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PubMed2004

Seckel syndrome exhibits cellular features demonstrating defects in the ATR-signalling pathway.

To date, the only reported genetic defect identified in the developmental disorder, Seckel syndrome, is a mutation in ataxia telangiectasia and Rad3-related protein (ATR). Seckel syndrome is clinically and genetically heterogeneous and whether defects in ATR significantly contribute to Seckel syndrome is unclear. Firstly, we characterize ATR-Seckel cells for their response to DNA damage. ATR-Seckel cells display impaired phosphorylation of ATR-dependent substrates, impaired G2/M checkpoint arrest and elevated micronucleus (MN) formation following exposure to UV and agents that cause replication stalling. We describe a novel phenotype, designated nuclear fragmentation (NF), that occurs following replication arrest. Finally, we report that ATR-Seckel cells have an endogenously increased number of centrosomes in mitotic cells demonstrating a novel role for ATR in regulating centrosome stability. We exploit these phenotypes to examine cell lines derived from additional unrelated Seckel syndrome patients. We show that impaired phosphorylation of ATR-dependent substrates is a common but not invariant feature of Seckel syndrome cell lines. In contrast, all cell lines displayed defective G2/M arrest, increased levels of NF and MN formation following exposure to agents that cause replication stalling. All the Seckel syndrome cell lines examined showed increased endogenous centrosome numbers. Though ATR cDNA can complement the defects in ATR-Seckel cells, it failed to complement any of the additional cell lines. We conclude that Seckel syndrome represents a further damage response disorder that is uniquely associated with defects in the ATR-signalling pathway resulting in failed checkpoint arrest following exposure to replication fork stalling.

Ataxia Telangiectasia Mutated Proteins↗

PubMed2003

A tumour-derived mutant allele of XRCC2 preferentially suppresses homologous recombination at DNA replication forks.

Homologous recombination repair (HRR) is required for both the repair of DNA double strand breaks (DSBs) and the maintenance of the integrity of DNA replication forks. To determine the effect of a mutant allele of the RAD51 paralog XRCC2 (342delT) found in an HRR-defective tumour cell line, 342delT was introduced into HRR proficient cells containing a recombination reporter substrate. In one set of transfectants, expression of 342delT conferred sensitivity to thymidine and mitomycin C and suppressed HRR induced at the recombination reporter by thymidine but not by DSBs. In a second set of transfectants, the expression of 342delT was accompanied by a decreased level of the full-length XRCC2. These cells were defective in the induction of HRR by either thymidine or DSBs. Thus 342delT suppresses recombination induced by thymidine in a dominant negative manner while recombination induced by DSBs appears to depend upon the level of XRCC2 as well as the expression of the mutant XRCC2 allele. These results suggest that HRR pathways responding to stalled replication forks or DSBs are genetically distinguishable. They further suggest a critical role for XRCC2 in HRR at replication forks, possibly in the loading of RAD51 onto gapped DNA.

Base Pair Mismatch↗

PubMed2006

Complex formation with Rev1 enhances the proficiency of Saccharomyces cerevisiae DNA polymerase zeta for mismatch extension and for extension opposite from DNA lesions.

Rev1, a Y family DNA polymerase (Pol) functions together with Polzeta, a B family Pol comprised of the Rev3 catalytic subunit and Rev7 accessory subunit, in promoting translesion DNA synthesis (TLS). Extensive genetic studies with Saccharomyces cerevisiae have indicated a requirement of both Polzeta and Rev1 for damage-induced mutagenesis, implicating their involvement in mutagenic TLS. Polzeta is specifically adapted to promote the extension step of lesion bypass, as it proficiently extends primer termini opposite DNA lesions, and it is also a proficient extender of mismatched primer termini on undamaged DNAs. Since TLS through UV-induced lesions and various other DNA lesions does not depend upon the DNA-synthetic activity of Rev1, Rev1 must contribute to Polzeta-dependent TLS in a nonenzymatic way. Here, we provide evidence for the physical association of Rev1 with Polzeta and show that this binding is mediated through the C terminus of Rev1 and the polymerase domain of Rev3. Importantly, a rev1 mutant that lacks the C-terminal 72 residues which inactivate interaction with Rev3 exhibits the same high degree of UV sensitivity and defectiveness in UV-induced mutagenesis as that conferred by the rev1Delta mutation. We propose that Rev1 binding to Polzeta is indispensable for the targeting of Polzeta to the replication fork stalled at a DNA lesion. In addition to this structural role, Rev1 binding enhances the proficiency of Polzeta for the extension of mismatched primer termini on undamaged DNAs and for the extension of primer termini opposite DNA lesions.

Base Pair Mismatch↗

PubMed1992

Replication forks are not found in a Drosophila minichromosome demonstrating a gradient of polytenization.

Differential DNA replication is widely held to influence polytene chromosome structure by causing the dramatic reductions in heterochromatic DNA content that are characteristic of most endopolyploid cells. The "underreplication model" of heterochromatic sequence underrepresentation predicts that replication intermediates should populate regions of DNA between fully polytenized euchromatic sequences and underpolytenized heterochromatic sequences. We directly tested this prediction using Dp1187, a 1300 kb Drosophila minichromosome containing well-defined heterochromatic regions. DNA from a euchromatic/heterochromatic junction region of Dp1187, demonstrating a significant gradient of underrepresentation in larval salivary glands, lacked the stalled replication forks predicted by the underreplication model. We consider an alternative mechanism leading to heterochromatic sequence underrepresentation involving a process of DNA elimination.

Animals↗

PubMed2006

DNA damage responses and their many interactions with the replication fork.

The cellular response to DNA damage is composed of cell cycle checkpoint and DNA repair mechanisms that serve to ensure proper replication of the genome prior to cell division. The function of the DNA damage response during DNA replication in S-phase is critical to this process. Recent evidence has suggested a number of interrelationships of DNA replication and cellular DNA damage responses. These include S-phase checkpoints which suppress replication initiation or elongation in response to DNA damage. Also, many components of the DNA damage response are required either for the stabilization of, or for restarting, stalled replication forks. Further, translesion synthesis permits DNA replication to proceed in the presence of DNA damage and can be coordinated with subsequent repair by homologous recombination (HR). Finally, cohesion of sister chromatids is established coincident with DNA replication and is required for subsequent DNA repair by homologous recombination. Here we review these processes, all of which occur at, or are related to, the advancing replication fork. We speculate that these multiple interdependencies of DNA replication and DNA damage responses integrate the many steps necessary to ensure accurate duplication of the genome.

Animals↗

PubMed2003

Elg1 forms an alternative RFC complex important for DNA replication and genome integrity.

Genome-wide synthetic genetic interaction screens with mutants in the mus81 and mms4 replication fork-processing genes identified a novel replication factor C (RFC) homolog, Elg1, which forms an alternative RFC complex with Rfc2-5. This complex is distinct from the DNA replication RFC, the DNA damage checkpoint RFC and the sister chromatid cohesion RFC. As expected from its genetic interactions, elg1 mutants are sensitive to DNA damage. Elg1 is redundant with Rad24 in the DNA damage response and contributes to activation of the checkpoint kinase Rad53. We find that elg1 mutants display DNA replication defects and genome instability, including increased recombination and mutation frequencies, and minichromosome maintenance defects. Mutants in elg1 show genetic interactions with pathways required for processing of stalled replication forks, and are defective in recovery from DNA damage during S phase. We propose that Elg1-RFC functions both in normal DNA replication and in the DNA damage response.

Carrier Proteins↗

PubMed2005

Differences in the association of p53 phosphorylated on serine 15 and key enzymes of homologous recombination.

Phosphorylation of p53 on serine 15 by ATM or ATR is a frequent modification and initiates a cascade of post-translational modifications. To identify possible mechanisms that modulate p53 functions in recombination surveillance, we compared the nuclear localization of p53 phosphorylated on serine 15 (p53pSer15) and the key enzymes of homologous recombination (HR) after replication fork stalling. We demonstrate an almost mutually exclusive subcompartmentalization with Rad52, while p53pSer15 was colocalizing with 40-60% of the Rad51 and Mre11 foci. Therefore, possible sites of p53pSer15-dependent regulation seem to be sites of Rad51- rather than Rad52-dependent HR processes. Remarkably, the association of p53pSer15 with repair complexes containing Rad51 or Mre11 was transient, because less than 20% of the Rad51 and Mre11 foci overlapped with p53pSer15 after 6 h. When we examined colocalization and co-immunoprecipitation of p53pSer15 and the RecQ helicase BLM with recombination surveillance and proapoptotic functions, we observed colocalization within a fraction of approximately 70% of the BLM foci and stable physical interactions until 6 h after replication arrest. Our data suggest that p53pSer15 plays a dual role in the functional interactions with early complexes of Rad51-dependent recombination and with BLM-associated surveillance and signalling complexes within distinct nuclear subcompartments.

Adenosine Triphosphatases↗

PubMed1999

Duplex opening by primosome protein PriA for replisome assembly on a recombination intermediate.

PriA and other primosome assembly proteins of Escherichia coli recruit the major replicative helicase DnaB for replisome assembly during bacteriophage Mu transposition and replication. MuA transposase catalyzes the transfer of Mu ends to target DNA, forming a potential replication fork that provides the assembly site for the replisome. However, this fork lacks the single-stranded DNA needed to load DnaB. Although no pre-existing primosome assembly sites that bind PriA were found within the Mu end sequences, PriA was able to bind to the forked DNA structure created by MuA. The helicase activity of PriA could then open the duplex to create the DnaB binding site. In a tightly coupled reaction on synthetic forked substrates, PriA promoted both the unwinding of the lagging strand arm and preprimosome assembly to load DnaB onto the lagging strand template. PriA apparently translocated 3' to 5' along the lagging strand template until sufficient single-stranded DNA was exposed for binding of DnaB, which then translocated 5' to 3' in the opposite direction. Mutant PriA lacking helicase activity was unable to promote this process, and loss of PriA helicase impaired Mu DNA replication in vivo and in vitro. This suggests that the opening of the duplex by PriA helicase is a critical step in the initiation of Mu DNA replication. Concerted helicase and primosome assembly functions would allow PriA to act as initiator on recombination intermediates and stalled replication forks. As part of the replisome, PriA may act as a mobile initiator that minimizes interruptions in chromosomal replication.

Bacterial Proteins↗

PubMed2004

Co-localization in replication foci and interaction of human Y-family members, DNA polymerase pol eta and REVl protein.

The progress of replicative DNA polymerases along the replication fork may be impeded by the presence of lesions in the genome. One way to circumvent such hurdles involves the recruitment of specialized DNA polymerases that perform limited incorporation of nucleotides in the vicinity of the damaged site. This process entails DNA polymerase switch between replicative and specialized DNA polymerases. Five eukaryotic proteins can carry out translesion synthesis (TLS) of damaged DNA in vitro, DNA polymerases zeta, eta, iota, and kappa, and REV1. To identify novel proteins that interact with hpol eta, we performed a yeast two-hybrid screen. In this paper, we show that hREV1 interacts with hpol eta as well as with hpol kappa and poorly with hpol iota. Furthermore, cellular localization analysis demonstrates that hREV1 is present, with hpol eta in replication factories at stalled replication forks and is tightly associated with nuclear structures. This hREV1 nuclear localization occurs independently of the presence of hpol eta. Taken together, our data suggest a central role for hREV1 as a scaffold that recruits DNA polymerases involved in TLS.

Base Sequence↗

PubMed2006

The histone chaperone ASF1 localizes to active DNA replication forks to mediate efficient DNA replication.

The packaging of the eukaryotic genome into chromatin is likely to regulate all processes that occur on the DNA template. The assembly and disassembly of chromatin structures from histone proteins and DNA are mediated by histone chaperones, including the histone H3/H4 chaperone anti-silencing function 1 (ASF1). To address the function of ASF1 in metazoan cells, we used RNA interference-mediated knockdown of Drosophila melanogaster ASF1 (dASF1). Cells lacking dASF1 accumulate in S phase of the cell cycle as determined by flow cytometry analysis of DNA content and quantitation of the proportion of cells with replication foci. In agreement, bromodeoxyuridine (BrdU) pulse-chase analysis demonstrates that the absence of ASF1 leads to delayed progression through S-phase. Furthermore, the absence of ASF1 leads to a reduced ability to incorporate the nucleoside analog BrdU, indicating that ASF1 is required for efficient DNA replication. We have also found that dASF1 colocalizes with DNA replication foci throughout S phase by immunofluorescence analysis and that these dASF1 foci are disrupted upon inhibition of DNA replication by treatment of cells with hydroxyurea. As such, these results demonstrate that dASF1 is present at active, but not stalled, replication forks. We propose that dASF1 has a direct role in modifying chromatin structure during DNA replication and that this function of dASF1 is important for the processivity of the replication machinery.

Animals↗

PubMed2004

Actinomycin D induces histone gamma-H2AX foci and complex formation of gamma-H2AX with Ku70 and nuclear DNA helicase II.

Formation of gamma-H2AX foci is a P. O.cellular response to genotoxic stress, such as DNA double strand breaks or stalled replication forks. Here we show that gamma-H2AX foci were also formed when cells were incubated with 0.5 microg/ml DNA intercalating agent actinomycin D. In untreated cells, gamma-H2AX co-immunoprecipitated with Ku70, a subunit of DNA-dependent protein kinase, as well as with nuclear DNA helicase II (NDH II), a DEXH family helicase also known as RNA helicase A or DHX9. This association was increased manifold after actinomycin D treatment. DNA degradation diminished the amount of Ku70 associated with gamma-H2AX but not that of NDH II. In vitro binding studies with recombinant NDH II and H2AX phosphorylated by DNA-dependent protein kinase confirmed a direct physical interaction between NDH II and gamma-H2AX. Thereby, the NDH II DEXH domain alone, i.e. its catalytic core, was able to support binding to gamma-H2AX. Congruently, after actinomycin D treatment, NDH II accumulated in RNA-containing nuclear bodies that predominantly co-localized with gamma-H2AX foci. Taken together, these results suggest that histone gamma-H2AX promotes binding of NDH II to transcriptionally stalled sites on chromosomal DNA.

Adenosine Triphosphatases↗

PubMed2006

Differential usage of non-homologous end-joining and homologous recombination in double strand break repair.

Repair of DNA double strand breaks (DSBs) plays a critical role in the maintenance of the genome. DSB arise frequently as a consequence of replication fork stalling and also due to the attack of exogenous agents. Repair of broken DNA is essential for survival. Two major pathways, homologous recombination (HR) and non-homologous end-joining (NHEJ) have evolved to deal with these lesions, and are conserved from yeast to vertebrates. Despite the conservation of these pathways, their relative contribution to DSB repair varies greatly between these two species. HR plays a dominant role in any DSB repair in yeast, whereas NHEJ significantly contributes to DSB repair in vertebrates. This active NHEJ requires a regulatory mechanism to choose HR or NHEJ in vertebrate cells. In this review, we illustrate how HR and NHEJ are differentially regulated depending on the phase of cell cycle and on the nature of the DSB.

Animals↗

PubMed2005

DNA polymerases eta and kappa are responsible for error-free translesion DNA synthesis activity over a cis-syn thymine dimer in Xenopus laevis oocyte extracts.

In translesion synthesis (TLS), specialized DNA polymerases (pols) facilitate progression of replication forks stalled by DNA damage. Although multiple TLS pols have been identified in eukaryotes, little is known about endogenous TLS pols and their relative contributions to TLS in vivo because of their low cellular abundance. Taking advantage of Xenopus laevis oocyte cells, with their extraordinary size and abundant enzymes involved in DNA metabolism, we have identified and characterized endogenous TLS pols for DNA damage induced by ultraviolet (UV) irradiation. We designed a TLS assay which monitors primer elongation on a synthetic oligomer template over a single UV-induced lesion, either a cys-syn cyclobutane pyrimidine dimer (CPD) or a pyrimidine (6-4) pyrimidone photoproduct. Four distinct TLS activities (TLS1-TLS4) were identified in X. laevis oocyte extracts, using three template/primer (T/P) DNA substrates having various sites at which primer extension is initiated relative to the lesion. TLS1 and TLS2 activities appear to be sequence-dependent. TLS3 and TLS4 extended the primers over the CPD in an error-free manner irrespective of sequence context. Base insertion opposite the CPD of the T/P substrate in which the 3'-end of the primer is placed one base upstream of the lesion was observed only with TLS3. TLS3 and TLS4 showed primer extension with similar efficiencies on the T/P substrate whose 3'-primer terminal dinucleotide (AA) was complementary to the CPD lesion. Investigations with antibodies and recombinant pols revealed that TLS3 and TLS4 were most likely attributable to pol eta and pol kappa, respectively. These results indicate that error-free insertion in CPD bypass is due mainly to pol eta (TLS3) in the extracts, and suggest that pol kappa (TLS4) may assist pol eta (TLS3) in error-free extension during CPD bypass.

Animals↗

PubMed2006

DNA damage-induced RPA focalization is independent of gamma-H2AX and RPA hyper-phosphorylation.

Replication protein A (RPA) is the major eukaryotic single stranded DNA binding protein that plays a central role in DNA replication, repair and recombination. Like many DNA repair proteins RPA is heavily phosphorylated (specifically on its 32 kDa subunit) in response to DNA damage. Phosphorylation of many repair proteins has been shown to be important for their recruitment to DNA damage-induced intra-nuclear foci. Further, phosphorylation of H2AX (gamma-H2AX) has been shown to be important for either the recruitment or stable retention of DNA repair proteins to these intra-nuclear foci. We address here the relationship between DNA damage-induced hyper-phosphorylation of RPA and its intra-nuclear focalization, and whether gamma-H2AX is required for RPA's presence at these foci. Using GFP-conjugated RPA, we demonstrate the formation of extraction-resistant RPA foci induced by DNA damage or stalled replication forks. The strong DNA damage-induced RPA foci appear after phosphorylated histone H2AX and Chk1, but earlier than the appearance of hyper-phosphorylated RPA. We demonstrate that while the functions of phosphoinositol-3-kinase-related protein kinases are essential for DNA damage-induced H2AX phosphorylation and RPA hyper-phosphorylation, they are dispensable for the induction of extraction-resistant RPA and RPA foci. Furthermore, in mouse cells genetically devoid of H2AX, DNA damage-induced extraction-resistant RPA appears with the same kinetics as in normal mouse cells. These results demonstrate that neither RPA hyper-phosphorylation nor H2AX are required for the formation in RPA intra-nuclear foci in response to DNA damage/replicational stress and are consistent with a role for RPA as a DNA damage sensor involved in the initial recognition of damaged DNA or blocked replication forks.

Aminoglycosides↗

PubMed2000

Analysis of conserved basic residues associated with DNA binding (Arg69) and catalysis (Lys76) by the RusA holliday junction resolvase.

Holliday junctions are key intermediates in both homologous recombination and DNA repair, and are also formed from replication forks stalled at lesions in the template strands. Their resolution is critical for chromosome segregation and cell viability, and is mediated by a class of small, homodimeric endonucleases that bind the structure and cleave the DNA. All the enzymes studied require divalent metal ions for strand cleavage and their active centres are characterised by conserved aspartate/glutamate residues that provide ligands for metal binding. Sequence alignments reveal that they also contain a number of conserved basic residues. We used site-directed mutagenesis to investigate such residues in the RusA resolvase. RusA is a 120 amino acid residue polypeptide that can be activated in Escherichia coli to promote recombination and repair in the absence of the Ruv proteins. The RuvA, RuvB and RuvC proteins form a complex on Holliday junction DNA that drives coupled branch migration (RuvAB) and resolution (RuvC) reactions. In contrast to RuvC, the RusA resolvase does not interact directly with a branch migration motor, which simplifies analysis of its resolution activity. Catalysis depends on three highly conserved acidic residues (Asp70, Asp72 and Asp91) that define the catalytic centre. We show that Lys76, which is invariant in RusA sequences, is essential for catalysis, but not for DNA binding, and that an invariant asparagine residue (Asn73) is required for optimal activity. Analysis of DNA binding revealed that RusA may interact with one face of an open junction before manipulating its conformation in the presence of Mg(2+) as part of the catalytic process. A well-conserved arginine residue (Arg69) is linked with this critical stage. These findings provide the first insights into the roles played by basic residues in DNA binding and catalysis by a Holliday junction resolvase.

Amino Acid Sequence↗

PubMed2001

Genetic analysis of an archaeal Holliday junction resolvase in Escherichia coli.

The study of genes and proteins in heterologous model systems provides a powerful approach to the analysis of common processes in biology. Here, we show how the bacterium Escherichia coli can be exploited to analyse genetically and biochemically the activity and function of a Holliday junction resolving enzyme from an archaeal species. We have purified and characterised a member of the newly discovered Holliday junction cleaving (Hjc) family of resolvases from the moderately thermophilic archaeon Methanobacterium thermoautotrophicum and demonstrate that it promotes DNA repair in resolvase-deficient ruv mutants of E. coli. The data presented provide the first direct evidence that such archaeal enzymes can promote DNA repair in vivo, and support the view that formation and resolution of Holliday junctions are key to the interplay between DNA replication, recombination and repair in all organisms. We also show that Hjc promotes DNA repair in E. coli in a manner that requires the presence of the RecG branch migration protein. These results support models in which RecG acts at a replication fork stalled at a lesion in the DNA, catalysing fork regression and forming a Holliday junction that can then be acted upon by Hjc.

Archaeal Proteins↗

PubMed1995

Competition between parental and recombinant plasmids affects the measure of recombination frequencies.

Recombination frequencies in multicopy plasmids are generally deduced from the rate of appearance of cells expressing a recombinant phenotype (i.e., "recombinant cells"). Detection of these cells requires not only the formation of a recombinant molecule but also the establishment of this molecule in the presence of the resident incompatible parental plasmid. Differences in fitness between parental and recombinant molecules will affect this establishment and could have great consequences for plasmid recombination measurements. To test this hypothesis, we compared recombination frequencies when the recombinant plasmid has or does not have a replication advantage over the parental plasmid. We used pBR322-derived plasmids which carry or lack the replication terminator TerB; recombination took place between directly repeated sequences of 16 bp and deleted TerB from the plasmid. The rate of appearance of recombinant cells strongly increased when the Tus/Ter system was active; however, we found no evidence for direct stimulation of recombination between direct repeats by replication fork stalling. The main factor responsible for the increase in the rate of appearance of recombinant cells when the parental plasmid carries TerB is the facilitated establishment of the recombinant plasmid since: (i) the transformation efficiency of the recombinant plasmid is higher in cells containing the Ter+ parent than in cells containing the Ter- parent; (ii) most recombinant plasmids did not lead to the appearance of recombinant cells when pBR322 was was not blocked by Tus, whereas the presence of TerB allows the detection of most events; and (iii) decreasing the parental plasmid copy number without modification of the recombinant plasmid leads to an exponential increase in the rate of appearance of recombinant cells. Our results show that the level of competition between parent and recombinant plasmids can greatly affect plasmid recombination frequencies deduced from the measure of recombinant cells. This effect can be as high as several orders of magnitude.

Base Sequence↗

PubMed1994

RecA, Tus protein and constitutive stable DNA replication in Escherichia coli rnhA mutants.

Constitutive stable DNA replication (cSDR), which uniquely occurs in Escherichia coli rnhA mutants deficient in ribonuclease HI activity, requires RecA function. The recA428 mutation, which inactivates the recombinase activity but imparts a constitutive coprotease activity, blocks cSDR in rnhA mutants. The result indicates that the recombinase activity of RecA, which promotes homologous pairing and strand exchange, is essential for cSDR. Despite the requirement for RecA recombinase activity, mutations in recB, recD, recJ, ruvA and ruvC neither inhibit nor stimulate cSDR. It was proposed that the property of RecA essential for homologous pairing and strand exchange is uniquely required for initiation of cSDR in rnhA mutants without involving the homologous recombination process. The possibility that RecA protein is necessary to counteract the action of Tus protein, a contra-helicase which stalls replication forks in the ter region of the chromosome, was ruled out because introduction of the tus::kan mutation, which inactivates Tus protein, did not alleviate the RecA requirement for cSDR.

Bacterial Proteins↗