Multiple replication factors augment DNA synthesis by the two eukaryotic DNA polymerases, alpha and delta.
Toshiki Tsurimoto,Bruce Stillman +1 more
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It is suggested that co‐ordinated synthesis of these strands requires dynamic protein‐protein interactions between these replication factors and the two DNA polymerases.Abstract:
DNA synthesis by two eukaryotic DNA polymerases, alpha and delta, was studied using a single-strand M13 DNA template primed at a unique site. In the presence of low amounts of either DNA polymerase alpha or delta, DNA synthesis was limited and short DNA strands of approximately 100 bases were produced. Addition of replication factors RF-A, PCNA and RF-C, which were previously shown to be required for SV40 DNA replication in vitro, differentially stimulated the activity of both DNA polymerases. RF-A and RF-C independently stimulated DNA polymerase alpha activity 4- to 6-fold, yielding relatively short DNA strands (less than 1 kb) and PCNA had no effect. In contrast, polymerase delta activity was stimulated co-operatively by PCNA, RF-A and RF-C approximately 25- to 30-fold, yielding relatively long DNA strands (up to 4 kb). Neither RF-C nor RF-A appear to correspond to known polymerase stimulatory factors. RF-A was previously shown to be required for initiation of DNA replication at the SV40 origin. Results presented here suggest that it also functions during elongation. The differential effects of these three replication factors on DNA polymerases alpha and delta is consistent with the model that the polymerases function at the replication fork on the lagging and leading strand templates respectively. We further suggest that co-ordinated synthesis of these strands requires dynamic protein-protein interactions between these replication factors and the two DNA polymerases.read more
Citations
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Journal ArticleDOI
REPLICATION PROTEIN A: A Heterotrimeric, Single-Stranded DNA-Binding Protein Required for Eukaryotic DNA Metabolism
TL;DR: Replication protein A (RPA) is a single-stranded DNA-binding protein that is required for multiple processes in eukaryotic DNA metabolism, including DNA replication, DNA repair, and recombination.
Journal ArticleDOI
The dna replication fork in eukaryotic cells
Shou Waga,Bruce Stillman +1 more
TL;DR: 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.
Journal ArticleDOI
Recombinant replication protein A: expression, complex formation, and functional characterization.
TL;DR: Recombinant human RPA has the same subunit composition and the same activities as the authentic complex from human cells and is capable of supporting SV40 DNA replication in vitro.
Journal ArticleDOI
Anatomy of a DNA replication fork revealed by reconstitution of SV40 DNA replication in vitro
Shou Waga,Bruce Stillman +1 more
TL;DR: Complete enzymatic replication of DNA from the simian virus 40 origin has been reconstituted with T antigen and highly purified cellular proteins to replicate both strands of the double helix conjointly.
Journal ArticleDOI
Replication Protein A (RPA): The Eukaryotic SSB
TL;DR: Evidence indicates that RPA utilizes a multistep pathway to bind single-stranded DNA involving a particular molecular polarity of RPA, a mechanism that is apparently used to facilitate origin denaturation.
References
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Book
Molecular Cloning: A Laboratory Manual
TL;DR: Molecular Cloning has served as the foundation of technical expertise in labs worldwide for 30 years as mentioned in this paper and has been so popular, or so influential, that no other manual has been more widely used and influential.
Book
Animal Cell Dna Polymerases
Michael Fry,Lawrence A. Loeb +1 more
TL;DR: The authors present: General Introduction.
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