Reconstitution of simian virus 40 DNA replication with purified proteins.

Weinberg, David H.; Collins, Kathleen L.; Simancek, Pamela; Russo, Alicia A.; Wold, Marc S.; Virshup, David M.; Kelly, Thomas J.

doi:10.1073/pnas.87.22.8692

Cited by 156 publications

(100 citation statements)

References 37 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…This was an important point to consider, as another explanation for the resistance of the full NER reaction to inhibition by p21 could be that di erent DNA polymerases are normally used for NER and for replication, with one polymerase particularly resistant to inhibition by p21. There is good evidence that replication of SV40 viral DNA in vitro requires pol d (Tsurimoto et al, 1990;Weinberg et al, 1990;Hurwitz et al, 1990), and that mammalian chromosomal replication requires both pol d and pol e (Zlotkin et al, 1996). It would be possible, for example, that NER normally functions with pol e and this enzyme is especially refractory to p21 inhibition.…”

Section: Resistance Of Ner In Human Cell Extracts To Inhibition By P21mentioning

confidence: 99%

Resistance of human nucleotide excision repair synthesis in vitro to p21Cdn1

et al. 1998

View full text Add to dashboard Cite

The p21 Cdn1 protein (cip1/waf1/sdi1) plays an important role as an inhibitor of mammalian cell proliferation in response to DNA damage. By interacting with and inhibiting the function of cyclin-Cdk complexes, p21 can block entry into S phase. p21 can also directly inhibit replicative DNA synthesis by binding to the DNA polymerase sliding clamp factor PCNA. When cells are damaged and p21 is induced, DNA nucleotide excision repair (NER) continues, even though this pathway is PCNA-dependent. We investigated features of p21-resistant NER using human cell extracts. A direct endlabelling approach was used to measure the excision of damaged oligonucleotides by NER and no inhibition by p21 was found. By contrast, ®lling of the *30 nt gaps created by NER could be inhibited by pre-binding p21 to PCNA, but only when gap ®lling was uncoupled from incision. Binding p21 to PCNA could also inhibit ®lling of model 30 nt gaps by both puri®ed DNA polymerases d and e. When p21 was incubated in a cell extract before addition of PCNA, inhibition of repair synthesis was gradually relieved with time. This incubation gives p21 the opportunity to associate with other targets. As p21 blocks association of DNA polymerases with PCNA but does not prevent loading of PCNA onto DNA, repair gap ®lling can occur rapidly as soon as p21 dissociates from PCNA. A synthetic PCNA-binding p21 peptide was an e cient inhibitor of NER synthesis in cell extracts.

show abstract

Section: Resistance Of Ner In Human Cell Extracts To Inhibition By P21mentioning

confidence: 99%

Resistance of human nucleotide excision repair synthesis in vitro to p21Cdn1

et al. 1998

View full text Add to dashboard Cite

show abstract

“…Biochemical studies using the SV40 DNA replication system reconstituted with purified proteins (21,24,26,54,190,191) have shown that two different polymerases, pol α/primase and pol δ, are involved in DNA synthesis and that pol δ is involved in the synthesis of both the leading and lagging strands. The switching from pol α/primase to pol δ occurs during priming of the leading strand (24) and during synthesis of every Okazaki fragment (26).…”

Section: Polymerase Switchingmentioning

confidence: 99%

The Dna Replication Fork in Eukaryotic Cells

Waga

Stillman

1998

Annu. Rev. Biochem.

739

653

View full text Add to dashboard Cite

Replication of the two template strands at eukaryotic cell DNA replication forks is a highly coordinated process that ensures accurate and efficient genome duplication. Biochemical studies, principally of plasmid DNAs containing the Simian Virus 40 origin of DNA replication, and yeast genetic studies have uncovered the fundamental mechanisms of replication fork progression. At least two different DNA polymerases, a single-stranded DNA-binding protein, a clamp-loading complex, and a polymerase clamp combine to replicate DNA. Okazaki fragment synthesis involves a DNA polymerase-switching mechanism, and maturation occurs by the recruitment of specific nucleases, a helicase, and a ligase. The process of DNA replication is also coupled to cell-cycle progression and to DNA repair to maintain genome integrity. CONTENTS

show abstract

Section: Introductionmentioning

confidence: 99%

“…Pol ␣/primase can only synthesize short primers that must then be extended by the activity of a processive DNA polymerase(s). Biochemical analysis of simian virus 40 (SV40) DNA replication, which has been extensively used as a model system for eukaryotic DNA replication, has shown that primers synthesized by Pol ␣ can be extended by Pol ␦ and that these two polymerases are sufficient for SV40 replication in vitro (Weinberg et al, 1990;Waga et al, 1994).A second processive DNA polymerase, Pol ⑀, is required for cell viability and chromosomal DNA replication in both fission (D'Urso and Nurse, 1997) and budding yeast (Morrison et al, 1990;Araki et al, 1992;Budd and Campbell, 1993). Pol ⑀ has also been implicated in both DNA repair (Jessberger et al, 1993;Wang et al, 1993;Shivji et al, 1995;Holmes, 1999) and cell cycle checkpoint control in eukaryotic cells (Navas et al, 1995).…”

mentioning

confidence: 99%

Schizosacchromyces pombeDpb2 Binds to Origin DNA Early in S Phase and Is Required for Chromosomal DNA Replication

Feng

Rodriguez-Menocal

Tolun

et al. 2003

MBoC

View full text Add to dashboard Cite

Genetic evidence suggests that DNA polymerase epsilon (Pol ⑀) 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 ⑀ 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 ⑀ 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 ⑀ is not dependent on its DNA synthesis activity. INTRODUCTIONChromosomal DNA replication in eukaryotic cells requires the activity of at least three DNA polymerases: ␣, ␦, and ⑀ (Waga and Stillman, 1998;Hubscher et al., 2000;Bell and Dutta, 2002). Pol ␣ is tightly associated with a primase activity capable of de novo DNA synthesis, suggesting that this enzyme is responsible for initiation of both leading and lagging strands (Waga and Stillman, 1998). Pol ␣/primase can only synthesize short primers that must then be extended by the activity of a processive DNA polymerase(s). Biochemical analysis of simian virus 40 (SV40) DNA replication, which has been extensively used as a model system for eukaryotic DNA replication, has shown that primers synthesized by Pol ␣ can be extended by Pol ␦ and that these two polymerases are sufficient for SV40 replication in vitro (Weinberg et al., 1990;Waga et al., 1994).A second processive DNA polymerase, Pol ⑀, is required for cell viability and chromosomal DNA replication in both fission (D'Urso and Nurse, 1997) and budding yeast (Morrison et al., 1990;Araki et al., 1992;Budd and Campbell, 1993). Pol ⑀ has also been implicated in both DNA repair (Jessberger et al., 1993;Wang et al., 1993;Shivji et al., 1995;Holmes, 1999) and cell cycle checkpoint control in eukaryotic cells (Navas et al., 1995). Recently, we have shown that the DNA polymerase and exonuclease domains of Pol ⑀ are dispensable for cell viability in fission yeast (Feng and D'Urso, 2001). Similar observations have been made for the evolutionarily distant yeast, Saccharomyces cerevisiae (Kesti et al., 1999;Dua et al., 2000). These findings have raised important questions regarding the essential role of this replicative enzyme in DNA synthesis. Although the N-terminal catalytic domains are dispensable, the C-terminal half of the enzyme is essential for cell viability and chromosomal replication in fission...

show abstract

Reconstitution of simian virus 40 DNA replication with purified proteins.

Cited by 156 publications

References 37 publications

Resistance of human nucleotide excision repair synthesis in vitro to p21Cdn1

Resistance of human nucleotide excision repair synthesis in vitro to p21Cdn1

The Dna Replication Fork in Eukaryotic Cells

Schizosacchromyces pombeDpb2 Binds to Origin DNA Early in S Phase and Is Required for Chromosomal DNA Replication

Contact Info

Product

Resources

About