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Role of DNA polymerase alpha and DNA primase in simian virus 40 DNA replication in vitro.

Abstract：

:The role of DNA polymerase alpha (pol alpha) and DNA primase has been investigated in the simian virus 40 (SV40) DNA replication system in vitro. Removal of pol alpha and primase activities from crude extracts of HeLa cells or monkey cells by use of an anti-pol alpha immunoaffinity column resulted in the loss of replication activity. The addition of purified pol alpha-primase complex isolated from HeLa cells or monkey cells restored the replication activity of depleted extracts. In contrast, the pol alpha-primase complex isolated from either mouse cells or calf thumus did not. Extracts prepared from mouse cells (a source that does not support replication of SV40) did not replicate SV40 DNA. However, the addition of purified pol alpha-primase complex isolated from HeLa cells activated mouse cell extracts. pol alpha and primase from HeLa cells were extensively purified and separated by a one-step immunoaffinity adsorption and elution procedure. Both activities were required to restore DNA synthesis; the addition of pol alpha or primase alone supported replication poorly. Crude extracts of HeLa cells that were active in SV40 replication catalyzed the synthesis of full-length linear double-stranded (RFIII) DNA in reaction mixtures containing poly(dT)-tailed pBR322 RFIII. Maximal activity was dependent on the addition of oligo(dA), ATP, and creatine phosphate and was totally inhibited by aphidicolin. Since pol alpha alone could not replicate this substrate and since there was no degradation of input DNA, we propose that other enzymatic activities associate with pol alpha, displace the non-template strand, and allow the enzyme to replicate through duplex regions.

journal_name

Proc Natl Acad Sci U S A

journal_title

Proceedings of the National Academy of Sciences of the United States of America

authors

Murakami Y,Wobbe CR,Weissbach L,Dean FB,Hurwitz J

doi

10.1073/pnas.83.9.2869

subject

Has Abstract

pub_date

1986-05-01 00:00:00

pages

2869-73

issue

9

eissn

0027-8424

issn

1091-6490

journal_volume

83

pub_type

杂志文章

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