Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “stalled replication fork”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 235 records · Page 13Linked to original sources
PubMed2002

Substrate specificity of RusA resolvase reveals the DNA structures targeted by RuvAB and RecG in vivo.

RusA endonuclease cleaves Holliday junctions by introducing paired strand incisions 5' to CC dinucleotides. Coordinated catalysis is achieved when both subunits of the homodimer interact simultaneously with cleavage sites located symmetrically. This requirement confers Holliday junction specificity. Uncoupled catalysis occurs when binding interactions are disturbed. Genetic studies indicate that uncoupling occurs rarely in vivo, and DNA cleavage is therefore restricted to Holliday junctions. We exploited the specificity of RusA to identify the DNA substrates targeted by the RuvAB and RecG branch-migration proteins in vivo. We present evidence that replication restart in UV-irradiated cells relies on the processing of stalled replication forks by RecG helicase and of Holliday junctions by the RuvABC resolvasome, and that RuvAB alone may not promote repair.

Bacterial Proteins↗
PubMed2003

Genotoxicity and mutagenicity of chromium(VI)/ascorbate-generated DNA adducts in human and bacterial cells.

Reduction of carcinogenic Cr(VI) by vitamin C generates ascorbate-Cr(III)-DNA cross-links, binary Cr(III)-DNA adducts, and can potentially cause oxidative DNA damage by intermediate reaction products. Here, we examined the mutational spectrum and the importance of different forms of DNA damage in genotoxicity and mutagenicity of Cr(VI) activated by physiological concentrations of ascorbate. Reduction of Cr(VI) led to a dose-dependent formation of both mutagenic and replication-blocking DNA lesions as detected by propagation of the pSP189 plasmids in human fibroblasts. Disruption of Cr-DNA binding abolished mutagenic responses and normalized the yield of replicated plasmids, indicating that Cr-DNA adducts were responsible for both mutagenicity and genotoxicity of Cr(VI). The absence of DNA breaks and abasic sites confirmed the lack of a significant production of hydroxyl radicals and Cr(V)-peroxo complexes in Cr(VI)-ascorbate reactions. Ascorbate-Cr(III)-DNA cross-links were much more mutagenic than smaller Cr(III)-DNA adducts and accounted for more than 90% of Cr(VI) mutagenicity. Ternary adducts were also several times more potent in the inhibition of replication than binary complexes. The Cr(VI)-induced mutational spectrum consisted of an approximately equal number of deletions and G/C-targeted point mutations (51% G/C --> T/A and 30% G/C --> A/T). In Escherichia coli cells, Cr(VI)-induced DNA adducts were only highly genotoxic but not mutagenic under either normal or SOS-induced conditions. Lower toxicity and high mutagenicity of ascorbate-Cr(III)-DNA adducts in human cells may result from the recruitment of an error-prone bypass DNA polymerase(s) to the stalled replication forks. Our results suggest that phosphotriester-type DNA adducts could play a more important role in human than bacterial mutagenesis.

Ascorbic Acid↗
PubMed2006

Replication-coupled repair of crotonaldehyde/acetaldehyde-induced guanine-guanine interstrand cross-links and their mutagenicity.

The repair of acetaldehyde/crotonaldehyde-induced guanine (N2)-guanine (N2) interstrand cross-links (ICLs), 3-(2-deoxyribos-1-yl)-5,6,7,8-(N2-deoxyguanosyl)-6(R or S)-methylpyrimido[1,2-alpha]purine-10(3H)-one, was studied using a shuttle plasmid bearing a site-specific ICL. Since the authentic ICLs can revert to monoadducts, a chemically stable model ICL, 1,3-bis(2'-deoxyguanos-N2-yl)butane derivative, was also employed to probe the ICL repair mechanism. Since the removal of ICL depends on the nucleotide excision repair (NER) mechanism in Escherichia coli, the plasmid bearing the model ICL failed to yield transformants in NER-deficient host cells, proving the stability of this ICL in cells. The authentic ICLs yielded transformants in the NER-deficient hosts; therefore, these transformants are produced by plasmid bearing spontaneously reverted monoadducts. In contrast, in NER-deficient human cells, the model ICL was removed by an NER-independent repair pathway, which is unique to higher eukaryotes. This repair did not associate with a transcriptional event, but with replication. The analysis of repaired molecules revealed that the authentic and model ICLs were repaired mostly (>94%) in an error-free manner in both hosts. The major mutations that were observed were G --> T transversions targeting the cross-linked dG located in the lagging strand template. These results support one of the current models for the mammalian NER-independent ICL repair mechanism, in which a DNA endonuclease(s) unhooks an ICL from the leading strand template at a stalled replication fork site by incising on both sides of the ICL and then translesion synthesis is conducted across the "half-excised" ICL attached to the lagging strand template to restore DNA synthesis.

Acetaldehyde↗
PubMed2000

The expanding polymerase universe.

Over the past year, the number of known prokaryotic and eukaryotic DNA polymerases has exploded. Many of these newly discovered enzymes copy aberrant bases in the DNA template over which 'respectable' polymerases fear to tread. The next step is to unravel their functions, which are thought to range from error-prone copying of DNA lesions, somatic hypermutation and avoidance of skin cancer, to restarting stalled replication forks and repairing double-stranded DNA breaks.

Animals↗
PubMed2023

Structural basis for stabilisation of the RAD51 nucleoprotein filament by BRCA2.

The BRCA2 tumour suppressor protein preserves genomic integrity via interactions with the DNA-strand exchange RAD51 protein in homology-directed repair. The RAD51-binding TR2 motif at the BRCA2 C-terminus is essential for protection and restart of stalled replication forks. Biochemical evidence shows that TR2 recognises filamentous RAD51, but existing models of TR2 binding to RAD51 lack a structural basis. Here we used cryo-electron microscopy and structure-guided mutagenesis to elucidate the mechanism of TR2 binding to nucleoprotein filaments of human RAD51. We find that TR2 binds across the protomer interface in the filament, acting as a brace for adjacent RAD51 molecules. TR2 targets an acidic-patch motif on human RAD51 that serves as a recruitment hub in fission yeast Rad51 for recombination mediators Rad52 and Rad55-Rad57. Our findings provide a structural rationale for RAD51 filament stabilisation by BRCA2 and reveal a common recruitment mechanism of recombination mediators to the RAD51 filament.

Humans↗
PubMed2004

Recovery from DNA damage checkpoint arrest by PP1-mediated inhibition of Chk1.

The G2 DNA damage checkpoint delays mitotic entry via the upregulation of Wee1 kinase and the downregulation of Cdc25 phosphatase by Chk1 kinase, and resultant inhibitory phosphorylation of Cdc2. While checkpoint activation is well understood, little is known about how the checkpoint is switched off to allow cell cycle re-entry. To identify proteins required for checkpoint release, we screened for genes in Schizosaccharomyces pombe that, when overexpressed, result in precocious mitotic entry in the presence of DNA damage. We show that overexpression of the type I protein phosphatase Dis2 sensitises S. pombe cells to DNA damage, causing aberrant mitoses. Dis2 abrogates Chk1 phosphorylation and activation in vivo, and dephosphorylates Chk1 and a phospho-S345 Chk1 peptide in vitro. dis2Delta cells have a prolonged chk1-dependent arrest and a compromised ability to downregulate Chk1 activity for checkpoint release. These effects are specific for the DNA damage checkpoint, because Dis2 has no effect on the chk1-independent response to stalled replication forks. We propose that inactivation of Chk1 by Dis2 allows mitotic entry following repair of DNA damage in the G2-phase.

Cell Cycle Proteins↗
PubMed2004

Nbs1 is required for ATR-dependent phosphorylation events.

Nijmegen breakage syndrome (NBS) is characterised by microcephaly, developmental delay, characteristic facial features, immunodeficiency and radiosensitivity. Nbs1, the protein defective in NBS, functions in ataxia telangiectasia mutated protein (ATM)-dependent signalling likely facilitating ATM phosphorylation events. While NBS shares overlapping characteristics with ataxia telangiectasia, it also has features overlapping with ATR-Seckel (ATR: ataxia-telangiectasia and Rad3-related protein) syndrome, a subclass of Seckel syndrome mutated in ATR. We show that Nbs1 also facilitates ATR-dependent phosphorylation. NBS cell lines show a similar defect in ATR phosphorylation of Chk1, c-jun and p-53 in response to UV irradiation- and hydroxyurea (HU)-induced replication stalling. They are also impaired in ubiquitination of FANCD2 after HU treatment, which is ATR dependent. Following HU-induced replication arrest, NBS and ATR-Seckel cells show similarly impaired G2/M checkpoint arrest and an impaired ability to restart DNA synthesis at stalled replication forks. Moreover, NBS cells fail to retain ATR in the nucleus following HU treatment and extraction. Our findings suggest that Nbs1 functions in both ATR- and ATM-dependent signalling. We propose that the NBS clinical features represent the result of these combined defects.

Animals↗
PubMed2004

Functional relationships of FANCC to homologous recombination, translesion synthesis, and BLM.

Some of the restarting events of stalled replication forks lead to sister chromatid exchange (SCE) as a result of homologous recombination (HR) repair with crossing over. The rate of SCE is elevated by the loss of BLM helicase or by a defect in translesion synthesis (TLS). We found that spontaneous SCE levels were elevated approximately 2-fold in chicken DT40 cells deficient in Fanconi anemia (FA) gene FANCC. To investigate the mechanism of the elevated SCE, we deleted FANCC in cells lacking Rad51 paralog XRCC3, TLS factor RAD18, or BLM. The increased SCE in fancc cells required Xrcc3, whereas the fancc/rad18 double mutant exhibited higher SCE than either single mutant. Unexpectedly, SCE in the fancc/blm mutant was similar to that in blm cells, indicating functional linkage between FANCC and BLM. Furthermore, MMC-induced formation of GFP-BLM nuclear foci was severely compromised in both human and chicken fancc or fancd2 cells. Our cell survival data suggest that the FA proteins serve to facilitate HR, but not global TLS, during crosslink repair.

Adenosine Triphosphatases↗
PubMed2005

Crystal structure and DNA-binding analysis of RecO from Deinococcus radiodurans.

The RecFOR pathway has been shown to be essential for DNA repair through the process of homologous recombination in bacteria and, recently, to be important in the recovery of stalled replication forks following UV irradiation. RecO, along with RecR, RecF, RecQ and RecJ, is a principal actor in this fundamental DNA repair pathway. Here we present the three-dimensional structure of a member of the RecO family. The crystal structure of Deinococcus radiodurans RecO (drRecO) reveals possible binding sites for DNA and for the RecO-binding proteins within its three discrete structural regions: an N-terminal oligonucleotide/oligosaccharide-binding domain, a helical bundle and a zinc-finger motif. Furthermore, drRecO was found to form a stable complex with RecR and to bind both single- and double-stranded DNA. Mutational analysis confirmed the existence of multiple DNA-binding sites within the protein.

Amino Acid Sequence↗
PubMed2006

Controlling the subcellular localization of DNA polymerases iota and eta via interactions with ubiquitin.

Y-family DNA polymerases have spacious active sites that can accommodate a wide variety of geometric distortions. As a consequence, they are considerably more error-prone than high-fidelity replicases. It is hardly surprising, therefore, that the in vivo activity of these polymerases is tightly regulated, so as to minimize their inadvertent access to primer-termini. We report here that one such mechanism employed by human cells relies on a specific and direct interaction between DNA polymerases iota and eta with ubiquitin (Ub). Indeed, we show that both polymerases interact noncovalently with free polyUb chains, as well as mono-ubiquitinated proliferating cell nuclear antigen (Ub-PCNA). Mutants of poliota (P692R) and poleta (H654A) were isolated that are defective in their interactions with polyUb and Ub-PCNA, whilst retaining their ability to interact with unmodified PCNA. Interestingly, the polymerase mutants exhibit significantly lower levels of replication foci in response to DNA damage, thereby highlighting the biological importance of the polymerase-Ub interaction in regulating the access of the TLS polymerases to stalled replication forks in vivo.

DNA-Directed DNA Polymerase↗
PubMed2003

Induction of a caffeine-sensitive S-phase cell cycle checkpoint by psoralen plus ultraviolet A radiation.

Induction of interstrand crosslinks (ICLs) in chromosomal DNA is considered a major reason for the antiproliferative effect of psoralen plus ultraviolet A (PUVA). It is unclear as to whether PUVA-induced cell cycle arrest is caused by ICLs mechanically stalling replication forks or by triggering cell cycle checkpoints. Cell cycle checkpoints serve to maintain genomic stability by halting cell cycle progression to prevent replication of damaged DNA templates or segregation of broken chromosomes. Here, we show that HaCaT keratinocytes treated with PUVA arrest with S-phase DNA content. Cells that had completed DNA replication were not perturbed by PUVA and passed through mitosis. Cells treated with PUVA during G1-phase continued traversing G1 until arresting in early S-phase. PUVA induced rapid phosphorylation of the Chk1 checkpoint kinase at Ser345 and a concomitant decrease in Cdc25A levels. Chk1 phosphorylation, decrease of Cdc25 A levels and S-phase arrest were abolished by caffeine, demonstrating that active checkpoint signaling rather than passive mechanical blockage by ICLs causes the PUVA-induced replication arrest. Overexpression of Cdc25A only partially overrode the S-phase arrest, suggesting that additional signaling events implement PUVA-induced S-phase arrest.

Animals↗
PubMed2003

Mechanisms of apoptosis induction by nucleoside analogs.

Nucleoside analogs are structurally, metabolically, and pharmacodynamically related agents that nevertheless have diverse biological actions and therapeutic consequences. This class of agents affects the structural integrity of DNA, generally after incorporation during replication or DNA excision repair synthesis, leading to stalled replication forks and chain termination. The DNA damage sensors ATM, ATR and DNA-PK recognize these events. These and other protein kinases activate checkpoint pathways that arrest cell cycle progression, and also signal for DNA repair. In addition, if these survival mechanisms are overwhelmed by the damage caused, a third function of these sensors is to activate signaling pathways that initiate apoptotic processes. A review of the spectrum of responses that are activated by clinically relevant nucleoside analogs begins to provide a mechanistic basis for diverse outcomes in cell viability. Such information, when coupled with an understanding of the intrinsic apoptotic potential of a tumor cell type may provide a rational basis for the design of therapeutic strategies.

Antimetabolites, Antineoplastic↗
PubMed2005

BLM helicase is activated in BCR/ABL leukemia cells to modulate responses to cisplatin.

Bloom protein (BLM) is a 3'-5' helicase, mutated in Bloom syndrome, which plays an important role in response to DNA double-strand breaks and stalled replication forks. Here, we show that BCR/ABL tyrosine kinase, which also modulates DNA repair capacity, is associated with elevated expression of BLM. Downregulation of BLM by antisense cDNA or dominant-negative mutant inhibits homologous recombination repair (HRR) and increases sensitivity to cisplatin in BCR/ABL-positive cells. Bone marrow cells from mice heterozygous for BLM mutation, BLM(Cin/+), transfected with BCR/ABL display increased sensitivity to cisplatin compared to those obtained from the wild-type littermates. BCR/ABL promotes interactions of BLM with RAD51, while simultaneous overexpression of BLM and RAD51 in normal cells increases drug resistance. These data suggest that BLM collaborates with RAD51 to facilitate HRR and promotes the resistance of BCR/ABL-positive leukemia cells to DNA-damaging agents.

Adenosine Triphosphatases↗
PubMed2001

The X philes: structure-specific endonucleases that resolve Holliday junctions.

Genetic recombination is a critical cellular process that promotes evolutionary diversity, facilitates DNA repair and underpins genome duplication. It entails the reciprocal exchange of single strands between homologous DNA duplexes to form a four-way branched intermediate commonly referred to as the Holliday junction. DNA molecules interlinked in this way have to be separated in order to allow normal chromosome transmission at cell division. This resolution reaction is mediated by structure-specific endonucleases that catalyse dual-strand incision across the point of strand cross-over. Holliday junctions can also arise at stalled replication forks by reversing the direction of fork progression and annealing of nascent strands. Resolution of junctions in this instance generates a DNA break and thus serves to initiate rather than terminate recombination. Junction resolvases are generally small, homodimeric endonucleases with a high specificity for branched DNA. They use a metal-binding pocket to co-ordinate an activated water molecule for phosphodiester bond hydrolysis. In addition, most junction endonucleases modulate the structure of the junction upon binding, and some display a preference for cleavage at specific nucleotide target sequences. Holliday junction resolvases with distinct properties have been characterized from bacteriophages (T4 endo VII, T7 endo I, RusA and Rap), Bacteria (RuvC), Archaea (Hjc and Hje), yeast (CCE1) and poxviruses (A22R). Recent studies have brought about a reappraisal of the origins of junction-specific endonucleases with the discovery that RuvC, CCE1 and A22R share a common catalytic core.

Archaea↗
PubMed2004

Host processing of branched DNA intermediates is involved in targeted transposition of IS911.

A simplified system using bacterial insertion sequence IS911 has been developed to investigate targeted insertion next to DNA sequences resembling IS ends. We show here that these IR-targeted events occur by an unusual mechanism. In the circular IS911 transposition intermediate the two IRs are abutted to form an IR/IR junction. IR-targeted insertion involves transfer of a single end of the junction to the target IR to generate a branched DNA structure. The single-end transfer (SET) intermediate, but not the final insertion product, can be detected in an in vitro reaction. SET intermediates must be processed by the bacterial host to obtain the final insertion products. Sequence analysis of these IR-targeted insertion products and of those obtained in vivo revealed high levels of DNA sequence conversion in which mutations from one IR were transferred to another. These sequence changes cannot be explained by the classic transposition pathway. A model is presented in which the four-way Holliday-like junction created by SET is processed by host-mediated branch migration, resolution, repair and replication. This pathway resembles those described for processing other branched DNA structures such as stalled replication forks.

DNA Transposable Elements↗
PubMed2004

Direct evidence that a conserved arginine in RuvB AAA+ ATPase acts as an allosteric effector for the ATPase activity of the adjacent subunit in a hexamer.

The Escherichia coli RuvA and RuvB protein complex promotes branch migration of Holliday junctions during recombinational repair and homologous recombination and at stalled replication forks. The RuvB protein belongs to the AAA(+) (ATPase associated with various cellular activities) ATPase family and forms a hexameric ring in an ATP-dependent manner. Studies on the oligomeric AAA(+) class ATPases suggest that a conserved arginine residue is located in close proximity to the ATPase site of the adjacent subunit and plays an essential role during ATP hydrolysis. This study presents direct evidence that Arg-174 of RuvB allosterically stimulates the ATPase of the adjacent subunit in a RuvB hexamer. RuvBR174A shows a dominant negative phenotype for DNA repair in vivo and inhibits the branch migration catalyzed by wild-type RuvB. A dominant negative phenotype was also observed with RuvBK68A (Walker A mutation). RuvB K68A-R174A double mutant demonstrates a more severe dominant negative effect than the single mutants RuvB K68A or R174A. Moreover, although RuvB K68A and R174A are totally defective in ATPase activity, ATPase activity is restored when these two mutant proteins are mixed at a 1:1 ratio. These results suggest that each of the two mutants has distinct functional defects and that restoration of the ATPase activity is brought by complementary interaction between the mutant subunits in the heterohexamers. This study demonstrates that R174 plays an intermolecular catalytic role during ATP hydrolysis by RuvB. This role may be a general feature of the oligomeric AAA/AAA(+) ATPases.

Adenosine Triphosphatases↗
PubMed2005

Observing spontaneous branch migration of Holliday junctions one step at a time.

Genetic recombination occurs between homologous DNA molecules via a four-way (Holliday) junction intermediate. This ancient and ubiquitous process is important for the repair of double-stranded breaks, the restart of stalled replication forks, and the creation of genetic diversity. Once formed, the four-way junction alone can undergo the stepwise exchange of base pairs known as spontaneous branch migration. Conventional ensemble assays, useful for finding average migration rates over long sequences, have been unable to examine the affect of sequence and structure on the migration process. Here, we present a single-molecule spontaneous branch migration assay with single-base pair resolution in a study of individual DNA junctions that can undergo one step of migration. Junctions exhibit markedly different dynamics of exchange between stacking conformers depending on the point of strand exchange, allowing the moment at which branch migration occurs to be detected. The free energy landscape of spontaneous branch migration is found to be highly nonuniform and governed by two types of sequence-dependent barriers, with unmediated local migration being up to 10 times more rapid than the previously deduced average rate.

Base Pairing↗
PubMed2005

Ubiquitinated proliferating cell nuclear antigen activates translesion DNA polymerases eta and REV1.

In response to DNA damage, the Rad6/Rad18 ubiquitin-conjugating complex monoubiquitinates the replication clamp proliferating cell nuclear antigen (PCNA) at Lys-164. Although ubiquitination of PCNA is recognized as an essential step in initiating postreplication repair, the mechanistic relevance of this modification has remained elusive. Here, we describe a robust in vitro system that ubiquitinates yeast PCNA specifically on Lys-164. Significantly, only those PCNA clamps that are appropriately loaded around effector DNA by its loader, replication factor C, are ubiquitinated. This observation suggests that, in vitro, only PCNA present at stalled replication forks is ubiquitinated. Ubiquitinated PCNA displays the same replicative functions as unmodified PCNA. These functions include loading onto DNA by replication factor C, as well as Okazaki fragment synthesis and maturation by the PCNA-coordinated actions of DNA polymerase delta, the flap endonuclease FEN1, and DNA ligase I. However, whereas the activity of DNA polymerase zeta remains unaffected by ubiquitination of PCNA, ubiquitinated PCNA specifically activates two key enzymes in translesion synthesis: DNA polymerase eta, the yeast Xeroderma pigmentosum ortholog, and Rev1, a deoxycytidyl transferase that functions in organizing the mutagenic DNA replication machinery. We propose that ubiquitination of PCNA increases its functionality as a sliding clamp to promote mutagenic DNA replication.

DNA↗