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At least 1,315 records · Page 73Linked to original sources
PubMed2000

The human homolog of Saccharomyces cerevisiae Mcm10 interacts with replication factors and dissociates from nuclease-resistant nuclear structures in G(2) phase.

Mcm10 (Dna43), first identified in Saccharomyces cerevisiae, is an essential protein which functions in the initiation of DNA synthesis. Mcm10 is a nuclear protein that is localized to replication origins and mediates the interaction of the Mcm2-7 complex with replication origins. We identified and cloned a human cDNA whose product was structurally homologous to the yeast Mcm10 protein. Human Mcm10 (HsMcm10) is a 98-kDa protein of 874 amino acids which shows 23 and 21% overall similarity to Schizosaccharomyces pombe Cdc23 and S. cerevisiae Mcm10, respectively. The messenger RNA level of HsMcm10 increased at the G(1)/S-boundary when quiescent human NB1-RGB cells were induced to proliferate as is the case of many replication factors. HsMcm10 associated with nuclease-resistant nuclear structures throughout S phase and dissociated from it in G(2) phase. HsMcm10 associated with human Orc2 protein when overexpressed in COS-1 cells. HsMcm10 also interacted with Orc2, Mcm2 and Mcm6 proteins in the yeast two-hybrid system. These results suggest that HsMcm10 may function in DNA replication through the interaction with Orc and Mcm2-7 complexes.

Amino Acid Sequence↗
PubMed1977

Construction of chimeric phages and plasmids containing the origin of replication of bacteriophage lambda.

Segments of the replication control region of bacteriophage lambda (lambda) and lambda mutants defective in replication were attached in vitro to the phi80 phage vector Charon 3 and to the plasmid vector mini Col El (pVH51). The chimeric phages and plasmids have been used to localize the origin of lambda DNA replication and to facilitate a structural analysis of the lambda replicator.

Chromosome Mapping↗
PubMed2002

Eclipse period without sequestration in Escherichia coli.

The classical Meselson-Stahl density shift experiment was used to determine the length of the eclipse period in Escherichia coli, the minimum time period during which no new initiation is allowed from a newly replicated origin of chromosome replication, oriC. Populations of bacteria growing exponentially in heavy ((15)NH(4)+ and (13)C(6)-glucose) medium were shifted to light ((14)NH(4)+ and (12)C(6)-glucose) medium. The HH-, HL- and LL-DNA were separated by CsCl density gradient centrifugation, and their relative amounts were determined using radioactive gene-specific probes. The eclipse period, estimated from the kinetics of conversion of HH-DNA to HL- and LL-DNA, turned out to be 0.60 generation times for the wild-type strain. This was invariable for widely varying doubling times (35, 68 and 112 min) and was independent of the chromosome locus at which the eclipse period was measured. For strains with seqA, dam and damseqA mutants, the length of the eclipse period was 0.16, 0.40 and 0.32 generation times respectively. Thus, initiations from oriC were repressed for a considerable proportion of the generation time even when the sequestration function seemed to be severely compromised. The causal relationship between the length of the eclipse period and the synchrony of initiations from oriC is discussed.

Bacterial Outer Membrane Proteins↗
PubMed2003

Thymine-rich single-stranded DNA activates Mcm4/6/7 helicase on Y-fork and bubble-like substrates.

The presence of multiple clusters of runs of asymmetric adenine or thymine is a feature commonly found in eukaryotic replication origins. Here we report that the helicase and ATPase activities of the mammalian Mcm4/6/7 complex are activated specifically by thymine stretches. The Mcm helicase is specifically activated by a synthetic bubble structure which mimics an activated replication origin, as well as by a Y-fork structure, provided that a single-stranded DNA region of sufficient length is present in the unwound segment or 3' tail, respectively, and that it carries clusters of thymines. Sequences derived from the human lamin B2 origin can serve as a potent activator for the Mcm helicase, and substitution of its thymine clusters with guanine leads to loss of this activation. At the fork, Mcm displays marked processivity, expected for a replicative helicase. These findings lead us to propose that selective activation by stretches of thymine sequences of a fraction of Mcm helicases loaded onto chromatin may be the determinant for selection of initiation sites on mammalian genomes.

Adenosine Triphosphatases↗
PubMed2003

Dynamic alterations of replication timing in mammalian cells.

BACKGROUND: The eukaryotic genome is divided into distinct replication timing domains, which are activated during S phase in a strictly conserved order. Cellular differentiation can alter replication timing in some loci, but recent experiments yielded conflicting data regarding the relationship between gene expression and replication timing. The genetic and epigenetic determinants of replication timing in mammalian cells have yet to be elucidated. RESULTS: We developed a mammalian experimental system in which the timing of DNA replication can be altered in a controlled manner. This system utilizes sequences from the human beta-globin locus that exhibit orientation-dependent transcriptional silencing when inserted into the murine genome. We found that before insertion, the murine target site replicated late during S phase. After insertion, replication timing depended on the orientation of the transgene. In a transcription-permissive orientation, the transgene and flanking sequences replicated early. In the reverse (silencing-prone) orientation, these sequences replicated late. Early replication correlated with histone modifications of the transgene chromatin but could be observed in the absence of the beta-globin promoter. Importantly, the replication timing switch did not require a replication origin within the transgene. CONCLUSIONS: Transgene insertions into mammalian heterochromatin can alter the timing of DNA replication at the insertion site. This differentiation-independent replication timing switch did not necessitate insertion of an active promoter or a replication origin. These observations suggest that the timing of DNA replication can be manipulated by changes in DNA sequence, but that the determinants of replication timing are distinct from the sequences that specify replication initiation sites.

Animals↗
PubMed1993

The replication functions of polyomavirus large tumor antigen are regulated by phosphorylation.

Polyomavirus (Py) large T antigen (T Ag) contains two clusters of phosphorylation sites within the amino-terminal half of the protein. To characterize possible regulatory effects of phosphorylation on viral DNA replication, Py T Ag was treated with calf intestinal alkaline phosphatase (CIAP). Incubation of the protein with a range of phosphatase concentrations caused progressive loss of phosphate without affecting its stability. Treatment with smaller quantities of CIAP stimulated the ability of the viral protein to mediate replication of constructs containing the viral replication origin, while higher concentrations of CIAP caused a marked diminution of this replication function. Several biochemical activities of Py T Ag were examined after CIAP treatment. Py T Ag DNA unwinding and nonspecific DNA binding were only slightly affected by dephosphorylation. However, as determined by DNase I footprinting experiments, treatment with smaller amounts of CIAP stimulated specific binding to the Py replication origin by Py T Ag, while treatment with larger amounts of CIAP caused marked inhibition of origin-specific binding by the viral protein. Phosphotryptic maps of Py T Ag before or after treatment with CIAP revealed changes in individual phosphopeptides that were uniquely associated with either the stimulation or the inhibition of replication. Our data therefore suggest that Py T Ag is regulated by both repressing and activating phosphates.

Alkaline Phosphatase↗
PubMed2006

Genome-wide analysis of re-replication reveals inhibitory controls that target multiple stages of replication initiation.

DNA replication must be tightly controlled during each cell cycle to prevent unscheduled replication and ensure proper genome maintenance. The currently known controls that prevent re-replication act redundantly to inhibit pre-replicative complex (pre-RC) assembly outside of the G1-phase of the cell cycle. The yeast Saccharomyces cerevisiae has been a useful model organism to study how eukaryotic cells prevent replication origins from reinitiating during a single cell cycle. Using a re-replication-sensitive strain and DNA microarrays, we map sites across the S. cerevisiae genome that are re-replicated as well as sites of pre-RC formation during re-replication. Only a fraction of the genome is re-replicated by a subset of origins, some of which are capable of multiple reinitiation events. Translocation experiments demonstrate that origin-proximal sequences are sufficient to predispose an origin to re-replication. Origins that reinitiate are largely limited to those that can recruit Mcm2-7 under re-replicating conditions; however, the formation of a pre-RC is not sufficient for reinitiation. Our findings allow us to categorize origins with respect to their propensity to reinitiate and demonstrate that pre-RC formation is not the only target for the mechanisms that prevent genomic re-replication.

Cell Cycle↗
PubMed1975

Interaction of P2 bacteriophage with the dnaB gene of Escherichia coli.

The dnaB gene product of Escherischia coli is required for multiplication of temperate phage P2. At 37 C in dnaB-ts mutnats, P2 will not plaque and gives a very small burst of progeny. P2 mutants have been isolated which can grow well enough to plaque under these conditions. This type of phage mutant is cis dominant, and one such mutant (P2rlb1) has been mapped near the left end of the early gene B and to the right of the cox4 (excision) mutation. The rlb1 mutation does not lie at the replication origin, but may affect transcription in the early region, which includes the replication origin. It may also represent a site on the P2 DNA which interacts with the dnaB gene product.

Chromosome Mapping↗
PubMed2001

Nucleosomes positioned by ORC facilitate the initiation of DNA replication.

The packaging of eukaryotic DNA into nucleosomes is a critical regulator of nuclear events. To address the interplay between chromatin and replication initiation, we have assessed the determinants and function of the nucleosomal configuration of S. cerevisiae replication origins. Using in vitro and in vivo assays, we demonstrate that the yeast initiator, the origin recognition complex (ORC), is required to maintain the nucleosomal configuration adjacent to origins. Disruption of the ORC-directed nucleosomal arrangement at an origin interferes with initiation of replication, but does not alter the association of ORC with the origin. Instead, the nucleosomes positioned by ORC are important for prereplicative complex formation. These findings suggest that origin-proximal nucleosomes facilitate replication initiation, and that local chromatin structure affects origin function.

Chromatin↗
PubMed2006

Possible multiple origins of replication in primate mitochondria: Alternative role of tRNA sequences.

DNA replication in vertebrate mitochondria is usually directional, leaving different portions of the genome single-stranded for different periods of time. During this time, mutations resulting from deaminations of cytosines to thymines and adenines to guanines accumulate on the heavy strand. Therefore, T/C and G/A ratios increase along mitochondrial genomes, proportionally to the time spent single-stranded during replication. Such trends exist at third codon positions for base ratios averaged across genes in individual genomes as well as for gene-specific and site-specific substitution frequencies estimated using phylogenetic methods. We use multiple regressions to test for the potential functioning of all 12 tRNA clusters in 19 primate mitochondrial genomes as alternative origins of light strand replication (OL). We provide a general algorithm for calculating time spent single stranded by a given site for any possible locations of the site and OL. For codon positions 1, 2, and 3, respectively, 23%, 9% and 35% of tRNA gene clusters have significant (p < 0.05) deamination gradients originating from them. The strength of the deamination gradient originating from tRNA gene clusters varies among species, and for five clusters, correlates with the tendency of tRNA genes in each of these clusters to form secondary structures that resemble the OL's structure. This is notably true for all codon positions for tRNA-Lys, which in absence of nuclear regulation, forms secondary structures resembling the hairpin structure of OL. For two tRNA gene clusters, correlations were statistically significant, but opposite to the direction expected by the known unidirectional replication, putatively compatible with bi-directional replication. Few substitutions in tRNA sequences can be neutral at the level of cloverleaf structure and function, yet significantly alter capacities to form OL-like structures, causing sudden evolution of genome-wide nucleotide contents.

Animals↗
PubMed1983

Alignment of the genome of monkey B-lymphotropic papovavirus to the genomes of simian virus 40 and BK virus.

We located the origin of DNA replication of African green monkey B-lymphotropic papovavirus DNA by analyzing pulse-labeled form I DNA. With the replication origin used as a reference point, the B-lymphotropic papovavirus genome was aligned with the genomes of simian virus 40 and BK virus from DNA homology between specific fragments hybridized under low-stringency conditions. From the results of these experiments, it was possible to deduce the correlation between the physical and functional maps of the B-lymphotropic papovavirus genome.

BK Virus↗
PubMed1983

Regulatory sequences and virus-cell interactions of JC virus.

A set of recombinant DNAs, representing several isolates of JCV, has been constructed. Some of these cloned DNAs transform nonpermissive primary hamster brain cells. Analysis of the early viral mRNAs and proteins isolated from these transformants revealed similarities with the large and small T messages and proteins of SV40. The region of the JCV genome from 0.63 to 0.73 map units was sequenced by the Maxam-Gilbert technique. This segment of DNA specifies several regulatory elements and the amino-terminal portion of the early viral proteins. Comparisons with the analogous regions in the polyomaviruses SV40 and BKV, confirm the close evolutionary relationship of these three viruses. Similarities include palindromic and symmetrical sequences near the origins of DNA replication, binding sites for the large T proteins, and AT-rich region (the Goldberg-Hogness or TATA box), and a large tanden duplication or triplication to the late side of the replication origin (however, these sequences differ). Homology between the sequences coding for the early proteins is also evident (27 of the first 33 amino acids are shared). Of greater interest are features of the JCV genome which differ from those of other polyomaviruses. Absent in JCV and BKV are sequences which resemble the third T antigen-binding site of SV40. In addition, a set of sequences present in JCV and BKV DNA (33 nucleotides in JCV, 22 nucleotides in BKV), and located near a 17 base pair palindrome shared by all three viruses, is missing in SV40 DNA. Another sequence, GGGXGGAG, which is repeated several times in many polyomaviruses and adenoviruses, and which is thought to play a role in DNA replication and/or transcription, is not found in the JCV sequence presented. Finally, the tandem repeat of JCV, unlike those of BKV and SV40, includes the Goldberg-Hogness sequence.

Animals↗
PubMed1989

The effect of DnaA protein levels and the rate of initiation at oriC on transcription originating in the ftsQ and ftsA genes: in vivo experiments.

The DnaA protein of Escherichia coli, essential for initiation at oriC, binds at a defined sequence which occurs at the chromosomal origin, near plasmid replication origins and in the promoters of the dnaA and mioC genes. This sequence also occurs at many other sites on the E. coli chromosome including three sites within the essential cell division genes ftsQ and A. Using an fts-lac fusion phage, lambda JFL100, we show here that fts gene expression responds both to reduced and increased intracellular levels of DnaA protein in a manner consistent with the hypothesis that DnaA protein regulates fts gene expression. Experiments using dnaC and dnaB-ts strains, however, suggest that DnaA control of fts transcription may be indirect, at least in part, with fts responding to the rate of initiation at oriC as well as to changes in DnaA protein level per se. It differs in this respect from dnaA gene expression which is unaffected when initiation of replication is inhibited by DnaB or DnaC inactivation. Strains integratively suppressed with pKN500 behave anomalously; neither fts nor dnaA transcription is significantly increased when DnaA is inactivated in these strains.

Bacterial Proteins↗
PubMed1991

Factors contributing to the restricted DNA replicating activity of JC virus.

The basis for the restricted host range behavior of JC virus (JCV) in vitro was investigated by focusing on its DNA replicating activity and comparing it to that of simian virus 40 (SV40). Prototype, mutant, and hybrid JCV and SV40 DNAs were tested for their replicating activity in cells permissive for one or both of the viruses. Results from these experiments indicated that, relative to its SV40 counterpart, the JCV T antigen functioned less efficiently and was more specific in its interactions with polyomavirus DNA replication origins. The JCV T antigen exhibited a lower specific DNA binding activity than did the SV40 T antigen, which might contribute to this virus' reduced DNA replicating activity. However, the JCV protein did bind to both the JCV and SV40 replication origins with similar efficiency, indicating that the ability of the JCV T antigen to discriminate between the JCV and SV40 origins involved a step subsequent to specific DNA binding. The results also suggested that the failure of JCV to replicate to detectable levels in monkey kidney cells was due to the inefficient interactions of its T protein with the viral origin and the host replication machinery. The inability of the JCV T antigen to carry out one or more of these DNA replication functions efficiently contributes to the restricted lytic behavior of this virus.

Animals↗
PubMed1993

cdc2 phosphorylation of threonine 124 activates the origin-unwinding functions of simian virus 40 T antigen.

Phosphorylation of simian virus 40 (SV40) T antigen on threonine 124 activates viral DNA replication in vivo and in vitro. We have manipulated the modification of T-antigen residue 124 both genetically and biochemically and have investigated individual replication functions of T antigen under conditions suitable for in vitro DNA replication. We find that the hexamer assembly, helicase, DNA polymerase alpha-binding, and transcriptional-autoregulation functions are independent of phosphorylation of threonine 124. In contrast, neither T antigen with an alanine mutation of threonine 124 made in human cells nor unphosphorylated T antigen made in Escherichia coli binds the SV40 replication origin as stably as phosphorylated wild-type T antigen does. Furthermore, modification of threonine 124 is essential for complete unwinding of the SV40 replication origin. We conclude that phosphorylation of threonine 124 enhances specific interactions of T antigen with SV40 origin DNA. Our findings do not exclude the possibility that phosphorylation of threonine 124 may affect additional undefined steps in DNA replication. We also show that DNase footprinting and KMnO4 modification assays are not as stringent as immunoprecipitation and origin-dependent strand displacement assays for detecting defects in the origin-binding and -unwinding functions of T antigen. Differences in the assays may explain discrepancies in previous reports on the role of T-antigen phosphorylation in DNA binding.

Amino Acid Sequence↗
PubMed2001

Endogenous isolation of replicon probes for assessing plasmid ecology of marine sediment microbial communities.

Six functional replication origins (repGA14, repGA33, repGA70, repSD41, repSD164 and repSD172), obtained from endogenously isolated, broad-host-range (BHR) marine plasmids ranging in size from 5 to 60 kb, were used to determine plasmid occurrence in three coastal marine sediment sites (in California, Georgia and South Carolina, USA). The plasmid-specific replicons were isolated from plasmid-bearing marine sediment bacteria belonging to the alpha and gamma subclasses of the Proteobacteria. The plasmid sources of the endogenous replicons were considered to be cryptic due to a lack of identifiable phenotypic traits. The putative Rep proteins from a number of these replicons showed similarity to replicons of two recognized families: RCR group III (repSD164) and the FIA family of theta group A (repSD41, repSD121, repGA33 and repGA14). Plasmids isolated from marine bacteria belonging to the genera Pseudoalteromonas, Shewanella and Vibrio cultivated from geographically different coastal sites exhibited homology to two of the marine plasmid replicons, repSD41 and repGA70, obtained from a Vibrio sp. The repGA33 plasmid origin, obtained from a Shewanella sp. isolated from coastal Georgia, was detected in 7% of the Georgia marine sediment Shewanella sp. isolates. Microbial community DNA extracted from marine sediments was also screened for the presence of the plasmid replication sequences. Community DNA samples amplified by PCR yielded a positive signal for the repSD172 and repGA14 replication sequences. The replication origin of BHR plasmid RK2 (IncP) was also detected in marine Vibrio sp. and microbial community DNA extracted from the three coastal sites. These findings provide molecular evidence that marine sediment bacteria harbour an untapped population of BHR plasmids.

Alphaproteobacteria↗
PubMedDate not supplied

Intraspecific nucleotide sequence variability surrounding the origin of replication in human mitochondrial DNA.

We have cloned the major noncoding region of human mitochondrial DNA (mtDNA) from 11 human placentas. Partial nucleotide sequences of five of these clones have been determined and they share a maximum of 900 bp around the origin of H-strand replication. Alignment of these sequences with others previously determined has revealed a striking pattern of nucleotide substitutions and insertion/deletion events. The level of sequence divergence significantly exceeds the reported estimates of divergence in coding regions. Two particularly hypervariable regions have also been defined. More than 96% of the base changes are transitions, and length alterations have occurred exclusively by addition or deletion of mono-or dinucleotide segments within serially repeating stretches. This region of the mitochondrial genome, which contains the initiation sites for replication and transcription, is the least conserved among species with respect to both sequence and length (Anderson et al., 1981; Walberg and Clayton, 1981). Despite this overall lack of primary sequence conservation, several consistencies appear among the available mammalian mtDNA sequences within this region. Between species, a conserved linear array of characteristic stretches exists which nonetheless differ in primary sequence. Among humans, several conserved blocks of nucleotides appear within domains deleted from the mtDNA of other species. These observations are consistent with both a species-specificity of nucleotide sequence, and a preservation of the necessary genetic functions among species. This provides a model for the evolution of protein-nucleic acid interactions in mammalian mitochondria.

Amino Acid Sequence↗
PubMed2003

Evidence for preferential mismatch repair of lagging strand DNA replication errors in yeast.

Duplex DNA is replicated in the 5'-3' direction by coordinated copying of leading and lagging strand templates with somewhat different proteins and mechanics, providing the potential for differences in the fidelity of replication of the two strands. We previously showed that in Saccharomyces cerevisiae, active replication origins establish a strand bias in the rate of base substitutions resulting from replication of unrepaired 8-oxo-guanine (GO) in DNA. Lower mutagenesis was associated with replicating lagging strand templates. Here, we test the hypothesis that this bias is due to more efficient repair of lagging stand mismatches by measuring mutation rates in ogg1 strains with a reporter allele in two orientations at loci on opposite sides of a replication origin on chromosome III. We compare a MMR-proficient strain to strains deleted for the MMR genes MSH2, MSH6, MLH1, or EXOI. Loss of MMR reduces the strand bias by preferentially increasing mutagenesis for lagging strand replication. We conclude that GO-A mismatches generated during lagging strand replication are more efficiently repaired. This is consistent with the hypothesis that 5' ends of Okazaki fragments and PCNA, present at high density during lagging strand replication, are used as strand discrimination signals for mismatch repair in vivo.

Base Pair Mismatch↗