Characterization of a stable, major DNA polymerase α species devoid of DNA primase activity

Kaiserman, H. B.; Benbow, Robert M.

doi:10.1093/nar/15.24.10249

Cited by 15 publications

(8 citation statements)

References 47 publications

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“…7A and B). In considering the report that the same monoclonal antibody inhibited DNA polymerase a purified from Xenopus ovaries by only 60% at saturating doses (34), it is conceivable that injection of a more-specific antibody against Xenopus DNA polymerase a, if available, would result in 100% inhibition of replication. We must therefore assume that inhibition of one species of DNA polymerase causes at least partial blockage of replication carried out by the other species.…”

Section: Resultsmentioning

confidence: 99%

Involvement of proliferating cell nuclear antigen (cyclin) in DNA replication in living cells.

Zuber¹,

Tan²,

Ryoji³

1989

Mol. Cell. Biol.

107

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Proliferating cell nuclear antigen (PCNA) (also called cyclin) is known to stimulate the activity of DNA polymerase 8 but not the other DNA polymerases in vitro. We injected a human autoimmune antibody against PCNA into unfertilized eggs of Xenopus laevis and examined the effects of this antibody on the replication of injected plasmid DNA as well as egg chromosomes. The anti-PCNA antibody inhibited plasmid replication by up to 67%, demonstrating that PCNA is involved in plasmid replication in living cells. This result further implies that DNA polymerase 8 is necessary for plasmid replication in vivo. Anti-PCNA antibody alone did not block plasmid replication completely, but the residual replication was abolished by coinjection of a monoclonal antibody against DNA polymerase a. Anti-DNA polymerase a alone inhibited plasmid replication by 63%. Thus, DNA polymerase a is also required for plasmid replication in this system. In similar studies on the replication of egg chromosomes, the inhibition by anti-PCNA antibody was only 30%, while anti-DNA polymerase a antibody blocked 73% of replication. We concluded that the replication machineries of chromosomes and plasmid differ in their relative content of DNA polymerase 8. In addition, we obtained evidence through the use of phenylbutyl deoxyguanosine, an inhibitor of DNA polymerase a, that the structure of DNA polymerase a holoenzyme for chromosome replication is significantly different from that for plasmid replication.Proliferating cell nuclear antigen (PCNA) was initially recognized as a nuclear autoantigen which reacts with autoimmune sera from a certain population of patients with systemic lupus erythematosus (47; for recent reviews, see references 60 and 61). This antigen is an acidic nuclear protein with a molecular weight of about 36,000 (13,42,51,58). It is readily extractable from whole-cell or nuclear preparations and is expressed mainly in mitotically active cells of various tissues and cell lines (58). Independently, Celis and his colleagues identified, by two-dimensional gel electrophoresis, a protein called cyclin which is abundant in proliferating cells (for reviews, see references 13 and 16). It was later shown that cyclin and PCNA are identical (42). The complete amino acid sequence of this protein was determined by cDNA cloning in two independent laboratories (1,43). Immunofluorescence studies with human autoantibodies against PCNA have demonstrated that PCNA expression increases during late Gl and early S phase, immediately preceding the onset of DNA synthesis (14,36,48,55,58). During S phase, the distribution of PCNA changes dramatically within the nucleus (5,7,14,36,41,55,58,61), following closely the sites of chromosomes which are pulselabeled with [3H]thymidine or bromodeoxyuridine (8, 41).These observations strongly suggest an association of PCNA with the DNA replication apparatus. It has also been proposed that PCNA might be involved in DNA repair synthesis, because UV damage of DNA caused an increase in the nuclear PCNA staining of non-S-phas...

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Section: Resultsmentioning

confidence: 99%

Involvement of proliferating cell nuclear antigen (cyclin) in DNA replication in living cells.

Zuber¹,

Tan²,

Ryoji³

1989

Mol. Cell. Biol.

107

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“…Studies employing aphidicolin suggested that the DNA synthesis we observed was carried out by DNA polymerase at or 6. In light of the report by others that only ca-type activities were detected in the oocyte extract (50), DNA polymerase ca may be the major species carrying out the observed DNA synthesis. This tentative conclusion is also consistent with the observation that the repair reaction is weakly retarded by DMSO (Fig.…”

Section: Discussionmentioning

confidence: 70%

Minichromosome assembly accompanying repair-type DNA synthesis inXenopusoocytes

1989

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Minichromosomes were assembled by injection of circular DNA into the nucleus of Xenopus oocytes. We observed that, in the course of DNA supercoiling and chromatin assembly, a small percentage of the injected DNA molecules incorporated a radioactive precursor. This DNA synthesis was carried out by aphidicolin-sensitive DNA polymerase, and generated short repair-like patches covalently linked to the injected DNA. We found that the DNA thus repaired was rapidly supercoiled almost to completion within 15 to 30 min after injection, whereas 60 to 120 min were required to supercoil the intact, bulk DNA molecules. Such differential supercoiling kinetics was also observed when UV-damaged DNA was injected. Chromatin assembly, which was characterized by DNA fragment sizes protected from micrococcal nuclease digestion, was consistent with the rapid DNA supercoiling and proceeded more efficiently on the repaired DNA. These results indicate that there are at least two kinetically distinct ways of assembling minichromosomes in the oocyte nucleus, and that the repaired DNA molecules preferentially follow the faster pathway.

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“…DNA polymerase assays were performed as described for assay 1 of Kaiserman and Benbow (1987). Incubations were for 30 min at 37 °C.…”

Section: Methodsmentioning

confidence: 99%

A DNA helicase from Xenopus laevis ovaries

Poll¹,

Benbow²

1988

Biochemistry

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A DNA helicase was extensively purified from Xenopus laevis ovaries. The most purified fraction was free of DNA topoisomerase, DNA polymerase, and nuclease activities. The enzyme had a Stokes radius of 54 A and a sedimentation coefficient of 6-7.3 S, from which a native molecular weight of 140,000-170,000 was calculated. DNA helicase activity required Mg2+ or Mn2+ and was dependent on hydrolysis of ATP or dATP. Monovalent cations, K+ and Na+, stimulated DNA unwinding with an optimum at 130 mM. DNA-dependent ATPase activity copurified with the X. laevis DNA helicase. Double-stranded and single-stranded DNA were both cofactors for the ATPase activity, but single-stranded DNA was more efficient. The molecular weight, monovalent cation dependence, cofactor requirements, and elution from single-stranded DNA-cellulose suggest that the X. laevis DNA helicase is different from previously described eukaryotic DNA helicases.

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Characterization of a stable, major DNA polymerase α species devoid of DNA primase activity

Cited by 15 publications

References 47 publications

Involvement of proliferating cell nuclear antigen (cyclin) in DNA replication in living cells.

Involvement of proliferating cell nuclear antigen (cyclin) in DNA replication in living cells.

Minichromosome assembly accompanying repair-type DNA synthesis inXenopusoocytes

A DNA helicase from Xenopus laevis ovaries

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