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PubMed2006

Formation and processing of stalled replication forks--utility of two-dimensional agarose gels.

Replication forks can be stalled by tightly bound proteins, DNA damage, nucleotide deprivation, or defects in the replication machinery. It is now appreciated that processing of stalled replication forks is critical for completion of DNA replication and maintenance of genome stability. In this chapter, we detail the use of two-dimensional (2D) agarose gels with Southern hybridization for the detection and analysis of blocked replication forks in vivo. This kind of 2D gel electrophoresis has been used extensively for analysis of replication initiation mechanisms for many years, and more recently has become a valuable tool for analysis of fork stalling. Although the method can provide valuable information when forks are stalled in random locations (e.g., after UV damage or nucleotide deprivation), it is even more informative with site-specific fork blockage, for example, blocks caused by tightly bound replication terminator proteins or by drug-stabilized topoisomerase cleavage complexes.

Blotting, Southern↗
PubMed2006

Stalled replication induces p53 accumulation through distinct mechanisms from DNA damage checkpoint pathways.

Stalled replication forks induce p53, which is required to maintain the replication checkpoint. In contrast to the well-established mechanisms of DNA damage-activated p53, the downstream effectors and upstream regulators of p53 during replication blockade remain to be deciphered. Hydroxyurea triggered accumulation of p53 through an increase in protein stability. The requirement of p53 accumulation for the replication checkpoint was not due to p21(CIP1/WAF1) as its down-regulation with short-hairpin RNA did not affect the checkpoint. Similar to DNA damage, stalled replication triggered the activation of the MRN-ataxia telangiectasia mutated (ATM)/ATM and Rad3-related-CHK1/CHK2 axis. Down-regulation of CHK1 or CHK2, however, reduced p53 basal expression but not the hydroxyurea-dependent induction. Moreover, p53 was still stabilized in ataxia telangiectasia cells or in cells treated with caffeine, suggesting that ATM was not a critical determinant. These data also suggest that the functions of ATM, CHK1, and CHK2 in the replication checkpoint were not through the p53-p21(CIP1/WAF1) pathway. In contrast, induction of p53 by hydroxyurea was defective in cells lacking NBS1 and BLM. In this connection, the impaired replication checkpoint in several other genetic disorders has little correlation with the ability to stabilize p53. These data highlighted the different mechanisms involved in the stabilization of p53 after DNA damage and stalled replication forks.

Acid Anhydride Hydrolases↗
PubMed2001

The tight linkage between DNA replication and double-strand break repair in bacteriophage T4.

Double-strand break (DSB) repair and DNA replication are tightly linked in the life cycle of bacteriophage T4. Indeed, the major mode of phage DNA replication depends on recombination proteins and can be stimulated by DSBs. DSB-stimulated DNA replication is dramatically demonstrated when T4 infects cells carrying two plasmids that share homology. A DSB on one plasmid triggered extensive replication of the second plasmid, providing a useful model for T4 recombination-dependent replication (RDR). This system also provides a view of DSB repair in T4-infected cells and revealed that the DSB repair products had been replicated in their entirety by the T4 replication machinery. We analyzed the detailed structure of these products, which do not fit the simple predictions of any of three models for DSB repair. We also present evidence that the T4 RDR system functions to restart stalled or inactivated replication forks. First, we review experiments involving antitumor drug-stabilized topoisomerase cleavage complexes. The results suggest that forks blocked at cleavage complexes are resolved by recombinational repair, likely involving RDR. Second, we show here that the presence of a T4 replication origin on one plasmid substantially stimulated recombination events between it and a homologous second plasmid that did not contain a T4 origin. Furthermore, replication of the second plasmid was increased when the first plasmid contained the T4 origin. Our interpretation is that origin-initiated forks become inactivated at some frequency during replication of the first plasmid and are then restarted via RDR on the second plasmid.

Bacteriophage T4↗
PubMed1996

Multiple DNA elements in ARS305 determine replication origin activity in a yeast chromosome.

A yeast autonomously replicating sequence, ARS305, shares essential components with a chromosome III replicator, ORI305. Known components include an ARS consensus sequence (ACS) element, presumed to bind the origin recognition complex (ORC), and a broad 3'-flanking sequence which contains a DNA unwinding element. Here linker substitution mutagenesis of ARS305 and analysis of plasmid mitotic stability identified three short sequence elements within the broad 3'-flanking sequence. The major functional element resides directly 3' of the ACS and the two remaining elements reside further downstream, all within non-conserved ARS sequences. To determine the contribution of the elements to replication origin function in the chromosome, selected linker mutations were transplaced into the ORI305 locus and two-dimensional gel electrophoresis was used to analyze replication bubble formation and fork directions. Mutation of the major functional element identified in the plasmid mitotic stability assay inactivated replication origin function in the chromosome. Mutation of each of the two remaining elements diminished both plasmid ARS and chromosomal origin activities to similar levels. Thus multiple DNA elements identified in the plasmid ARS are determinants of replication origin function in the natural context of the chromosome. Comparison with two other genetically defined chromosomal replicators reveals a conservation of functional elements known to bind ORC, but no two replicators are identical in the arrangement of elements downstream of ORC binding elements or in the extent of functional sequences adjacent to the ACS.

Base Sequence↗
PubMed2006

Transcription regulatory elements are punctuation marks for DNA replication.

Collisions between DNA replication and transcription significantly affect genome organization, regulation, and stability. Previous studies have described collisions between replication forks and elongating RNA polymerases. Although replication collisions with the transcription-initiation or -termination complexes are potentially even more important because most genes are not actively transcribed during DNA replication, their existence and mechanisms remained unproven. To address this matter, we have designed a bacterial promoter that binds RNA polymerase and maintains it in the initiating mode by precluding the transition into the elongation mode. By using electrophoretic analysis of replication intermediates, we have found that this steadfast transcription-initiation complex inhibits replication fork progression in an orientation-dependent manner during head-on collisions. Transcription terminators also appeared to attenuate DNA replication, but in the opposite, codirectional orientation. Thus, transcription regulatory signals may serve as "punctuation marks" for DNA replication in vivo.

AT Rich Sequence↗
PubMed1997

The Oct-1 POU homeodomain stabilizes the adenovirus preinitiation complex via a direct interaction with the priming protein and is displaced when the replication fork passes.

Initiation of adenovirus DNA replication is strongly enhanced by two cellular transcription factors, NFI and Oct-1, which bind to the auxiliary origin and tether the viral precursor terminal protein-DNA polymerase (pTP.pol) complex to the core origin. NFI acts through a direct contact with the DNA polymerase, but the mode of action of Oct 1 is unknown. Employing glutathione S-transferase-POU pull-down assays and protein affinity chromatography, we have established that the POU domain contacts pTP rather than pol. The POU homeodomain is responsible for this interaction. The protein-protein contacts lead to increased binding of pTP-pol to the core origin, which is caused by a reduced off-rate. The enhanced formation of a pTP.pol.POU complex on the origin correlates with stimulation of replication. Using an immobilized replication system, we have studied the kinetics of dissociation of the Oct-1 POU domain during replication. In contrast to NFI, which dissociates very early in initiation, Oct-1 dissociates only when the binding site is rendered single-stranded upon translocation of the replication fork. Our data indicate that NFI and Oct-1 enhance initiation synergistically by touching different targets in the preinitiation complex and dissociate independently after initiation.

Adenoviridae↗
PubMed1997

When helicase and topoisomerase meet!

Several examples of direct interactions between helicases and topoisomerases have recently been described. The data suggest a possible cooperation between these enzymes in major DNA events such as the progression of a replication fork, segregation of newly replicated chromosomes, disruption of nucleosomal structure, DNA supercoiling, and finally recombination, repair, and genomic stability. A first example is the finding of a strong interaction between T antigen and topoisomerase I in mammalian cells, that may trigger unwinding of the parental DNA strands at the replication forks of Simian Virus 40. A second example is the reverse gyrase from thermophilic prokaryotes, composed of a putative helicase domain, and a topoisomerase domain in the same polypeptide. This enzyme may be required to maintain genomic stability at high temperature. A third example is the finding of an interaction between type II topoisomerase and the helicase Sgs1 in yeast. This interaction possibly allows the faithful segregation of newly replicated chromosomes in eukaryotic cells. A fourth example is the interaction between the same helicase Sgs1 and topoisomerase III in yeast, that may control recombination level and genetic stability of repetitive sequences. Recently, in humans, mutations in genes similar to Sgs1 have been found to be responsible for Bloom's and Werner's syndromes. The cooperation between helicases and topoisomerases is likely to be extended to many aspects of DNA mechanisms including chromatin condensation/decondensation.

Archaea↗
PubMed2001

A DNA replication origin and a replication fork barrier used in vivo in the circular plasmid pKD1.

As in other yeasts, ARS-containing plasmids can be maintained extrachromosomally in Kluyveromyces lactis. Although some fragments of K. lactis DNA have ARS activity in both K. lactis and Saccharomyces cerevisiae, it appears that the sequences required for ARS activity in the two yeasts are different. As an approach to a better understanding of ARS structure and function in K. lactis, we analyzed the replication of the circular plasmid pKD1. We identified a 159-bp sequence able to promote autonomous replication of pKD1 in both yeasts; this fragments contains both a sequence related to the S. cerevisiae ARS consensus sequence and a region of 53% identity to the 40-bp sequence essential for K. lactis KARS101 function. By the analysis of in vivo replication intermediates we provide the first direct evidence that DNA replication initiates at or near the K. lactis ARS element. Replication terminates at the cisacting stability locus of pKD1, which functions as a replication fork barrier (RFB) and is necessary for proper plasmid segregation. RFB activity requires the pKDI gene products that are important for plasmid segregation, suggesting a link between DNA replication termination and plasmid segregation in a eukaryotic organism.

Base Sequence↗
PubMed1994

Yeast Saccharomyces cerevisiae as a model system to study the cytotoxic activity of the antitumor drug camptothecin.

Eukaryotic DNA topoisomerase I catalyzes the relaxation of positively and negatively supercoiled DNA and plays a critical role in processes involving DNA, such as DNA replication, transcription and recombination. The enzyme is encoded by the TOP1 gene and is highly conserved in its amino acid sequence and sensitivity to the anti-neoplatic agent camptothecin. This plant alkaloid specifically targets DNA topoisomerase I by reversibly stabilizing the covalent enzyme-DNA intermediate. Presumably, it is the interaction of these drug-stabilized adducts with other cellular components, such as replication forks, that actually produces the DNA lesions leading to cell death. A conservation of the mechanism(s) of camptothecin-induced cell killing is also implicit in studies of the yeast Saccharomyces cerevisiae, where the camptothecin sensitivity of delta TOP1 yeast cells can be restored by plasmids expressing either yeast or human TOP1 sequences. This genetically tractable system is currently being exploited to describe the specific molecular interactions required for the cytotoxic action of camptothecin. The results of mutational analyses of yeast and human DNA topoisomerase I are presented, as well as a genetic screen designed to identify genes, other than TOP1, that are required for the cytotoxic activity of camptothecin.

Amino Acid Sequence↗
PubMed2025

Enzymatic and Structural Roles of Candida albicans Rev1 in DNA Damage Response and Disseminated Candidiasis.

Translesion DNA synthesis (TLS) is a fundamental biological process that enables DNA replication through various lesions to ensure genome stability and to prevent cell death due to replication fork collapse. Rev1, a member of Y-family DNA polymerase (Pol), functions in concert with a B-family enzyme Polζ in promoting TLS through various lesions. Interestingly, for such a function, the catalytic activity of Rev1 seems to be dispensable in Saccharomyces cerevisiae. Unlike Polζ, which possesses robust DNA polymerase activity, biochemical assays suggest that Rev1 predominantly incorporates a "C" opposite any templating residues, but the biological relevance of this activity of Rev1 remains elusive. Here we characterized Rev1 from Candida albicans, an opportunistic fungal pathogen responsible for maximum casualties due to systemic candidiasis in immunosuppressed individuals. Concerted genetic analyses of several Rev1 mutants in various DNA-damaging conditions suggested that in most lesion bypasses except 4-NQO-induced DNA lesions, the catalytic role of Rev1 is not important. However, simultaneous interactions of BRCT and the C-terminal domain of Rev1 with PCNA and Polζ, respectively, enable Rev1 to be essential during TLS. DNA damage recovery and mutagenesis assays further confirmed the lesion-specific roles of various domains of Rev1. Contrary to ex vivo data, animal studies suggested that CaRev1 is dispensable for systemic candidiasis development. We discuss the possible involvement of other TLS DNA polymerases in DNA damage response while C. albicans replicates and establishes itself in the host.

Candida albicans↗
PubMed2002

The RuvABC resolvase is indispensable for recombinational repair in sbcB15 mutants of Escherichia coli.

The RuvABC proteins of Escherichia coli play an important role in the processing of Holliday junctions during homologous recombination and recombinational repair. Mutations in the ruv genes have a moderate effect on recombination and repair in wild-type strains but confer pronounced recombination deficiency and extreme sensitivity to DNA-damaging agents in a recBC sbcBC background. Genetic analysis presented in this work revealed that the (Delta)ruvABC mutation causes an identical DNA repair defect in UV-irradiated recBC sbcBC, sbcBC, and sbcB strains, indicating that the sbcB mutation alone is responsible for the extreme UV sensitivity of recBC sbcBC ruv derivatives. In experiments with gamma irradiation and in conjugational crosses, however, sbcBC (Delta)ruvABC and sbcB (Delta)ruvABC mutants displayed higher recombination proficiency than the recBC sbcBC (Delta)ruvABC strain. The frequency of conjugational recombination observed with the sbcB (Delta)ruvABC strain was quite similar to that of the (Delta)ruvABC single mutant, indicating that the sbcB mutation does not increase the requirement for RuvABC in a recombinational process starting from preexisting DNA ends. The differences between the results obtained in three experimental systems used suggest that in UV-irradiated cells, the RuvABC complex might act in an early stage of recombinational repair. The results of this work are discussed in the context of recent recombination models which propose the participation of RuvABC proteins in the processing of Holliday junctions made from stalled replication forks. We suggest that the mutant SbcB protein stabilizes these junctions and makes their processing highly dependent on RuvABC resolvase.

Bacterial Proteins↗
PubMed2002

Mutations in DNA replication genes reduce yeast life span.

Surprisingly, the contribution of defects in DNA replication to the determination of yeast life span has never been directly investigated. We show that a replicative yeast helicase/nuclease, encoded by DNA2 and a member of the same helicase subfamily as the RecQ helicases, is required for normal life span. All of the phenotypes of old wild-type cells, for example, extended cell cycle time, age-related transcriptional silencing defects, and nucleolar reorganization, occur after fewer generations in dna2 mutants than in the wild type. In addition, the life span of dna2 mutants is extended by expression of an additional copy of SIR2 or by deletion of FOB1, which also increase wild-type life span. The ribosomal DNA locus and the nucleolus seem to be particularly sensitive to defects in dna2 mutants, although in dna2 mutants extrachromosomal ribosomal circles do not accumulate during the aging of a mother cell. Several other replication mutations, such as rad27 Delta, encoding the FEN-1 nuclease involved in several aspects of genomic stability, also show premature aging. We propose that replication fork failure due to spontaneous, endogenous DNA damage and attendant genomic instability may contribute to replicative senescence. This may imply that the genomic instability, segmental premature aging symptoms, and cancer predisposition associated with the human RecQ helicase diseases, such as Werner, Bloom, and Rothmund-Thomson syndromes, are also related to replicative stress.

Adenosine Triphosphatases↗
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↗
PubMed2006

The Bloom's syndrome helicase can promote the regression of a model replication fork.

Homozygous inactivation of BLM gives rise to Bloom's syndrome, a disorder associated with genomic instability and cancer predisposition. BLM encodes a member of the RecQ DNA helicase family that is required for the maintenance of genome stability and the suppression of sister-chromatid exchanges. BLM has been proposed to function in the rescue of replication forks that have collapsed or stalled as a result of encountering lesions that block fork progression. One proposed mechanism of fork rescue involves regression in which the nascent leading and lagging strands anneal to create a so-called "chicken foot" structure. Here we have developed an in vitro system for analysis of fork regression and show that BLM, but not Escherichia coli RecQ, can promote the regression of a model replication fork. BLM-mediated fork regression is ATP-dependent and occurs processively, generating regressed arms of >250 bp in length. These data establish the existence of a eukaryotic protein that could promote replication fork regression in vivo and suggest a novel pathway through which BLM might suppress genetic exchanges.

Adenosine Triphosphatases↗
PubMed2006

Accumulation of FFA-1, the Xenopus homolog of Werner helicase, and DNA polymerase delta on chromatin in response to replication fork arrest.

Werner syndrome is a genetic disorder characterized by premature aging and cancer-prone symptoms, and is caused by mutation of the WRN gene. WRN is a member of the RecQ helicase family and is thought to function in processes implicated in DNA replication and repair to maintain genome stability; however, its precise function is still unclear. We found that replication fork arrest markedly enhances chromatin binding of focus-forming activity 1 (FFA-1), a Xenopus WRN homolog, in Xenopus egg extracts. In addition to FFA-1, DNA polymerase delta (Poldelta) and replication protein A, but not DNA polymerase epsilon and proliferating cell nuclear antigen, accumulated increasingly on replication-arrested chromatin. Elevated accumulation of these proteins was dependent on formation of pre-replicative complexes (pre-RCs). Double-strand break (DSB) formation also enhanced chromatin binding of FFA-1, but not Poldelta, independently of pre-RC formation. In contrast to FFA-1, chromatin binding of Xenopus Bloom syndrome helicase (xBLM) only slightly increased after replication arrest or DSB formation. Thus, WRN-specific, distinct processes can be reproduced in the in vitro system in egg extracts, and this system is useful for biochemical analysis of WRN functions during DNA metabolism.

Adenosine Triphosphatases↗
PubMed2003

Evidence that yeast SGS1, DNA2, SRS2, and FOB1 interact to maintain rDNA stability.

We and others have proposed that faulty processing of arrested replication forks leads to increases in recombination and chromosome instability in Saccharomyces cerevisiae. Now we use the ribosomal DNA locus, which is a good model for all stages of DNA replication, to test this hypothesis. We showed previously that DNA replication pausing at the ribosomal DNA replication fork barrier (RFB) is accompanied by the occurrence of double-strand breaks near the RFB. Both pausing and breakage are elevated in the hypomorphic dna2-2 helicase mutant. Deletion of FOB1 suppresses the elevated pausing and DSB formation. Our current work shows that mutation inactivating Sgs1, the yeast RecQ helicase ortholog, also causes accumulation of stalled replication forks and DSBs at the rDNA RFB. Either deletion of FOB1, which suppresses fork blocking and certain types of rDNA recombination, or an increase in SIR2 gene dosage, which suppresses rDNA recombination, reduces the number of forks persisting at the RFB. Although dna2-2 sgs1Delta double mutants are conditionally lethal, they do not show enhanced rDNA defects compared to sgs1Delta alone. However, surprisingly, the dna2-2 sgs1Delta lethality is suppressed by deletion of FOB1. On the other hand, the dna2-2 sgs1Delta lethality is only partially suppressed by deletion of rad51Delta. We propose that the replication-associated defects that we document in the rDNA are characteristic of similar events occurring either stochastically throughout the genome or at other regions where replication forks move slowly or stall, such as telomeres, centromeres, or replication slow zones.

Adenosine Triphosphatases↗
PubMed1993

[Poisons of DNA topoisomerases I and II].

Over the past decade, DNA topoisomerase I and II appeared to be the targets of some antitumor agents: CPT-11 and Topotecan derived from Camptothecin which interact with topoisomerase I; Actinomycin D, Adriamycin and Daunorubicin, Elliptinium Acetate, Mitoxantrone, Etoposide and Teniposide, Amsacrine which interact with topoisomerase II. The multiple functions of these enzymes are important as they play a role during replication, transcription, recombination, repair and chromatine organisation. Particularly, they relax torsional constraints which appear when intertwined DNA strands are separated while replication fork or RNA polymerases are moving. To some extent, topoisomerase I and II are structurally and functionally different. Moreover, topoisomerase I is not indispensable for a living cell whereas topoisomerase II is. Drug-topoisomerase interaction which probably leads to antitumoral effect of the compounds studied in this review is not a trivial inhibition of the enzyme but rather a poisoning due to stabilization of cleavable complexes between the enzyme and DNA. These stabilized complexes are likely to induce apoptosis-like programmed cell death, which is characterised by DNA fragmentation. However, it appears that it is the collision of the replication fork with the drug-stabilized cleavable complex that is responsible for the cytotoxicity of the drug: poisoning of topoisomerases by antitumor agents leads to a new concept of "dynamic toxicity". Although they interact with a common target, topoisomerase II poisons have differential effects on macromolecules syntheses, cell cycle and chromosome fragmentation; a few compounds may produce free radicals. Because of these differential effects in addition to quantitative and qualitative variations of stabilized cleavable complexes, in particular DNA sequences on which topoisomerase II is stabilized, these antitumor agents do not resemble each other. Cellular resistance to topoisomerases poisons results of two principal types of alteration: target and/or drug transport modification. Decreased ability to form the cleavable complex in resistant cells may be the consequence of both decreased amount of topoisomerase or altered enzyme. On the other hand, overexpression of membrane P-glycoprotein, which pumps drugs out of the cell by an energy dependent process provokes a decreased accumulation of these drugs. Cross resistances to other drugs are mainly under control of these two different mechanisms of resistance. A complete knowledge of their individual effects and mechanisms of resistance would allow a better clinical use of topoisomerases poisons, especially when administered in combination chemotherapy.

Antineoplastic Agents↗
PubMed2002

Recombinational DNA repair and human disease.

We review the genes and proteins related to the homologous recombinational repair (HRR) pathway that are implicated in cancer through either genetic disorders that predispose to cancer through chromosome instability or the occurrence of somatic mutations that contribute to carcinogenesis. Ataxia telangiectasia (AT), Nijmegen breakage syndrome (NBS), and an ataxia-like disorder (ATLD), are chromosome instability disorders that are defective in the ataxia telangiectasia mutated (ATM), NBS, and Mre11 genes, respectively. These genes are critical in maintaining cellular resistance to ionizing radiation (IR), which kills largely by the production of double-strand breaks (DSBs). Bloom syndrome involves a defect in the BLM helicase, which seems to play a role in restarting DNA replication forks that are blocked at lesions, thereby promoting chromosome stability. The Werner syndrome gene (WRN) helicase, another member of the RecQ family like BLM, has very recently been found to help mediate homologous recombination. Fanconi anemia (FA) is a genetically complex chromosomal instability disorder involving seven or more genes, one of which is BRCA2. FA may be at least partially caused by the aberrant production of reactive oxidative species. The breast cancer-associated BRCA1 and BRCA2 proteins are strongly implicated in HRR; BRCA2 associates with Rad51 and appears to regulate its activity. We discuss in detail the phenotypes of the various mutant cell lines and the signaling pathways mediated by the ATM kinase. ATM's phosphorylation targets can be grouped into oxidative stress-mediated transcriptional changes, cell cycle checkpoints, and recombinational repair. We present the DNA damage response pathways by using the DSB as the prototype lesion, whose incorrect repair can initiate and augment karyotypic abnormalities.

Ataxia Telangiectasia↗