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Results for “Replicability”

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At least 235 records · Page 13Linked to original sources

PubMed1998

DNA ligase I is recruited to sites of DNA replication by an interaction with proliferating cell nuclear antigen: identification of a common targeting mechanism for the assembly of replication factories.

In mammalian cells, DNA replication occurs at discrete nuclear sites termed replication factories. Here we demonstrate that DNA ligase I and the large subunit of replication factor C (RF-C p140) have a homologous sequence of approximately 20 amino acids at their N-termini that functions as a replication factory targeting sequence (RFTS). This motif consists of two boxes: box 1 contains the sequence IxxFF whereas box 2 is rich in positively charged residues. N-terminal fragments of DNA ligase I and the RF-C large subunit that contain the RFTS both interact with proliferating cell nuclear antigen (PCNA) in vitro. Moreover, the RFTS of DNA ligase I and of the RF-C large subunit is necessary and sufficient for the interaction with PCNA. Both subnuclear targeting and PCNA binding by the DNA ligase I RFTS are abolished by replacement of the adjacent phenylalanine residues within box 1. Since sequences similar to the RFTS/PCNA-binding motif have been identified in other DNA replication enzymes and in p21(CIP1/WAF1), we propose that, in addition to functioning as a DNA polymerase processivity factor, PCNA plays a central role in the recruitment and stable association of DNA replication proteins at replication factories.

Amino Acid Sequence↗

PubMed1999

Post-replicative base excision repair in replication foci.

Base excision repair (BER) is initiated by a DNA glycosylase and is completed by alternative routes, one of which requires proliferating cell nuclear antigen (PCNA) and other proteins also involved in DNA replication. We report that the major nuclear uracil-DNA glycosylase (UNG2) increases in S phase, during which it co-localizes with incorporated BrdUrd in replication foci. Uracil is rapidly removed from replicatively incorporated dUMP residues in isolated nuclei. Neutralizing antibodies to UNG2 inhibit this removal, indicating that UNG2 is the major uracil-DNA glycosylase responsible. PCNA and replication protein A (RPA) co-localize with UNG2 in replication foci, and a direct molecular interaction of UNG2 with PCNA (one binding site) and RPA (two binding sites) was demonstrated using two-hybrid assays, a peptide SPOT assay and enzyme-linked immunosorbent assays. These results demonstrate rapid post-replicative removal of incorporated uracil by UNG2 and indicate the formation of a BER complex that contains UNG2, RPA and PCNA close to the replication fork.

Amino Acid Sequence↗

PubMed2006

The 32 kDa subunit of replication protein A (RPA) participates in the DNA replication of Mung bean yellow mosaic India virus (MYMIV) by interacting with the viral Rep protein.

Mung bean yellow mosaic India virus (MYMIV) is a member of genus begomoviridae and its genome comprises of bipartite (two components, namely DNA-A and DNA-B), single-stranded, circular DNA of about 2.7 kb. During rolling circle replication (RCR) of the DNA, the stability of the genome and maintenance of the stem-loop structure of the replication origin is crucial. Hence the role of host single-stranded DNA-binding protein, Replication protein A (RPA), in the RCR of MYMIV was examined. Two RPA subunits, namely the RPA70 kDa and RPA32 kDa, were isolated from pea and their roles were validated in a yeast system in which MYMIV DNA replication has been modelled. Here, we present evidences that only the RPA32 kDa subunit directly interacted with the carboxy terminus of MYMIV-Rep both in vitro as well as in yeast two-hybrid system. RPA32 modulated the functions of Rep by enhancing its ATPase and down regulating its nicking and closing activities. The possible role of these modulations in the context of viral DNA replication has been discussed. Finally, we showed the positive involvement of RPA32 in transient replication of the plasmid DNA bearing MYMIV replication origin using an in planta based assay.

Biological Assay↗

PubMed2006

The replicative polymerases PolC and DnaE are required for theta replication of the Bacillus subtilis plasmid pBS72.

Plasmids are the tools of choice for studying bacterial functions involved in DNA maintenance. Here a genetic study on the replication of a novel, low-copy-number, Bacillus subtilis plasmid, pBS72, is reported. The results show that two plasmid elements, the initiator protein RepA and an iteron-containing origin, and at least nine host-encoded replication proteins, the primosomal proteins DnaB, DnaC, DnaD, DnaG and DnaI, the DNA polymerases DnaE and PolC, and the polymerase cofactors DnaN and DnaX, are required for pBS72 replication. On the contrary, the cellular initiators DnaA and PriA, the helicase PcrA and DNA polymerase I are dispensable. From this, it is inferred that pBS72 replication is of the theta type and is initiated by an original mechanism. Indirect evidence suggests that during this process the DnaC helicase might be delivered to the plasmid origin by the weakly active DnaD pathway stimulated by a predicted interaction between DnaC and a domain of RepA homologous to the major DnaC-binding domain of the cellular initiator DnaA. The plasmid pBS72 replication fork appears to require the same functions as the bacterial chromosome and the unrelated plasmid pAMbeta1. Most importantly, this replication machinery contains the two type C polymerases, PolC and DnaE. As the mechanism of initiation of the three genomes is substantially different, this suggests that both type C polymerases might be required in any Cairns replication in B. subtilis and presumably in other bacteria encoding PolC and DnaE.

Bacillus subtilis↗

PubMed2006

The role of replication initiation control in promoting survival of replication fork damage.

Dam methylase mutants were recovered in a screen for mutants sensitive to UV irradiation or mild inhibition of replication elongation. Dam's role in tolerance of DNA damage is to provide binding sites for SeqA, because seqA mutants showed similar sensitivity that was genetically epistatic to dam. The sensitivity of seqA mutants to UV irradiation and to the replication inhibitors hydroxyurea (HU) and azidothymidine (AZT) was suppressed by alleles of dnaA that reduce the efficiency of replication initiation. These results suggest that for survival of replication fork damage, SeqA's repression of replication initiation is more important than its effects on nucleoid organization. Convergence of forks upon DNA damage is a likely explanation for seqA mutant sensitivity, because its poor survival of UV was suppressed by reducing secondary initiation through minimal medium growth. Surprisingly, growth in minimal medium reduced the ability of seqA+ strains to form colonies in the presence of low levels of AZT. Double dnaA seqA mutants exhibited plating efficiencies much superior to wild-type strains during chronic low-level AZT exposure in minimal medium. This suggests that mild inhibition of replication fork progression may actively restrain initiation such that seqA+ strains fail to recover initiation capacity after sustained conditions of replication arrest.

Bacterial Outer Membrane Proteins↗

PubMed2006

The chromosome partitioning proteins Soj (ParA) and Spo0J (ParB) contribute to accurate chromosome partitioning, separation of replicated sister origins, and regulation of replication initiation in Bacillus subtilis.

Soj (ParA) and Spo0J (ParB) of Bacillus subtilis belong to a conserved family of proteins required for efficient plasmid and chromosome partitioning in many bacterial species. Unlike most Par systems, for which intact copies of both parA and parB are required for the Par system to function, inactivating soj does not cause a detectable chromosome partitioning phenotype whereas inactivating spo0J leads to a 100-fold increase in the production of anucleate cells. This suggested either that Soj does not function like other ParA homologues, or that a cellular factor might compensate for the absence of soj. We found that inactivating smc, the gene encoding the structural maintenance of chromosomes (SMC) protein, unmasked a role for Soj in chromosome partitioning. A soj null mutation dramatically enhanced production of anucleate cells in an smc null mutant. To look for effects of a soj null on other phenotypes perturbed in a spo0J null mutant, we analysed replication initiation and origin positioning in (soj-spo0J)+, Deltasoj, Deltaspo0J and Delta(soj-spo0J) cells. All of the mutations caused increased initiation of replication and, to varying extents, affected origin positioning. Using a new assay to measure separation of the chromosomal origins, we found that inactivating soj, spo0J or both led to a significant defect in separating replicated sister origins, such that the origins remain too close to be spatially resolved. Separation of a region outside the origin was not affected. These results indicate that there are probably factors helping to pair sister origin regions for part of the replication cycle, and that Soj and Spo0J may antagonize this pairing to contribute to timely separation of replicated origins. The effects of Deltasoj, Deltaspo0J and Delta(soj-spo0J) mutations on origin positioning, chromosome partitioning and replication initiation may be a secondary consequence of a defect in separating replicated origins.

Bacillus subtilis↗

PubMed1994

DNA replication studies with coliphage 186: the involvement of the Escherichia coli DnaA protein in 186 replication is indirect.

The inability of coliphage 186 to infect productively a dnaA(Ts) mutant at a restrictive temperature was confirmed. However, the requirement by 186 for DnaA is indirect, since 186 can successfully infect suppressed dnaA (null) strains. The block to 186 infection of a dnaA(Ts) strain at a restrictive temperature is at the level of replication but incompletely so, since some 20% of the phage specific replication seen with infection of a dnaA+ host does occur. A mutant screen, to isolate host mutants blocked in 186-specific replication but not in the replication of the close relative coliphage P2, which has no DnaA requirement, yielded a mutant whose locus we mapped to the rep gene. A 186 mutant able to infect this rep mutant was isolated, and the mutation was located in the phage replication initiation endonuclease gene A, suggesting direct interaction between the Rep helicase and phage endonuclease during replication. DNA sequencing indicated a glutamic acid-to-valine change at residue 155 of the 694-residue product of gene A. In the discussion, we speculate that the indirect need of DnaA function is at the level of lagging-strand synthesis in the rolling circle replication of 186.

Bacterial Proteins↗

PubMed2004

Replication of Enterococcus faecalis pheromone-responding plasmid pAD1: location of the minimal replicon and oriV site and RepA involvement in initiation of replication.

The hemolysin-determining plasmid pAD1 is a member of a widely disseminated family of highly conjugative elements commonly present in clinical isolates of Enterococcus faecalis. The determinants repA, repB, and repC, as well as adjacent iteron sequences, are believed to play important roles in pAD1 replication and maintenance. The repA gene encodes an initiator protein, whereas repB and repC encode proteins related to stability and copy number. The present study focuses specifically on repA and identifies a replication origin (oriV) within a central region of the repA determinant. A small segment of repA carrying oriV was able to support replication in cis of a plasmid vector otherwise unable to replicate, if an intact RepA was supplied in trans. We demonstrate that under conditions in which RepA is expressed from an artificial promoter, a segment of DNA carrying only repA is sufficient for stable replication in E. faecalis. We also show that RepA binds specifically to oriV DNA at several sites containing inverted repeat sequences (i.e., IR-1) and nonspecifically to single-stranded DNA, and related genetic analyses confirm that these sequences play an important role in replication. Finally, we reveal a relationship between the internal structure of RepA and its ability to recognize oriV. An in-frame deletion within repA resulting in loss of 105 nucleotides, including at least part of oriV, did not eliminate the ability of the altered RepA protein to initiate replication using an intact origin provided in trans. The relationship of RepA to other known initiator proteins is also discussed.

Amino Acid Sequence↗

PubMed2006

Rolling-circle replication of an animal circovirus genome in a theta-replicating bacterial plasmid in Escherichia coli.

A bacterial plasmid containing 1.75 copies of double-stranded porcine circovirus (PCV) DNA in tandem (0.8 copy of PCV type 1 [PCV1], 0.95 copy of PCV2) with two origins of DNA replication (Ori) yielded three different DNA species when transformed into Escherichia coli: the input construct, a unit-length chimeric PCV1(Rep)/PCV2(Cap) genome with a composite Ori but lacking the plasmid vector, and a molecule consisting of the remaining 0.75 copy PCV1(Cap)/PCV2(Rep) genome with a different composite Ori together with the bacterial plasmid. Replication of the input construct was presumably via the theta replication mechanism utilizing the ColE(1) Ori, while characteristics of the other two DNA species, including a requirement of two PCV Oris and the virus-encoded replication initiator Rep protein, suggest they were generated via the rolling-circle copy-release mechanism. Interestingly, the PCV-encoded Rep' protein essential for PCV DNA replication in mammalian cells was not required in bacteria. The fact that the Rep' protein function(s) can be compensated by the bacterial replication machinery to support the PCV DNA replication process echoes previous suggestions that circular single-stranded DNA animal circoviruses, plant geminiviruses, and nanoviruses may have evolved from prokaryotic episomal replicons.

Animals↗

PubMed1995

Mutational analysis of the 18-base-pair inverted repeat element at the bovine papillomavirus origin of replication: identification of critical sequences for E1 binding and in vivo replication.

Replication of bovine papillomavirus requires two viral proteins, E1 and E2-TA. Previously we demonstrated that sequences within an imperfect 18-bp inverted repeat (IR) element were sufficient to confer specific binding of the E1 protein to the origin region (S. E. Holt, G. Schuller, and V. G. Wilson, J. Virol. 68:1094-1102, 1994). To identify critical nucleotides for E1 binding and origin function, a series of individual point mutations was constructed at each nucleotide position in the 18-bp IR. Binding of E1 to these point mutations established that both the position of the mutation and the specific nucleotide change were important for the E1-DNA interaction. Equivalent mutations from each half of the IR exhibited similar binding, suggesting that the halves were functionally symmetric for E1 interactions. Each of these mutations was evaluated also for origin function in vivo by a transient-replication assay. No single point mutation eliminated replication capacity completely, though many mutants were severely impaired, demonstrating an important functional contribution for the E1 binding site. Furthermore, E1 binding was not sufficient for replication, as several origin mutants bound E1 well in vitro but replicated poorly in vivo. This suggests that certain nucleotides within the 18-bp IR may be involved in postbinding events necessary for replication initiation. The results with the point mutations suggest that E1-E1 interactions are important for stable complex formation and also indicate that there is some flexibility with regard to formation of a functional E1 replication complex at the origin.

Adenovirus E1A Proteins↗

PubMed2004

Proteomics of herpes simplex virus replication compartments: association of cellular DNA replication, repair, recombination, and chromatin remodeling proteins with ICP8.

In this study, we have used immunoprecipitation and mass spectrometry to identify over 50 cellular and viral proteins that are associated with the herpes simplex virus 1 (HSV-1) ICP8 single-stranded DNA-binding protein. Many of the coprecipitating cellular proteins are known members of large cellular complexes involved in (i) DNA replication or damage repair, including RPA and MSH6; (ii) nonhomologous and homologous recombination, including the catalytic subunit of the DNA-dependent protein kinase, Ku86, and Rad50; and (iii) chromatin remodeling, including BRG1, BRM, hSNF2H, BAF155, mSin3a, and histone deacetylase 2. It appears that DNA mediates the association of certain proteins with ICP8, while more direct protein-protein interactions mediate the association with other proteins. A number of these proteins accumulate in viral replication compartments in the infected cell nucleus, indicating that these proteins may have a role in viral replication. WRN, which functions in cellular recombination pathways via its helicase and exonuclease activities, is not absolutely required for viral replication, as viral yields are only very slightly, if at all, decreased in WRN-deficient human primary fibroblasts compared to control cells. In Ku70-deficient murine embryonic fibroblasts, viral yields are increased by almost 50-fold, suggesting that the cellular nonhomologous end-joining pathway inhibits HSV replication. We hypothesize that some of the proteins coprecipitating with ICP8 are involved in HSV replication and may give new insight into viral replication mechanisms.

Animals↗

PubMed1997

A lesion in the DNA replication initiation factor Mcm10 induces pausing of elongation forks through chromosomal replication origins in Saccharomyces cerevisiae.

We describe a new minichromosome maintenance factor, Mcm10, and show that this essential protein is involved in the initiation of DNA replication in Saccharomyces cerevisiae. The mcm10 mutant has an autonomously replicating sequence-specific minichromosome maintenance defect and arrests at the nonpermissive temperature with dumbbell morphology and 2C DNA content. Mcm10 is a nuclear protein that physically interacts with several members of the MCM2-7 family of DNA replication initiation factors. Cloning and sequencing of the MCM10 gene show that it is identical to DNA43, a gene identified independently for its putative role in replicating DNA. Two-dimensional DNA gel analysis reveals that the mcm10-1 lesion causes a dramatic reduction in DNA replication initiation at chromosomal origins, including ORI1 and ORI121. Interestingly, the mcm10-1 lesion also causes replication forks to pause during elongation through these same loci. This novel phenotype suggests a unique role for the Mcm10 protein in the initiation of DNA synthesis at replication origins.

Alleles↗

PubMed2003

Mechanical link between cohesion establishment and DNA replication: Ctf7p/Eco1p, a cohesion establishment factor, associates with three different replication factor C complexes.

CTF7/ECO1 is an essential yeast gene required for the establishment of sister chromatid cohesion. The findings that CTF7/ECO1, POL30 (PCNA), and CHL12/CTF18 (a replication factor C [RFC] homolog) genetically interact provided the first evidence that the processes of cohesion establishment and DNA replication are intimately coupled-a link now confirmed by other studies. To date, however, it is unknown how Ctf7p/Eco1p function is coupled to DNA replication or whether Ctf7p/Eco1p physically associates with any components of the DNA replication machinery. Here, we report that Ctf7p/Eco1p associates with proteins that perform partially redundant functions in DNA replication. Chl12p/Ctf18p combines with Rfc2p to Rfc5p to form one of three independent RFC complexes. By chromatographic methods, Ctf7p/Eco1p was found to associate with Chl12/Ctf18p and with Rfc2p, Rfc3p, Rfc4p, and Rfc5p. The association between Ctf7p/Eco1p and this RFC complex is biologically relevant in that (i) Ctf7p/Eco1p cosediments with Chl12p/Ctf18p in vivo and (ii) rfc5-1 mutant cells exhibit precocious sister separation. Previous studies revealed that Rfc1p or Rad24p associates with Rfc2p to Rfc5p to form two other RFC complexes independent of Ctf18p-RFC complexes. These Rfc1p-RFC and Rad24p-RFC complexes function in DNA replication or repair and DNA damage checkpoint pathways. Importantly, Ctf7p/Eco1p also associates with Rfc1p and Rad24p, suggesting that these RFC complexes also play critical roles in cohesion establishment. The associations between Ctf7p/Eco1p and RFC subunits provide novel evidence regarding the physical linkage between cohesion establishment and DNA replication. Furthermore, the association of Ctf7p/Eco1p with each of three RFC complexes supplies new insights into the functional redundancy of RFC complexes in cohesion establishment.

Acetyltransferases↗

PubMed2004

Replication protein A (RPA) phosphorylation prevents RPA association with replication centers.

Mammalian replication protein A (RPA) undergoes DNA damage-dependent phosphorylation at numerous sites on the N terminus of the RPA2 subunit. To understand the functional significance of RPA phosphorylation, we expressed RPA2 variants in which the phosphorylation sites were converted to aspartate (RPA2(D)) or alanine (RPA2(A)). Although RPA2(D) was incorporated into RPA heterotrimers and supported simian virus 40 DNA replication in vitro, the RPA2(D) mutant was selectively unable to associate with replication centers in vivo. In cells containing greatly reduced levels of endogenous RPA2, RPA2(D) again did not localize to replication sites, indicating that the defect in supporting chromosomal DNA replication is not due to competition with the wild-type protein. Use of phosphospecific antibodies demonstrated that endogenous hyperphosphorylated RPA behaves similarly to RPA2(D). In contrast, under DNA damage or replication stress conditions, RPA2(D), like RPA2(A) and wild-type RPA2, was competent to associate with DNA damage foci as determined by colocalization with gamma-H2AX. We conclude that RPA2 phosphorylation prevents RPA association with replication centers in vivo and potentially serves as a marker for sites of DNA damage.

Animals↗

PubMed2006

The Chinese hamster dihydrofolate reductase replication origin decision point follows activation of transcription and suppresses initiation of replication within transcription units.

Chinese hamster ovary (CHO) cells select specific replication origin sites within the dihydrofolate reductase (DHFR) locus at a discrete point during G1 phase, the origin decision point (ODP). Origin selection is sensitive to transcription but not protein synthesis inhibitors, implicating a pretranslational role for transcription in origin specification. We have constructed a DNA array covering 121 kb surrounding the DHFR locus, to comprehensively investigate replication initiation and transcription in this region. When nuclei isolated within the first 3 h of G1 phase were stimulated to initiate replication in Xenopus egg extracts, replication initiated without any detectable preference for specific sites. At the ODP, initiation became suppressed from within the Msh3, DHFR, and 2BE2121 transcription units. Active transcription was mostly confined to these transcription units, and inhibition of transcription by alpha-amanitin resulted in the initiation of replication within transcription units, indicating that transcription is necessary to limit initiation events to the intergenic region. However, the resumption of DHFR transcription after mitosis took place prior to the ODP and so is not on its own sufficient to suppress initiation of replication. Together, these results demonstrate a remarkable flexibility in sequence selection for initiating replication and implicate transcription as one important component of origin specification at the ODP.

Animals↗

PubMed1984

Gap filling and not replication fork progression is the rate-limiting step in the replication of UV-damaged simian virus 40 DNA.

We have analyzed the structural characteristics of simian virus 40 replicative intermediate DNA produced after UV irradiation and the kinetics of conversion of this intermediate DNA into form I DNA. Replicative intermediate DNA isolated at 30 or 60 min after UV irradiation consists primarily of two species of molecules that sediment in neutral sucrose gradients as either Cairns theta structures or relaxed monomeric circles. Replication forks on the Cairns intermediate DNA are symmetrically located with respect to the origin of replication, ruling out the possibility of asymmetric pauses or blocks to replication fork progression at damage sites. The relaxed circles contain at least one randomly located discontinuity in the daughter strand. Pulse-chase experiments demonstrated that a UV fluence-dependent fraction of the Cairns intermediate DNA progresses through the relaxed circular intermediate before being converted to completed form I molecules. Disappearance of Cairns intermediate DNA occurs at the same rate in irradiated and unirradiated cells, whereas completion of the relaxed circular intermediate DNA occurs at a slow rate, relatively independent of UV fluence. These data support a model for replication of UV-damaged DNA in which replication rapidly continues past damage sites via a gap formation event.

Animals↗

PubMed2006

Analysis of inhibition of DNA replication in irradiated cells using the SV40-based in vitro assay of DNA replication.

The mechanisms of inhibition of DNA replication after DNA damage through the activation of the S-phase checkpoint have been the focus of several investigations over the last 40 yr. Recent studies have identified several components of this checkpoint response and there is strong interest in its biochemical characterization. Helpful for the delineation of the mechanism of the S-phase checkpoint is the observation that factors inhibiting DNA replication in vivo can be found in active form in extracts prepared from irradiated cells, when these are tested using the simian virus 40 (SV40) assay for in vitro DNA replication. In this assay, replication of plasmids carrying the minimal origin of SV40 DNA replication is achieved in vitro using cytoplasmic cell extracts and SV40 large tumor antigen (TAg) as the only noncellular protein. Here, we describe protocols developed to measure in vitro DNA replication with the purpose of analyzing its regulation after exposure to DNA damage. The procedures include the preparation of components of the in vitro DNA replication reaction including cytoplasmic extracts from cells that have sustained DNA damage. The assay is powerful but is limited by the fact that initiation steps carried out by the TAg in vitro may have different cellular determinants.

Antigens, Polyomavirus Transforming↗

PubMed1998

The replication inhibition activity of the WT1 tumor suppressor protein resides in its N-terminal 298 amino acid region, and does not require specific binding of the protein to the replication origin sequence.

Using the simian virus 40 replication origin as the model, it was reported previously that the Wilms' tumor suppressor protein WT1 can inhibit DNA replication. In the present study, we found that a hybrid protein (termed GAL4-WT1AE Z) consisting of the DNA-binding domain of the yeast transcription factor GAL4 fused in-frame with the N-terminal 298 amino acid (aa) transcriptional regulatory region of WT1 retained the ability to inhibit replication. The hybrid protein and the full-length WT1 inhibited replication without regard to the presence or absence of their binding sites in the replication origin region, indicating that inhibition of replication by the proteins does not require their specific binding to the origin region. The inhibition efficiency of the hybrid protein was the same as that of WT1, indicating that the replication inhibition activity resides in the N-terminal 298 aa region, and that the C-terminal zinc finger-containing DNA-binding domain of WT1 is functionally dispensable for this effect.

Amino Acid Sequence↗