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At least 451 records · Page 25Linked to original sources
PubMed1994

Homologous recombination of monkey alpha-satellite repeats in an in vitro simian virus 40 replication system: possible association of recombination with DNA replication.

To study homologous recombination between repeated sequences in an in vitro simian virus 40 (SV40) replication system, we constructed a series of substrate DNAs that contain two identical fragments of monkey alpha-satellite repeats. Together with the SV40-pBR322 composite vector encoding Apr and Kmr, the DNAs also contain the Escherichia coli galactokinase gene (galK) positioned between two alpha-satellite fragments. The alpha-satellite sequence used consists of multiple units of tandem 172-bp sequences which differ by microheterogeneity. The substrate DNAs were incubated in an in vitro SV40 DNA replication system and used to transform the E. coli galK strain DH10B after digestion with DpnI. The number of E. coli galK Apr Kmr colonies which contain recombinant DNAs were determined, and their structures were analyzed. Products of equal and unequal crossovers between identical 172-bp sequences and between similar but not identical (homeologous) 172-bp sequences, respectively, were detected, although those of the equal crossover were predominant among all of the galK mutant recombinants. Similar products were also observed in the in vivo experiments with COS1 cells. The in vitro experiments showed that these recombinations were dependent on the presence of both the SV40 origin of DNA replication and SV40 large T antigen. Most of the recombinant DNAs were generated from newly synthesized DpnI-resistant DNAs. These results suggest that the homologous recombination observed in this SV40 system is associated with DNA replication and is suppressed by mismatches in heteroduplexes formed between similar but not identical sequences.

Animals↗
PubMed1975

Replication of colicinogenic factor E1 DNA in plasmolysed Escherichia coli cells. Coupling of DNA replication and RNA synthesis.

Plasmolysed chloramphenicol-treated Escherichia coli cells carrying the colicinogenic factor E1 utilize deoxynucleoside triphosphates for the semi-conservative synthesis of Col E1 DNA. Col E1 DNA replication in plasmolysed cells can be dissociated into two temporally separated processes: (a) a rifampicin-sensitive RNA synthesis, which is stimulated by adenosine 3':5'-monophosphate (cyclic AMP) and requires all four ribonucleoside triphosphates and (b) an ATP-dependent DNA synthesis, which is inhibited by arabinosylnucleoside triphosphates and sulfhydryl-blocking reagents. Thes two processes exhibit different sensitivities to inhibition by polyamines and actinomycin D.

Adenosine Triphosphate↗
PubMed2006

Evidence suggesting that Pif1 helicase functions in DNA replication with the Dna2 helicase/nuclease and DNA polymerase delta.

The precise machineries required for two aspects of eukaryotic DNA replication, Okazaki fragment processing (OFP) and telomere maintenance, are poorly understood. In this work, we present evidence that Saccharomyces cerevisiae Pif1 helicase plays a wider role in DNA replication than previously appreciated and that it likely functions in conjunction with Dna2 helicase/nuclease as a component of the OFP machinery. In addition, we show that Dna2, which is known to associate with telomeres in a cell-cycle-specific manner, may be a new component of the telomere replication apparatus. Specifically, we show that deletion of PIF1 suppresses the lethality of a DNA2-null mutant. The pif1delta dna2delta strain remains methylmethane sulfonate sensitive and temperature sensitive; however, these phenotypes can be suppressed by further deletion of a subunit of pol delta, POL32. Deletion of PIF1 also suppresses the cold-sensitive lethality and hydroxyurea sensitivity of the pol32delta strain. Dna2 is thought to function by cleaving long flaps that arise during OFP due to excessive strand displacement by pol delta and/or by an as yet unidentified helicase. Thus, suppression of dna2delta can be rationalized if deletion of POL32 and/or PIF1 results in a reduction in long flaps that require Dna2 for processing. We further show that deletion of DNA2 suppresses the long-telomere phenotype and the high rate of formation of gross chromosomal rearrangements in pif1Delta mutants, suggesting a role for Dna2 in telomere elongation in the absence of Pif1.

Adenosine Triphosphatases↗
PubMed1984

A temperature-sensitive (TS) mutant of frog virus 3 (FV3) is defective in second-stage DNA replication.

It has been suggested that FV3 DNA replication occurs in two stages [R. Goorha (1982) J. Virol. 43, 519-528]. First-stage DNA synthesis is restricted to the nucleus, where the replicating DNA ranges from genome to twice genome size; second-stage DNA replication occurs exclusively in the cytoplasm, and the replicating DNA is concatameric. A temperature-sensitive mutant (ts 12488) of FV3, at a nonpermissive temperature (30 degrees), synthesized DNA in the nucleus only, and the size of the replicative complex (as determined by neutral sucrose gradient analysis) was between genome and twice genome length. These characteristics establish that at nonpermissive temperature, ts 12488 is arrested in the first stage of DNA replication. Temperature shift-down (30 degrees----25 degrees) of ts 12488-infected cells at 4 hr postinfection showed that, within 30 min of the shift, the replicative complex became very large (more than 10 times genome size). Furthermore, newly synthesized DNA was now found in the cytoplasmic fraction also. These results suggest that ts 12488, upon shift-down, enters into the second stage of DNA replication where progeny DNA is synthesized as a large concatamer. In shift-down experiments, de novo protein synthesis was not required to initiate second-stage DNA replication, strongly suggesting that the thermosensitive protein is directly involved in second-stage DNA replication. This genetic evidence establishes the previous biochemical findings of a two-stage replication scheme for FV3 DNA.

Animals↗
PubMed1985

Initiation of DNA replication on single-stranded DNA templates catalyzed by purified replication proteins of bacteriophage lambda and Escherichia coli.

Initiation of bacteriophage lambda DNA replication at the chromosomal origin depends on the lambda O and P replication proteins. These two viral initiators, together with an Escherichia coli protein fraction, promote the replication in vitro of single-stranded circular DNA chromosomes such as that of bacteriophage M13. This nonspecific strand initiation reaction, which we have termed the "lambda single-strand replication reaction," has now been established with eight purified proteins, each of which is also required for replication of the phage lambda chromosome in vivo. An early rate-limiting step in the overall reaction is the ATP-dependent assembly of an activated nucleoprotein prepriming complex. In this step the lambda O and P initiators cooperate with the E. coli dnaJ and dnaK proteins to transfer the bacterial dnaB protein onto M13 DNA that is coated with the single-stranded DNA-binding protein. Multiple RNA primers are synthesized on each DNA circle when isolated prepriming complex is incubated with primase and rNTPs. In the complete system, DNA polymerase III holoenzyme extends the first primer synthesized into full-length complementary strands. Because the properties of this system are closely analogous to those found for the replication of phi X174 viral DNA by E. coli proteins, we infer that a mobile prepriming or priming complex (primosome) operates in the lambda single-strand replication reaction.

Bacterial Proteins↗
PubMed2001

Replicative DNA polymerases.

SUMMARY: Replicative DNA polymerases are essential for the replication of the genomes of all living organisms. On the basis of sequence similarities they can be classified into three types. Type A polymerases are homologous to bacterial polymerases I, Type B comprises archaebacterial DNA polymerases and eukaryotic DNA polymerase alpha, and the bacterial polymerase III class make up type C. Structures have been solved for several type A and B polymerases, which share a similar architecture. The structure of type C is not yet known. The catalytic mechanism of all three types involves two metal-ion-binding acidic residues in the active site. Replicative polymerases are constitutively expressed, but their activity is regulated through the cell cycle and in response to different growth conditions.

Animals↗
PubMed1991

Analysis of replicative intermediates produced during bacteriophage phi 29 DNA replication in vitro.

Replication of bacteriophage phi 29 DNA initiates at either end of its linear double-stranded DNA molecule and proceeds by a strand-displacement mechanism. In the present paper we have used an in vitro phi 29 DNA replication system to analyse by electron microscopy the replicative intermediates produced at different reaction times. Two types of replicative intermediates were observed: type I (full-length double-stranded phi 29 DNA molecules with one or more single-stranded DNA branches) and type II (full-length phi 29 DNA molecules formed by a double-stranded DNA portion of variable length from one end plus a single-stranded DNA portion spanning to the other end). Thus, the types of replicative intermediates produced in vivo were also formed in the in vitro phi 29 DNA replication system. Analysis of type I intermediates indicated that initiation of DNA replication occurs preferentially at both ends of the same DNA template, in a non-simultaneous manner. Type II intermediates appeared as early as two minutes after the reaction started, well before unit-length single-stranded phi 29 DNA molecules were synthesized. In addition, replication of recombinant phi 29 DNA templates lacking terminal protein at one end did not produce type II intermediates and led to an accumulation of full-length single-stranded phi 29 DNA molecules. These two observations strongly suggest that type II intermediates appear when two growing DNA chains, running from opposite ends, merge.

Bacillus subtilis↗
PubMed1994

Cdk-interacting protein 1 directly binds with proliferating cell nuclear antigen and inhibits DNA replication catalyzed by the DNA polymerase delta holoenzyme.

Cdk-interacting protein 1 (Cip1) is a p53-regulated 21-kDa protein that inhibits several members of the cyclin-dependent kinase (CDK) family. It was initially observed in complexes containing CDK4, cyclin D, and proliferating cell nuclear antigen (PCNA). PCNA, in conjunction with activator 1, acts as a processivity factor for eukaryotic DNA polymerase (pol) delta, and these three proteins constitute the pol delta holoenzyme. In this report, we demonstrate that Cip1 can also directly inhibit DNA synthesis in vitro by binding to PCNA. Cip1 efficiently inhibits simian virus 40 replication dependent upon pol alpha, activator 1, PCNA, and pol delta, and this inhibition can be overcome by additional PCNA. Simian virus 40 DNA replication, catalyzed solely by high levels of pol alpha-primase complex, is unaffected by Cip1. Using the surface plasmon resonance technique, a direct physical interaction of PCNA and Cip1 was detected. We have observed that Cip1 efficiently inhibits synthesis of long (7.2 kb) but not short (10 nt) templates, suggesting that its association with PCNA is likely to impair the processive movement of pol delta during DNA chain elongation, as opposed to blocking assembly of the pol delta holoenzyme. The implications of the Cip1-PCNA interaction with respect to regulation of DNA synthesis, cell cycle checkpoint control, and DNA repair are discussed.

CDC2-CDC28 Kinases↗
PubMed1982

Conserved sequences at the origin of adenovirus DNA replication.

The origin of adenovirus DNA replication lies within an inverted sequence repetition at either end of the linear, double-stranded viral DNA. Initiation of DNA replication is primed by a deoxynucleoside that is covalently linked to a protein, which remains bound to the newly synthesized DNA. We demonstrate that virion-derived DNA-protein complexes from five human adenovirus serological subgroups (A to E) can act as a template for both the initiation and the elongation of DNA replication in vitro, using nuclear extracts from adenovirus type 2 (Ad2)-infected HeLa cells. The heterologous template DNA-protein complexes were not as active as the homologous Ad2 DNA, most probably due to inefficient initiation by Ad2 replication factors. In an attempt to identify common features which may permit this replication, we have also sequenced the inverted terminal repeated DNA from human adenovirus serotypes Ad4 (group E), Ad9 and Ad10 (group D), and Ad31 (group A), and we have compared these to previously determined sequences from Ad2 and Ad5 (group C), Ad7 (group B), and Ad12 and Ad18 (group A) DNA. In all cases, the sequence around the origin of DNA replication can be divided into two structural domains: a proximal A . T-rich region which is partially conserved among these serotypes, and a distal G . C-rich region which is less well conserved. The G . C-rich region contains sequences similar to sequences present in papovavirus replication origins. The two domains may reflect a dual mechanism for initiation of DNA replication: adenovirus-specific protein priming of replication, and subsequent utilization of this primer by host replication factors for completion of DNA synthesis.

Adenoviruses, Human↗
PubMed2003

Adenovirus DNA replication.

Replication of the adenovirus genome is catalysed by adenovirus DNA polymerase in which the adenovirus preterminal protein acts as a protein primer. DNA polymerase and preterminal protein form a heterodimer which, in the presence of the cellular transcription factors NFI/CTFI and NFIII/Oct-1, binds to the origin of DNA replication. DNA replication is initiated by DNA polymerase mediated transfer of dCMP onto preterminal protein. Further DNA synthesis is catalysed by DNA polymerase in a strand displacement mechanism which also requires adenovirus DNA binding protein. Here, we discuss the role of individual proteins in this process as revealed by biochemical analysis, mutagenesis and molecular modelling.

Adenoviridae↗
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↗
PubMed1992

ORS12, a mammalian autonomously replicating DNA sequence, is present at the centromere of CV-1 cell chromosomes.

ors12, an 812-bp-long sequence, previously isolated by extrusion of nascent DNA from replication bubbles active at the onset of S phase (G. Kaufmann, M. Zannis-Hadzopoulus, and R. G. Martin Mol. Cell. Biol. 5, 721-727, 1985), has been shown to function as an origin of DNA replication in autonomously replicating plasmids (L. Frappier and M. Zannis-Hadjopoulos Proc. Natl. Acad. Sci. USA 84, 6668-6672, 1987) and in a cell-free system (C. E. Pearson, L. Frappier, and M. Zannis-Hadzopoulos Biochim. Biophys. Acta 1090, 156-166, 1991). A portion of ors12 (nucleotides 1-168) consists of the highly reiterated alpha-satellite sequence (B. S. Rao et al. Gene 87, 233-242, 1990). We have estimated the copy number of the non-alpha-satellite portion of ors12 in CV-1 cells to be < 9 copies per haploid genome and have used it as a probe to generate a genomic map of ors12 on CV-1 DNA. In situ hybridization of CV-1 metaphase chromosomes, using a biotinylated probe of the entire ors12 sequence, positively identified the centromeres of all chromosomes. However, when the non-alpha-satellite portion of ors12 was used as a probe, it positively identified the centromeric region of only six chromosomes, namely, B4, C11, D14, D24, E25, and E27, as well as that of a marker chromosome. The results suggest that ors12 represents a centromeric putative replication origin that is present on a subset of CV-1 chromosomes and is activated at the onset of S phase.

Animals↗
PubMed1996

3'-->5' exonucleases of DNA polymerases epsilon and delta correct base analog induced DNA replication errors on opposite DNA strands in Saccharomyces cerevisiae.

The base analog 6-N-hydroxylaminopurine (HAP) induces bidirectional GC-->AT and AT-->GC transitions that are enhanced in DNA polymerase epsilon and delta 3'-->5' exonuclease-deficient yeast mutants, pol2-4 and pol3-01, respectively. We have constructed a set of isogenic strains to determine whether the DNA polymerases delta and epsilon contribute equally to proofreading of replication errors provoked by HAP during leading and lagging strand DNA synthesis. Site-specific GC-->AT and AT-->GC transitions in a Pol+, pol2-4 or pol3-01 genetic background were scored as reversions of ura3 missense alleles. At each site, reversion was increased in only one proofreading-deficient mutant, either pol2-4 or pol3-01, depending on the DNA strand in which HAP incorporation presumably occurred. Measurement of the HAP-induced reversion frequency of the ura3 alleles placed into chromosome III near to the defined active replication origin ARS306 in two orientations indicated that DNA polymerases epsilon and delta correct HAP-induced DNA replication errors on opposite DNA strands.

Adenine↗
PubMed1992

A deletion in the simian virus 40 large T antigen impairs lytic replication in monkey cells in vivo but enhances DNA replication in vitro: new complementation function of T antigen.

We describe a new complementation function within the simian virus 40 (SV40) A gene. This function is required for viral DNA replication and virus production in vivo but, surprisingly, does not affect any of the intrinsic enzymatic functions of T antigen directly required for in vitro DNA replication. Other well-characterized SV40 T-antigen mutants, whether expressed stably from integrated genomes or in cotransfection experiments, complement these mutants for in vivo DNA replication and plaque formation. These new SV40 mutants were isolated and cloned from human cells which stably carry the viral DNA. The alteration in the large-T-antigen gene was shown by marker rescue and nucleotide sequence analysis to be a deletion of 322 bp spanning the splice-donor site of the first exon, creating a 14-amino-acid deletion in the large T antigen. The mutant gene was expressed in H293 human cells from an adenovirus vector, and the protein was purified by immunoaffinity chromatography. The mutant protein directs greater levels of DNA replication in vitro than does the wild-type protein. Moreover, the mutant protein reduces the lag time for in vitro DNA synthesis and can be diluted to lower levels than wild-type T antigen and still promote good replication, which is in clear contrast to the in vivo situation. These biochemical features of the protein are independent of the source of the cellular replication factors (i.e., HeLa, H293, COS 7, or CV1 cells) and the cells from which the T antigens were purified. The mutant T antigen does not transform Rat-2 cells. Several different models which might reconcile the differences observed in vivo and in vitro are outlined. We propose that the function of T antigen affected prepares cells for SV40 replication by activation of a limiting cellular replication factor. Furthermore, a link between the induction of a cellular replication factor and transformation by SV40 is discussed.

Amino Acid Sequence↗
PubMed1998

DNA ligase I selectively affects DNA synthesis by DNA polymerases delta and epsilon suggesting differential functions in DNA replication and repair.

The joining of single-stranded breaks in double-stranded DNA is an essential step in many important processes such as DNA replication, DNA repair, and genetic recombination. Several data implicate a role for DNA ligase I in DNA replication, probably coordinated by the action of other enzymes and proteins. Since both DNA polymerases delta and epsilon show multiple functions in different DNA transactions, we investigated the effect of DNA ligase I on various DNA synthesis events catalyzed by these two essential DNA polymerases. DNA ligase I inhibited replication factor C-independent DNA synthesis by polymerase delta. Our results suggest that the inhibition may be due to DNA ligase I interaction with proliferating cell nuclear antigen (PCNA) and not to a direct interaction with the DNA polymerase delta itself. Strand displacement activity by DNA polymerase delta was also affected by DNA ligase I. The DNA polymerase delta holoenzyme (composed of DNA polymerase delta, PCNA, and replication factor C) was inhibited in the same way as the DNA polymerase delta core, strengthening the hypothesis of a PCNA interaction. Contrary to DNA polymerase delta, DNA synthesis by DNA polymerase epsilon was stimulated by DNA ligase I in a PCNA-dependent manner. We conclude that DNA ligase I displays different influences on the two multipotent DNA polymerases delta and epsilon through PCNA. This might be of importance in the selective involvement in DNA transactions such as DNA replication and various mechanisms of DNA repair.

DNA↗
PubMed2003

Chromatin assembly factor 1 is essential and couples chromatin assembly to DNA replication in vivo.

De novo chromatin assembly maintains histone density on the daughter strands in the wake of the replication fork. The heterotrimer chromatin assembly factor 1 (CAF-1) couples DNA replication to histone deposition in vitro, but is not essential for yeast cell proliferation. Depletion of CAF-1 in human cell lines demonstrated that CAF-1 was required for efficient progression through S-phase. Cells lacking CAF-1 accumulated in early and mid S-phase and replicated DNA slowly. The checkpoint kinase Chk1, but not Chk2, was phosphorylated in response to CAF-1 depletion, consistent with a DNA replication defect. CAF-1-depleted cell extracts completely lacked DNA replication-coupled chromatin assembly activity, suggesting that CAF-1 is required for efficient S-phase progression in human cells. These results indicate that, in contrast to yeast, human CAF-1 is necessary for coupling chromatin assembly with DNA replication.

Base Sequence↗