 preferentially formed intrastrand adducts with guanines separated by three nucleotide base pairs. Interstrand and intrastrand adducts were formed with duplexes both longer and shorter than the preferred target sequences. ELB-21 elicited rapid bactericidal effects against prophage-carrying and prophage-free S. aureus strains; cell lysis occurred following activation and release of resident prophages. Killing appeared to be due to irreparable damage to bacterial DNA and susceptibility to ELB-21 was governed by the capacity of staphylococci to repair DNA lesions through induction of the SOS DNA damage response mediated by the RecA-LexA pathway.ConclusionsThe data support the contention that ELB-21 arrests DNA replication, eliciting formation of ssDNA-RecA filaments that inactivate LexA, the SOS repressor, and phage repressors such as Cl, resulting in activation of the DNA damage response and de-repression of resident prophages. Above the MIC threshold, DNA repair is ineffective.

Hydroxyurea Induces Genome-Wide Epigenetic Changes In Sickle Cell Disease

AbstractAbstract 2670 Introduction:Hydroxyurea (HU) is currently the only pharmacologic agent widely used to ameliorate the symptoms of sickle cell disease (SCD). The clinical effects of HU are diverse, including the well-known increase in levels of fetal hemoglobin (HbF), effects on leukocyte and platelet counts, and down-regulation of red cell adhesion despite increased expression of some adhesion receptors. However, the precise mechanisms by which HU exerts its ameliorative and pleiotropic actions are not well understood. HU is primarily thought to inhibit DNA replication and cause cell cycle arrest due to inhibition of ribonucleotide reductase (Yarbro, 1992). This mechanism is likely how HU exhibits its ameliorative effects in myeloproliferative conditions. But it is not as clear how this action results in an increase in the percentage of red blood cells that express a large percentage of HbF (Platt, 2008). Previous evidence suggests that HU may induce epigenetic (specifically, hypermethylation) changes to DNA (Nyce, 1989). Thus, we hypothesized that HU may increase HbF levels and cause other systemic changes through epigenetic mechanisms. Methods:To test the hypothesis that HU usage is associated with alterations in DNA methylation, we examined DNA samples from 24 adult patients with SCD, 12 of whom were taking HU at the time of DNA collection and 12 of whom were not. DNA from each individual was pre-treated with bisulfite (Zymo Research) and assessed for methylation levels at 27,578 CpG sites in 14,495 genes using the Illumina HumanMethylation27 BeadChip. One sample (off HU) failed to undergo a successful bisulfite DNA conversion and was subsequently removed from analysis. The relative levels of methylation (β) were calculated as the ratio of methylated probe signal to total locus signal intensity. Linear regression (PROC GLM, SAS version 9.1.3, Cary, NC) was used to test for differences in methylation (β) as a function of HU usage, controlling for sex and age. Results:On average, patients using HU had higher mean levels of methylation genome-wide compared with patients not taking HU, although the difference was not statistically significant, likely due to the small number of patients examined. Using the Benjamini-Hochberg false discovery correction for multiple testing and setting a stringent corrected p-value threshold of 0.05, we identified 247 out of the approximately 27,000 CpG sites that were differentially methylated as a function of HU usage. Eight CpG sites met a very stringent Bonferroni correction and represented a functionally diverse set of genes, including ones that encode a phosphatase, influence neural outgrowth, and play a role in vertebral development. CpG sites meeting the false discovery correction (n=247) were subsequently subjected to gene network and ontology analysis using DAVID pathway software (http://david.abcc.ncifcrf.gov/) to determine if specific biological pathways were statistically enriched for differences in DNA methylation. Several of these sites were associated with pathways involved in cell growth, senescence and differentiation, as might be expected given the known effects of HU on hematopoiesis. However, the TGFβ pathway was also significantly represented in this subset of CpG sites (pathway analysis p<0.05). Discussion:Consistent with findings by Nyce (1989), we observed a trend in which HU usage was associated with hypermethylation, so that patients taking HU had on average more methylation genome-wide than patients not taking HU. Genetic variation in the TGFβ pathway has been implicated in the occurrence of several SCD complications, including pulmonary hypertension (Ashley-Koch et al., 2008), risk for stroke (Sebastiani et al., 2005), leg ulcers (Nolan et al., 2006), bacteremia (Adewoye et al., 2006), and priapism (Elliott et al., 2007). Therefore, identification of this pathway as affected by HU is especially interesting. In summary, we have evidence that DNA epigenetic differences occur in sickle cell patients as a result of HU usage and are associated a wide variety of gene pathways, consistent with the diverse array of clinical and laboratory changes observed in patients on HU. Future studies will include confirmation of the epigenetic differences in the specific genes implicated among a larger cohort of SCD patients on and off HU, as well as functional studies of these genes. Disclosures:No relevant conflicts of interest to declare.

DDK Phosphorylates Checkpoint Clamp Component Rad9 and Promotes Its Release from Damaged Chromatin

When inappropriate DNA structures arise, they are sensed by DNA structure-dependent checkpoint pathways and subsequently repaired. Recruitment of checkpoint proteins to such structures precedes recruitment of proteins involved in DNA metabolism. Thus, checkpoints can regulate DNA metabolism. We show that fission yeast Rad9, a 9-1-1 heterotrimeric checkpoint-clamp component, is phosphorylated by Hsk1(Cdc7), the Schizosaccharomyces pombeDbf4-dependent kinase (DDK) homolog, in response to replication-induced DNA damage. Phosphorylation of Rad9 disrupts its interaction with replication protein A (RPA) and is dependent on 9-1-1 chromatin loading, the Rad9-associated protein Rad4/Cut5(TopBP1), and prior phosphorylation by Rad3(ATR). rad9 mutants defective in DDK phosphorylation show wild-type checkpoint responses but abnormal DNA repair protein foci and decreased viability after replication stress. We propose that Rad9 phosphorylation by DDK releases Rad9 from DNA damage sites to facilitate DNA repair.

IQGAP1 translocates to the nucleus in early S-phase and contributes to cell cycle progression after DNA replication arrest

IQGAP1 is a plasma membrane-associated protein and an important regulator of the actin cytoskeleton, contributing to cell migration, polarity and adhesion. In this study, we demonstrate the nuclear translocation of IQGAP1 using confocal microscopy and cell fractionation. Moreover, we identify a specific pool of IQGAP1 that accumulates in the nucleus during late G1-early S phase of the cell cycle. The nuclear targeting of IQGAP1 was facilitated by N- and C-terminal sequences, and its ability to slowly shuttle between nucleus and cytoplasm/membrane was partly regulated by the CRM1 export receptor. The inhibition of GSK-3β also stimulated nuclear localization of IQGAP1. The dramatic nuclear accumulation of IQGAP1 observed when cells were arrested in G1/S phase suggested a possible role in cell cycle regulation. In support of this, we used immunoprecipitation assays to show that the nuclear pool of IQGAP1 in G1/S-arrested cells associates with DNA replication complex factors RPA32 and PCNA. More important, the siRNA-mediated silencing of IQGAP1 significantly delayed cell cycle progression through S phase and G2/M in NIH 3T3 cells released from thymidine block. Our findings reveal an unexpected regulatory pathway for IQGAP1, and show that a pool of this cytoskeletal regulator translocates into the nucleus in late G1/early S phase to stimulate DNA replication and progression of the cell cycle.

A Senescence-Like Cell-Cycle Arrest Occurs During Megakaryocytic Maturation: Implications for Physiological and Pathological Megakaryocytic Proliferation

Author SummaryMegakaryocytes are huge bone marrow cells that shed platelets into the blood stream to promote clotting at sites of injury. Mature megakaryocytes differentiate from precursor cells in response to a hormone called thrombopoetin. Here, we show that as part of this normal differentiation process mature megakaryocytes enter a state called senescence in which cell division stops—a feature normally associated with cell aging and death. By studying megakaryocytes in culture, we were able to determine the biochemical pathway induced by thrombopoetin that leads to gene activation associated with senescence. We conclude that thrombopoietin acts differently at two steps in megakaryocyte differentiation: in the early stages it induces megakaryocyte proliferation, and at a latter stage it arrests the cell division cycle leading to platelet production by these cells. Interestingly, certain malignant megakaryocytes did not undergo senescence in response to thrombopoetin, which might explain the abnormal proliferation of these cancerous cells.

The Human WRN and BLM RecQ Helicases Differentially Regulate Cell Proliferation and Survival after Chemotherapeutic DNA Damage

Loss-of-function mutations in the human RecQ helicase genes WRN and BLM respectively cause the genetic instability/cancer predisposition syndromes Werner syndrome and Bloom syndrome. To identify common and unique functions of WRN and BLM, we systematically analyzed cell proliferation, cell survival, and genomic damage in isogenic cell lines depleted of WRN, BLM, or both proteins. Cell proliferation and survival were assessed before and after treatment with camptothecin, cis-diamminedichloroplatinum(II), hydroxyurea, or 5-fluorouracil. Genomic damage was assessed, before and after replication arrest, by gamma-H2AX staining, which was quantified at the single-cell level by flow cytometry. Cell proliferation was affected strongly by the extent of WRN and/or BLM depletion, and more strongly by BLM than by WRN depletion (P = 0.005). The proliferation of WRN/BLM-codepleted cells, in contrast, did not differ from BLM-depleted cells (P = 0.34). BLM-depleted and WRN/BLM-codepleted cells had comparably impaired survival after DNA damage, whereas WRN-depleted cells displayed a distinct pattern of sensitivity to DNA damage. BLM-depleted and WRN/BLM-codepleted cells had similar, significantly higher gamma-H2AX induction levels than did WRN-depleted cells. Our results provide new information on the role of WRN and BLM in determining cell proliferation, cell survival, and genomic damage after chemotherapeutic DNA damage or replication arrest. We also provide new information on functional redundancy between WRN and BLM. These results provide a strong rationale for further developing WRN and BLM as biomarkers of tumor chemotherapeutic responsiveness.

381 Functional studies of gene products and signal transduction mechanisms involved in cancer progression and metastasis – influence of extracellular S100A4

DNA damage tolerance pathways, which include translesion DNA synthesis (TLS) and template switching, are crucial for prevention of DNA replication arrest and maintenance of genomic stability. However, these pathways utilize error-prone DNA polymerases or template exchange between sister DNA strands, and consequently have the potential to induce mutations or chromosomal rearrangements.Post-translational modifications of proliferating cell nuclear antigen (PCNA) play important roles in controlling these pathways. For example, TLS is mediated by mono-ubiquitination of PCNA at lysine 164, for which RAD6–RAD18 is the primary E2–E3 complex. Elaborate protein–protein interactions between mono-ubiquitinated PCNA and Y-family DNA polymerases constitute the core of the TLS regulatory system, and enhancers of PCNA mono-ubiquitination and de-ubiquitinating enzymes finely regulate TLS and suppress TLS-mediated mutagenesis. The template switching pathway is promoted by K63-linked poly-ubiquitination of PCNA at lysine 164. Poly-ubiquitination is achieved by a coupled reaction mediated by two sets of E2–E3 complexes, RAD6–RAD18 and MMS2–UBC13–HTLF/SHPRH. In addition to these mono- and poly-ubiquitinations, simultaneous mono-ubiquitinations on multiple units of the PCNA homotrimeric ring promote an unidentified damage tolerance mechanism that remains to be fully characterized. Furthermore, SUMOylation of PCNA in mammalian cells can negatively regulate recombination. Other modifications, including ISGylation, acetylation, methylation, or phosphorylation, may also play roles in DNA damage tolerance and control of genomic stability.

Crystal Structure of Human REV7 in Complex with a Human REV3 Fragment and Structural Implication of the Interaction between DNA Polymerase ζ and REV1

DNA polymerase zeta (Polzeta) is an error-prone DNA polymerase involved in translesion DNA synthesis. Polzeta consists of two subunits: the catalytic REV3, which belongs to B family DNA polymerase, and the noncatalytic REV7. REV7 also interacts with REV1 polymerase, which is an error-prone Y family DNA polymerase and is also involved in translesion DNA synthesis. Cells deficient in one of the three REV proteins and those deficient in all three proteins show similar phenotype, indicating the functional collaboration of the three REV proteins. REV7 interacts with both REV3 and REV1 polymerases, but the structure of REV7 or REV3, as well as the structural and functional basis of the REV1-REV7 and REV3-REV7 interactions, remains unknown. Here we show the first crystal structure of human REV7 in complex with a fragment of human REV3 polymerase (residues 1847-1898) and reveal the mechanism underlying REV7-REV3 interaction. The structure indicates that the interaction between REV7 and REV3 creates a structural interface for REV1 binding. Furthermore, we show that the REV7-mediated interactions are responsible for DNA damage tolerance. Our results highlight the function of REV7 as an adapter protein to recruit Polzeta to a lesion site. REV7 is alternatively called MAD2B or MAD2L2 and also involved in various cellular functions such as signal transduction and cell cycle regulation. Our results will provide a general structural basis for understanding the REV7 interaction.

Schizosaccharomyces pombe Cds1Chk2 regulates homologous recombination at stalled replication forks through the phosphorylation of recombination protein Rad60

The Schizosaccharomyces pombe rad60 gene is essential for cell growth and is involved in repairing DNA double-strand breaks. Rad60 physically interacts with, and is functionally related to, the structural maintenance of chromosomes 5 and 6 protein complex (Smc5/6). Rad60 is phosphorylated in response to hydroxyurea (HU)-induced DNA replication arrest in a Cds1(Chk2)-dependent manner. Rad60 localizes in nucleus in unchallenged cells, but becomes diffused throughout the cell in response to HU. To understand the role of Rad60 phosphorylation, we mutated the putative phosphorylation target motifs of Cds1(Chk2) and have identified two Cds1(Chk2) target residues responsible for Rad60 dispersal in response to HU. We show that the phosphorylation-defective rad60 mutation partially suppresses HU sensitivity and the elevated recombination frequency of smc6-X. Our data suggest that Rad60 phosphorylation is required to regulate homologous recombination at stalled replication forks, probably by regulating Smc5/6.

TOR Complex 2 Controls Gene Silencing, Telomere Length Maintenance, and Survival under DNA-Damaging Conditions

The Target Of Rapamycin (TOR) kinase belongs to the highly conserved eukaryotic family of phosphatidylinositol-3-kinase-related kinases (PIKKs). TOR proteins are found at the core of two distinct evolutionarily conserved complexes, TORC1 and TORC2. Disruption of TORC1 or TORC2 results in characteristically dissimilar phenotypes. TORC1 is a major cell growth regulator, while the cellular roles of TORC2 are not well understood. In the fission yeast Schizosaccharomyces pombe, Tor1 is a component of the TORC2 complex, which is particularly required during starvation and various stress conditions. Our genome-wide gene expression analysis of Deltator1 mutants indicates an extensive similarity with chromatin structure mutants. Consistently, TORC2 regulates several chromatin-mediated functions, including gene silencing, telomere length maintenance, and tolerance to DNA damage. These novel cellular roles of TORC2 are rapamycin insensitive. Cells lacking Tor1 are highly sensitive to the DNA-damaging drugs hydroxyurea (HU) and methyl methanesulfonate, similar to mutants of the checkpoint kinase Rad3 (ATR). Unlike Rad3, Tor1 is not required for the cell cycle arrest in the presence of damaged DNA. Instead, Tor1 becomes essential for dephosphorylation and reactivation of the cyclin-dependent kinase Cdc2, thus allowing reentry into mitosis following recovery from DNA replication arrest. Taken together, our data highlight critical roles for TORC2 in chromatin metabolism and in promoting mitotic entry, most notably after recovery from DNA-damaging conditions. These data place TOR proteins in line with other PIKK members, such as ATM and ATR, as guardians of genome stability.

Chk1 C-terminal regulatory phosphorylation mediates checkpoint activation by de-repression of Chk1 catalytic activity

Chk1 is phosphorylated within its C-terminal regulatory domain by the upstream ATM/ ATR kinases during checkpoint activation, however how this modulates Chk1 function is poorly understood. Here, we show that Chk1 kinase activity is rapidly stimulated in a cell cycle phase-specific manner in response to both DNA damage and replication arrest, and that the extent and duration of activation correlates closely with regulatory phosphorylation at serines (S) S317, S345, and S366. Despite their evident co-regulation, substitutions of individual Chk1 regulatory sites with alanine (A) residues have differential effects on checkpoint proficiency and kinase activation. Thus, whereas Chk1 S345 is essential for all functions tested, mutants lacking S317 or S366 retain partial proficiency for G2/ M and S/ M checkpoint arrests triggered by DNA damage or replication arrest. These phenotypes reflect defects in Chk1 kinase induction, since the mutants are either partially (317A, 366A) or completely (345A) resistant to kinase activation. Importantly, S345 phosphorylation is impaired in Chk1 S317A and S366A mutants, suggesting that modification of adjacent SQ sites promotes this key regulatory event. Finally, we provide biochemical evidence that Chk1 catalytic activity is stimulated via a de-repression mechanism.

Cdc28/Cdk1 positively and negatively affects genome stability in S. cerevisiae

We studied the function of the cyclin-dependent kinase Cdc28 (Cdk1) in the DNA damage response and maintenance of genome stability using Saccharomyces cerevisiae. Reduced Cdc28 activity sensitizes cells to chronic DNA damage, but Cdc28 is not required for cell viability upon acute exposure to DNA-damaging agents. Cdc28 is also not required for activation of the DNA damage and replication checkpoints. Chemical–genetic analysis reveals that CDC28 functions in an extensive network of pathways involved in maintenance of genome stability, including homologous recombination, sister chromatid cohesion, the spindle checkpoint, postreplication repair, and telomere maintenance. In addition, Cdc28 and Mre11 appear to cooperate to prevent mitotic catastrophe after DNA replication arrest. We show that reduced Cdc28 activity results in suppression of gross chromosomal rearrangements (GCRs), indicating that Cdc28 is required for formation or recovery of GCRs. Thus, we conclude that Cdc28 functions in a genetic network that supports cell viability during DNA damage while promoting the formation of GCRs.

Cell cycle synchronization of Escherichia coli using the stringent response, with fluorescence labeling assays for DNA content and replication

We describe a method for synchronization of the cell cycle in the bacterium Escherichia coli. Treatment of asynchronous cultures with the amino acid analog, dl-serine hydroxamate, induces the stringent response, with concomitant arrest of DNA replication at initiation. Following release of the stringent response, cells initiate DNA replication in synchrony, as determined by flow cytometry for DNA content, Southern blotting and microscopy. This method has the advantage that it can be used in fully wild-type cells, at different growth rates, and may be applicable to other bacterial species with replication control by the stringent response. We also elaborate other methods useful for establishing cell cycle parameters in bacterial populations. We describe flow cytometric methods for analyzing bacterial populations for DNA content using the DNA-specific dye PicoGreen, readily detected by most commercial flow cytometers. We also present an method for incorporation of the nucleotide ethynyl-deoxyuridine, EdU, followed by “click” labeling with fluorescent dyes, which allows us to measure and visualize newly replicated DNA in fixed E. coli K-12 cells under non-denaturing conditions.

GINS Inactivation Phenotypes Reveal Two Pathways for Chromatin Association of Replicative α and ε DNA Polymerases in Fission Yeast

The tetrameric GINS complex, consisting of Sld5-Psf1-Psf2-Psf3, plays an essential role in the initiation and elongation steps of eukaryotic DNA replication, although its biochemical function is unclear. Here we investigate the function of GINS in fission yeast, using fusion of Psf1 and Psf2 subunits to a steroid hormone-binding domain (HBD) to make GINS function conditional on the presence of beta-estradiol. We show that inactivation of Psf1-HBD causes a tight but rapidly reversible DNA replication arrest phenotype. Inactivation of Psf2-HBD similarly blocks premeiotic DNA replication and leads to loss of nuclear localization of another GINS subunit, Psf3. Inactivation of GINS has distinct effects on the replication origin association and chromatin binding of two of the replicative DNA polymerases. Inactivation of Psf1 leads to loss of chromatin binding of DNA polymerase epsilon, and Cdc45 is similarly affected. In contrast, chromatin association of the catalytic subunit of DNA polymerase alpha is not affected by defective GINS function. We suggest that GINS functions in a pathway that involves Cdc45 and is necessary for DNA polymerase epsilon chromatin binding, but that a separate pathway sets up the chromatin association of DNA polymerase alpha.

DNA Replication Arrest in Response to Genotoxic Stress Provokes Early Activation of Stress-Activated Protein Kinases (SAPK/JNK)

The impact of DNA damage-induced replication blockage for early activation of stress kinases [stress-activated protein kinase (SAPK)/c-Jun N-terminal kinase (JNK)] is largely unknown. Here, we show that induction of dual phosphorylation of SAPK/JNK by the DNA polymerase inhibitor aphidicolin was not ameliorated by additional exposure to ultraviolet (UV) light, indicating that overlapping mechanisms participate in signaling to SAPK/JNK triggered by both agents. UV-induced DNA replication blockage, cyclobutane pyrimidine dimer formation and DNA strand break induction coincided with SAPK/JNK phosphorylation at early (≤30 min) but not late (≥2 h) time points after exposure. Genotoxin-stimulated SAPK/JNK activation was attenuated in nonproliferating cells, indicating that S phase-dependent mechanisms are involved in signaling to SAPK/JNK. Correspondingly, UV-induced phosphorylation of SAPK/JNK was higher in S-phase cells as compared with G1-phase cells. Activation of SAPK/JNK by genotoxins was below detection limit in nonproliferating human peripheral blood lymphocytes, whereas peripheral blood lymphocytes stimulated to proliferation displayed clear SAPK/JNK activation. UV-induced phosphorylation of SAPK/JNK was attenuated in XPC-defective cells, ameliorated in BRCA2 mutated cells and not changed in cells lacking ATM, DNA-PK, CSB, XPA, p53, ERCC1 or PARP as compared with the corresponding wild types. Based on these data, we suggest that DNA replication blockage caused by genotoxin-induced DNA damage contributes to early activation of SAPK/JNK.

Replication fork stalling in WRN-deficient cells is overcome by prompt activation of a MUS81-dependent pathway

Failure to stabilize and properly process stalled replication forks results in chromosome instability, which is a hallmark of cancer cells and several human genetic conditions that are characterized by cancer predisposition. Loss of WRN, a RecQ-like enzyme mutated in the cancer-prone disease Werner syndrome (WS), leads to rapid accumulation of double-strand breaks (DSBs) and proliferating cell nuclear antigen removal from chromatin upon DNA replication arrest. Knockdown of the MUS81 endonuclease in WRN-deficient cells completely prevents the accumulation of DSBs after fork stalling. Also, MUS81 knockdown in WS cells results in reduced chromatin recruitment of recombination enzymes, decreased yield of sister chromatid exchanges, and reduced survival after replication arrest. Thus, we provide novel evidence that WRN is required to avoid accumulation of DSBs and fork collapse after replication perturbation, and that prompt MUS81-dependent generation of DSBs is instrumental for recovery from hydroxyurea-mediated replication arrest under such pathological conditions.

DNA replication checkpoint promotes G 1 -S transcription by inactivating the MBF repressor Nrm1

The cell cycle transcriptional program imposes order on events of the cell-cycle and is a target for signals that regulate cell-cycle progression, including checkpoints required to maintain genome integrity. Neither the mechanism nor functional significance of checkpoint regulation of the cell-cycle transcription program are established. We show that Nrm1, an MBF-specific transcriptional repressor acting at the transition from G(1) to S phase of the cell cycle, is at the nexus between the cell cycle transcriptional program and the DNA replication checkpoint in fission yeast. Phosphorylation of Nrm1 by the Cds1 (Chk2) checkpoint protein kinase, which is activated in response to DNA replication stress, promotes its dissociation from the MBF transcription factor. This leads to the expression of genes encoding components that function in DNA replication and repair pathways important for cell survival in response to arrested DNA replication.

ERCC1 is required for FANCD2 focus formation

The rare genetic disorder Fanconi anemia, caused by a deficiency in any of at least thirteen identified genes, is characterized by cellular sensitivity to DNA interstrand crosslinks and genome instability. The excision repair cross complementing protein, ERCC1, first identified as a participant in nucleotide excision repair, appears to also act in crosslink repair, possibly in incision and at a later stage. We have investigated the relationship of ERCC1 to the Fanconi anemia pathway, using depletion of ERCC1 by siRNA in transformed normal human fibroblasts and fibroblasts from Fanconi anemia patients. We find that depletion of ERCC1 does not hinder formation of double strand breaks in crosslink repair as indexed by γH2AX. However, the monoubiquitination of FANCD2 protein in response to MMC treatment is decreased and the localization of FANCD2 to nuclear foci is eliminated. Arrest of DNA replication by hydroxyurea, producing double strand breaks without crosslinks, also requires ERRC1 for FANCD2 localization to nuclear foci. Our results support a role for ERCC1 after creation of a double strand break for full activation of the Fanconi anemia pathway.

Reverse transcriptase-PCR differential display analysis of meningococcal transcripts during infection of human cells: Up-regulation of priA and its role in intracellular replication

BackgroundIn vitro studies with cell line infection models are beginning to disclose the strategies that Neisseria meningitidis uses to survive and multiply inside the environment of the infected host cell. The goal of this study was to identify novel virulence determinants that are involved in this process using an in vitro infection system.ResultsBy using reverse transcriptase-PCR differential display we have identified a set of meningococcal genes significantly up-regulated during residence of the bacteria in infected HeLa cells including genes involved in L-glutamate transport (gltT operon), citrate metabolism (gltA), disulfide bond formation (dsbC), two-partner secretion (hrpA-hrpB), capsulation (lipA), and DNA replication/repair (priA). The role of PriA, a protein that in Escherichia coli plays a central role in replication restart of collapsed or arrested DNA replication forks, has been investigated. priA inactivation resulted in a number of growth phenotypes that were fully complemented by supplying a functional copy of priA. The priA-defective mutant exhibited reduced viability during late logarithmic growth phase. This defect was more severe when it was incubated under oxygen-limiting conditions using nitrite as terminal electron acceptors in anaerobic respiration. When compared to wild type it was more sensitive to hydrogen peroxide and the nitric oxide generator sodium nitroprusside. The priA-defective strain was not affected in its ability to invade HeLa cells, but, noticeably, exhibited severely impaired intracellular replication and, at variance with wild type and complemented strains, it co-localized with lysosomal associated membrane protein 1.ConclusionIn conclusion, our study i.) demonstrates the efficacy of the experimental strategy that we describe for discovering novel virulence determinants of N. meningitidis and ii.) provides evidence for a role of priA in preventing both oxidative and nitrosative injury, and in intracellular meningococcal replication.

A Ubiquitin-Proteasome Pathway for the Repair of Topoisomerase I-DNA Covalent Complexes

Reversible topoisomerase I (Top1)-DNA cleavage complexes are the key DNA lesion induced by anticancer camptothecins (e.g. topotecan and irinotecan) as well as structurally perturbed DNAs (e.g. oxidatively damaged DNA, UV-irradiated DNA, alkylated DNA, uracil-substituted DNA, mismatched DNA, gapped and nicked DNA, and DNA with abasic sites). Top1 cleavage complexes arrest transcription and trigger transcription-dependent degradation of Top1, a phenomenon termed Top1 down-regulation. In the current study, we have investigated the role of Top1 down-regulation in the repair of Top1 cleavage complexes. Using quiescent (serum-starved) human WI-38 cells, camptothecin (CPT) was shown to induce Top1 down-regulation, which paralleled the induction of DNA single-strand breaks (SSBs) (assayed by comet assays) and ATM autophosphorylation (at Ser-1981). Interestingly, Top1 down-regulation, induction of DNA SSBs and ATM autophosphorylation were all abolished by the proteasome inhibitor MG132. Furthermore, studies using immunoprecipitation and dominant-negative ubiquitin mutants have suggested a specific requirement for the assembly of Lys-48-linked polyubiquitin chains for CPT-induced Top1 down-regulation. In contrast to the effect of proteasome inhibition, inactivation of PARP1 was shown to increase the amount of CPT-induced SSBs and the level of ATM autophosphorylation. Together, these results support a model in which Top1 cleavage complexes arrest transcription and activate a ubiquitin-proteasome pathway leading to the degradation of Top1 cleavage complexes. Degradation of Top1 cleavage complexes results in the exposure of Top1-concealed SSBs for repair through a PARP1-dependent process.