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At least 523 records · Page 29Linked to original sources
PubMed1991

Mouse DNA primase plays the principal role in determination of permissiveness for polyomavirus DNA replication.

We have investigated the species-specific replication of polyomavirus DNA in the cell-free system that was established previously (Y. Murakami, T. Eki, M. Yamada, C. Prives, and J. Hurwitz, Proc. Natl. Acad. Sci. USA 83:6347-6351, 1986). Extracts from various species of cells supported polyomavirus DNA replication in a species-specific manner that was consistent with the host range specificity of polyomavirus; extracts prepared from mouse and hamster cells were active, whereas extracts prepared from human, monkey, and insect cells were inactive. The addition of DNA polymerase alpha-primase purified from mouse cells induced the replication of polyomavirus DNA in a cell-free system containing polyomavirus large tumor antigen and nonpermissive cell extracts, such as human and insect cell extracts. Isolated mouse DNA primase alone also induced polyomavirus DNA replication in human cell extracts but not in insect cell extracts, indicating that mouse DNA primase plays the principal role in determining permissiveness for polyomavirus DNA replication.

Animals↗
PubMed1975

The viral DNA replication machinery of adenovirus 12.

The viral DNA replication complex was found in the M band after fractionation. However, it did not associate with the nuclear membrane, but was found in association with types II and IV inclusions. It is suggested, therefore, that the viral DNA replication complex is the inclusions types II and IV. The analysis with Ad12 ts mutants showed the involvement of three viral genes in initiation of viral DNA replication, in formation of the viral DNA replication complex and the inclusions types II and IV. Temperature sensitivity of the gene products involved in viral DNA replication was also examined. From the data presented, the following sequence of viral DNA replication in Ad12-infected cells is suggested: (1) Synthesis of a protein (gene A product), which may be an acceptor or may modify the nuclear constituent to be an acceptor for parental viral DNA, to form the viral DNA replication complex; (2) synthesis of a protein (gene B product), which may be required for the parental viral DNA to associate with the acceptor and to form a type II inclusion-like structure (precursor of type II inclusion?); (3) synthesis of a protein (gene C product), which may be required for activation of the complex or type II inclusion and to alter type II inclusion to type IV inclusion. Further studies are necessary to substantiate the suggestion.

Adenoviridae↗
PubMed1991

Multicomponent origin of cytomegalovirus lytic-phase DNA replication.

Cytomegalovirus (CMV) lytic-phase DNA replication requires both trans-acting factors, such as the virus-coded DNA polymerase, and a previously undefined cis-acting element, the origin, within which initiation occurs. We have located a candidate origin of CMV lytic-phase DNA replication, oriLyt, in both simian and human strains by assessing the ability of cloned restriction fragments to mediate phosphonoformic acid-sensitive DNA replication after transfection into human fibroblasts when required trans-acting factors were supplied by infection. In initial experiments the simian CMV-like strain Colburn EcoRI D fragment directed DNA replication; this fragment contains all of the single-stranded DNA-binding protein gene (dbp) and about 7 kbp of upstream sequence. A larger region upstream of human CMV dbp also mediated replication in transient assays. Subsequent subcloning and deletion analyses defined a CMV strain Colburn region sufficient for origin function, spanning about 1,300 bp in the apparently noncoding region upstream of dbp. The nucleotide sequence of this region revealed four distinct domains, containing (i) a 9-bp repeated sequence, (ii) an A+T-rich segment, (iii) an 11-bp direct repeat, and (iv) a 47-bp direct repeat. At least some part of each of these domains was required for origin function. Therefore, like the Epstein-Barr virus lytic-phase origin of DNA replication, CMV oriLyt appears to be structurally complex.

Base Sequence↗
PubMed2003

UV- and MMS-induced mutagenesis of lambdaO(am)8 phage under nonpermissive conditions for phage DNA replication.

Mutagenesis in Escherichia coli, a subject of many years of study is considered to be related to DNA replication. DNA lesions nonrepaired by the error-free nucleotide excision repair (NER), base excision repair (BER) and recombination repair (RR), stop replication at the fork. Reinitiation needs translesion synthesis (TLS) by DNA polymerase V (UmuC), which in the presence of accessory proteins, UmuD', RecA and ssDNA-binding protein (SSB), has an ability to bypass the lesion with high mutagenicity. This enables reinitiation and extension of DNA replication by DNA polymerase III (Pol III). We studied UV- and MMS-induced mutagenesis of lambdaO(am)8 phage in E. coli 594 sup+ host, unable to replicate the phage DNA, as a possible model for mutagenesis induced in nondividing cells (e.g. somatic cells). We show that in E. coli 594 sup+ cells UV- and MMS-induced mutagenesis of lambdaO(am)8 phage may occur. This mutagenic process requires both the UmuD' and C proteins, albeit a high level of UmuD' and low level of UmuC seem to be necessary and sufficient. We compared UV-induced mutagenesis of lambdaO(am)8 in nonpermissive (594 sup+) and permissive (C600 supE) conditions for phage DNA replication. It appeared that while the mutagenesis of lambdaO(am)8 in 594 sup+ requires the UmuD' and C proteins, which can not be replaced by other SOS-inducible protein(s), in C600 supE their functions may be replaced by other inducible protein(s), possibly DNA polymerase IV (DinB). Mutations induced under nonpermissive conditions for phage DNA replication are resistant to mismatch repair (MMR), while among those induced under permissive conditions, only about 40% are resistant.

Bacteriophage lambda↗
PubMed2000

Inhibition of DNA methyltransferase inhibits DNA replication.

Ectopic expression of DNA methyltransferase transforms vertebrate cells, and inhibition of DNA methyltransferase reverses the transformed phenotype by an unknown mechanism. We tested the hypothesis that the presence of an active DNA methyltransferase is required for DNA replication in human non-small cell lung carcinoma A549 cells. We show that the inhibition of DNA methyltransferase by two novel mechanisms negatively affects DNA synthesis and progression through the cell cycle. Competitive polymerase chain reaction of newly synthesized DNA shows decreased origin activity at three previously characterized origins of replication following DNA methyltransferase inhibition. We suggest that the requirement of an active DNA methyltransferase for the functioning of the replication machinery has evolved to coordinate DNA replication and inheritance of the DNA methylation pattern.

Animals↗
PubMed2001

The Bacillus subtilis phage phi 29 protein p16.7, involved in phi 29 DNA replication, is a membrane-localized single-stranded DNA-binding protein.

The functional role of the phi 29-encoded integral membrane protein p16.7 in phage DNA replication was studied using a soluble variant, p16.7A, lacking the N-terminal membrane-spanning domain. Because of the protein-primed mechanism of DNA replication, the bacteriophage phi 29 replication intermediates contain long stretches of single-stranded DNA (ssDNA). Protein p16.7A was found to be an ssDNA-binding protein. In addition, by direct and functional analysis we show that protein p16.7A binds to the stretches of ssDNA of the phi 29 DNA replication intermediates. Properties of protein p16.7A were compared with those of the phi 29-encoded single-stranded DNA-binding protein p5. The results obtained show that both proteins have different, non-overlapping functions. The likely role of p16.7 in attaching phi 29 DNA replication intermediates to the membrane of the infected cell is discussed. Homologues of gene 16.7 are present in phi 29-related phages, suggesting that the proposed role of p16.7 is conserved in this family of phages.

Bacillus Phages↗
PubMed2003

Schizosacchromyces pombe Dpb2 binds to origin DNA early in S phase and is required for chromosomal DNA replication.

Genetic evidence suggests that DNA polymerase epsilon (Pol epsilon) has a noncatalytic essential role during the early stages of DNA replication initiation. Herein, we report the cloning and characterization of the second largest subunit of Pol epsilon in fission yeast, called Dpb2. We demonstrate that Dpb2 is essential for cell viability and that a temperature-sensitive mutant of dpb2 arrests with a 1C DNA content, suggesting that Dpb2 is required for initiation of DNA replication. Using a chromatin immunoprecipitation assay, we show that Dpb2, binds preferentially to origin DNA at the beginning of S phase. We also show that the C terminus of Pol epsilon associates with origin DNA at the same time as Dpb2. We conclude that Dpb2 is an essential protein required for an early step in DNA replication. We propose that the primary function of Dpb2 is to facilitate assembly of the replicative complex at the start of S phase. These conclusions are based on the novel cell cycle arrest phenotype of the dpb2 mutant, on the previously uncharacterized binding of Dpb2 to replication origins, and on the observation that the essential function of Pol epsilon is not dependent on its DNA synthesis activity.

Base Sequence↗
PubMed1996

Plasmid-like replicative intermediates of the Epstein-Barr virus lytic origin of DNA replication.

During the lytic phase of herpesviruses, intermediates of viral DNA replication are found as large concatemeric molecules in the infected cells. It is not known, however, what the early events in viral DNA replication that yield these concatemers are. In an attempt to identify these early steps of DNA replication, replicative intermediates derived from the lytic origin of Epstein-Barr virus, oriLyt, were analyzed. As shown by density shift experiments with bromodeoxyuridine, oriLyt replicated semiconservatively soon after induction of the lytic cycle and oriLyt-containing DNA is amplified to yield monomeric plasmid progeny DNA (besides multimeric forms and high-molecular-weight DNA). A new class of plasmid progeny DNA which have far fewer negative supercoils than do plasmids extracted from uninduced cells is present only in cells undergoing the lytic cycle of Epstein-Barr virus. This finding is consistent with plasmid DNAs having fewer nucleosomes before extraction. The newly replicated plasmid DNAs are dependent on a functional oriLyt in cis and support an efficient marker transfer into Escherichia coli as monomeric plasmids. Multimeric forms of presumably circular progeny DNA of oriLyt, as well as detected recombination events, indicate that oriLyt-mediated DNA replication is biphasic: an early theta-like mode is followed by a complex pattern which could result from rolling-circle DNA replication.

Cell Line↗
PubMedDate not supplied

Maintaining transcriptional states through DNA replication.

The timing of DNA replication has been implicated in gene regulation based on observations that actively transcribed genes generally replicate earlier in S-phase than their inactive counterparts. However, we recently showed that differentiation-induced gene silencing in lymphocytes does not generally result in silenced genes switching from early to late replication, but instead alters the onset of separation of newly synthesised sister-chromatids. Our findings may provide novel insights into the mechanisms that allow inactive chromatin structure to be propagated through DNA replication.

Animals↗
PubMed1997

CDC45 is required in conjunction with CDC7/DBF4 to trigger the initiation of DNA replication.

The initiation of DNA replication in Saccharomyces cerevisiae requires the protein product of the CDC45 gene. We report that although Cdc45p is present at essentially constant levels throughout the cell cycle, it completes its initiation function in late G1, after START and prior to DNA synthesis. Shortly after mitosis, cells prepare for initiation by assembling prereplicative complexes at their replication origins. These complexes are then triggered at the onset of S phase to commence DNA replication. Cells defective for CDC45 are incapable of activating the complexes to initiate DNA replication. In addition, Cdc45p and Cdc7p/Dbf4p, a kinase implicated in the G1/S phase transition, are dependent on one another for function. These data indicate that CDC45 functions in late G1 phase in concert with CDC7/DBF4 to trigger initiation at replication origins after the assembly of the prereplicative complexes.

Carrier Proteins↗
PubMed2000

Interactions between Mcm10p and other replication factors are required for proper initiation and elongation of chromosomal DNA replication in Saccharomyces cerevisiae.

BACKGROUND: MCM10 is essential for the initiation of chromosomal DNA replication in Saccharomyces cerevisiae. Previous work showed that Mcm10p interacts with the Mcm2-7 protein complex that may be functioning as the replication-licensing factor. In addition, Mcm10p is required during origin activation and disassembly of the prereplicative complex, which allows smooth passage of replication forks. RESULTS: We show that an mcm10 mutation causes a slow progression of DNA synthesis and a loss of chromosome integrity during the S phase and prevents entry into mitosis, despite apparent completion of chromosomal DNA replication at nonpermissive temperatures. Furthermore, Mcm10p interacts genetically with the origin recognition complex (ORC) and various replication elongation factors, including a subunit of DNA polymerases epsilon and delta. Mcm10p is an abundant protein (approximately 4 x 10(4) copies per haploid cell) that is almost exclusively localized in the chromatin and/or nuclear matrix fractions during all phases of the cell cycle. When it is visualized by the chromosome-spreading method followed by immunostaining, Mcm10p forms punctate foci on chromatin throughout the cell cycle and these foci mostly overlap with those of Orc1p, a component of ORC. CONCLUSIONS: These results suggest that Mcm10p, like the Mcm2-7 proteins, is a critical component of the prereplication chromatin and acts together with ORC during the initiation of chromosomal DNA replication; in addition, Mcm10p plays an important role during the elongation of DNA replication.

Cell Cycle Proteins↗
PubMed1983

Transcription from the SV40 early-early and late-early overlapping promoters in the absence of DNA replication.

Transcription for a hybrid SV40 promoter-beta globin coding sequence recombinant initiates from both early-early (EE) and late-early (LE) SV40 start sites (EES and LES) in the absence of DNA replication. The 72-bp repeat is essential to potentiate the elements of the two overlapping EE and LE promoters (EEP and LEP). Two current models, which can account for the EE to LE shift in RNA chain initiation during the SV40 replication cycle, are that LE transcription is linked to replication and occurs on newly replicated DNA molecules or that there are two promoter elements, a stronger EEP and a weaker LEP, T antigen repressing the EEP late in infection. Our results support the second model. A 5'-TATTTAT-3' to 5'-TATCGAT-3' mutation in the putative SV40 TATA box decreases transcription from EES, increases transcription from LES, and inhibits DNA replication. Therefore, this element acts as a classical TATA box for transcription, and yet is also important for DNA replication.

Animals↗
PubMed1985

Overproduction and purification of protein P6 of Bacillus subtilis phage phi 29: role in the initiation of DNA replication.

A phi 29 DNA fragment containing gene 6, required for DNA replication, has been cloned in plasmid pPLc28 under the control of the PL promoter of phage lambda. A polypeptide with an electrophoretic mobility close to that of p6 was labelled with 35S-methionine after heat induction. This protein, representing about 4% of the total E. coli protein after 1 h of induction, was obtained in a highly purified form. The protein was characterized as p6 by amino acid analysis and NH2-and COOH-terminal sequence determination. Protein p6 has an apparent molecular weight of 23,600, suggesting that the native form of the protein is a dimer. The purified protein p6 stimulated the protein-primed initiation of phi 29 DNA replication when added to purified proteins p2 (phi 29-coded DNA polymerase) and p3 (terminal protein).

Amino Acid Sequence↗
PubMed1998

Cellular proteins required for adeno-associated virus DNA replication in the absence of adenovirus coinfection.

We previously reported the development of an in vitro adeno-associated virus (AAV) DNA replication system. The system required one of the p5 Rep proteins encoded by AAV (either Rep78 or Rep68) and a crude adenovirus (Ad)-infected HeLa cell cytoplasmic extract to catalyze origin of replication-dependent AAV DNA replication. However, in addition to fully permissive DNA replication, which occurs in the presence of Ad, AAV is also capable of partially permissive DNA replication in the absence of the helper virus in cells that have been treated with genotoxic agents. Limited DNA replication also occurs in the absence of Ad during the process of establishing a latent infection. In an attempt to isolate uninfected extracts that would support AAV DNA replication, we discovered that HeLa cell extracts grown to high density can occasionally display as much in vitro replication activity as Ad-infected extracts. This finding confirmed previous genetic analyses which suggested that no Ad-encoded proteins were absolutely essential for AAV DNA replication and that the uninfected extracts should be useful for studying the differences between helper-dependent and helper-independent AAV DNA replication. Using specific chemical inhibitors and monoclonal antibodies, as well as the fractionation of uninfected HeLa extracts, we identified several of the cellular enzymes involved in AAV DNA replication. They were the single-stranded DNA binding protein, replication protein A (RFA), the 3' primer binding complex, replication factor C (RFC), and proliferating cell nuclear antigen (PCNA). Consistent with the current model for AAV DNA replication, which requires only leading-strand DNA synthesis, we found no requirement for DNA polymerase alpha-primase. AAV DNA replication could be reconstituted with purified Rep78, RPA, RFC, and PCNA and a phosphocellulose chromatography fraction (IIA) that contained DNA polymerase activity. As both RFC and PCNA are known to be accessory proteins for polymerase delta and epsilon, we attempted to reconstitute AAV DNA replication by substituting either purified polymerase delta or polymerase epsilon for fraction IIA. These attempts were unsuccessful and suggested that some novel cellular protein or modification was required for AAV DNA replication that had not been previously identified. Finally, we also further characterized the in vitro DNA replication assay and demonstrated by two-dimensional (2D) gel electrophoresis that all of the intermediates commonly seen in vivo are generated in the in vitro system. The only difference was an accumulation of single-stranded DNA in vivo that was not seen in vitro. The 2D data also suggested that although both Rep78 and Rep68 can generate dimeric intermediates in vitro, Rep68 is more efficient in processing dimers to monomer duplex DNA. Regardless of the Rep that was used in vitro, we found evidence of an interaction between the elongation complex and the terminal repeats. Nicking at the terminal repeats of a replicating molecule appeared to be inhibited until after elongation was complete.

Adenoviridae↗
PubMed2002

Histone acetyl transferases: a role in DNA repair and DNA replication.

In eukaryotic cells DNA is associated with proteins to form a complex known as chromatin. The dominant proteins within this chromatin complex are the histones, which are subject to a wide variety of covalent and reversible posttranslational modifications such as acetylation. A specialized family of enzymes, the histone acetyl transferases, catalyzes the transfer of acetyl groups from their cosubstrate acetyl-coenzyme A to lysine residues of histones. Acetylation of histone N-terminal lysine residues induces chromosomal changes and results in the loss of chromosomal repression that allows the successful transcription of the underlying genes. Analogously, in DNA repair and also DNA replication the chromosomal repression is thought to be relieved by such mechanisms. Recently several publications have provided evidence that histone acetyl transferases also modify nonhistone proteins and thereby regulate their activities. This review discusses various aspects of histone acetyl transferases and summarizes recent findings which suggest a role for histone acetyl transferases in DNA repair and DNA replication.

Acetyltransferases↗
PubMed1991

Cytometric analysis of DNA replication inhibited by emetine and cyclosporin A.

DNA staining methods based on aspecific interactions with dye molecules have been replaced by an immunofluorescent approach to measure DNA replication. Biotin-11-dUTP was incorporated into permeable thymocytes isolated after emetine or cyclosporin A treatment of mice. Active sites of DNA replication were amplified based on biotin-avidin interaction and verified under fluorescent microscope. Cytometry of fluorescent images allow the direct measurement of replicating DNA without aspecific detection of total cellular DNA. Cytometric analysis of replication revealed that emetine acts at the early S phase, while cyclosporin A blocks in vivo DNA synthesis at mid S phase.

Animals↗
PubMed1998

Effects of bulky polycyclic aromatic hydrocarbon adducts on DNA replication by exonuclease-deficient T7 and T4 DNA polymerases.

In vitro DNA replication by exonuclease-deficient T7 DNA polymerase (Sequenase) and an exonuclease deficient T4 DNA polymerase was examined on a 244-nucleotide DNA template treated with three electrophilic polycyclic aromatic hydrocarbon (PAH) metabolites: racemic trans-7,8-dihydroxy-anti-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BaPDE), trans-2,3-dihydroxy-anti-1,10b-epoxy-10b,1,2,3-tetrahydrofluoranthene (FADE), or 3,4-epoxy-3,4-dihydrocyclopenta[cd]pyrene (CPPE). The DNA replication terminated opposite template guanines and, to a lesser extent, at template adenines, as expected, as purines were modified preferentially by the chemical treatments. Analysis of the products synthesized on the damaged templates indicated that bypass replication by Sequenase proceeded in three steps: (1) replication first terminated one base 3' to each adduct; (2) a nucleotide was then incorporated opposite the PAH-modified base; and (3) replication continued at some sites to give full bypass of the lesions. The rate of lesion bypass was affected by the type of chemical adduct, the sequence context of the adduct, and the concentration of deoxynucleoside triphosphates. Short DNA repeats appeared to facilitate translesion replication.

Bacteriophage T4↗
PubMed1983

Studies on the replication of Escherichia coli phage lambda DNA. I. The kinetics of DNA replication and requirements for the generation of rolling circles.

Escherichia coli phage lambda DNA has been isolated from infected bacteria using a new technique by which virtually all phage DNA is recovered. Isolated DNA is examined by electron microscopy. Addition of phi X174 RF1 molecules as a counting standard enables us to determine the average number of lambda DNA molecules present in an infected cell. In this study, we have followed the kinetics of lambda DNA replication and examined rolling circle replication. The most important findings are the following: (1) Rolling circle replication is initiated at roughly the same time as is theta replication, indicating that the rolling circle is not solely a late-replicating form. (2) theta replication stops at about 16 min after infection. (3) Early in infection the number of DNA molecules per cell doubles every 2-3 min until theta replication stops, at which point most DNA synthesis consists of growth of the tails of about three rolling circles per cell. (4) Neither the timing of rolling circle replication nor the number of molecules is affected by the activity of the lambda red genes. (5). The red genes are responsible for the production of oligomeric circles late in infection.

Bacteriophage lambda↗