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PMID: 1318195 Published · ppublish English Journal Article Research Support, Non-U.S. Gov't Research Support, U.S. Gov't, P.H.S.

cdc2 family kinases phosphorylate a human cell DNA replication factor, RPA, and activate DNA replication.

The EMBO journal ·Vol. 11 ·No. 6 ·1992-06-00 ·Pages 2189-99

Dutta A, Stillman B

Abstract

RPA is a single-stranded DNA binding protein complex purified from human cells and is essential for the initiation and elongation stages of SV40 DNA replication in vitro. In both human and yeast cells, the 34 kDa polypeptide subunit of RPA is phosphorylated in the S and G2 phases of the cell cycle and not in G1. One of the major RPA kinases present in extracts of human cells was purified and shown to be the cyclin B-cdc2 complex. This purified kinase, and a closely related cyclin A associated cdc2-like kinase, phosphorylated RPA p34 on a subset of the chymotryptic peptides that were phosphorylated in vivo at the G1-S transition. Two serines near the N-terminus of RPA p34 were identified as possible sites of phosphorylation by cdc2 kinase. These same serines were necessary for RPA phosphorylation in vivo. The purified cdc2 kinase stimulated SV40 DNA replication in vitro when added to G1 cell extracts. The kinase also stimulated unwinding at the origin of replication, one of the earliest steps in DNA replication requiring RPA, but only in the presence of an additional factor present in G1 cell extracts. Thus, one or more members of the cyclin-cdc2 kinase family may be required for the initiation and maintenance of S phase, in part due to their ability to phosphorylate and activate a cellular DNA replication factor, RPA.

MeSH Terms
Adenovirus Early Proteins Amino Acid Sequence CDC2 Protein Kinase/metabolism Cell Cycle/physiology Cell Line, Transformed Cyclins/metabolism DNA Replication DNA-Binding Proteins/genetics,isolation & purification,metabolism G1 Phase/physiology HeLa Cells Humans Kinetics Molecular Sequence Data Mutagenesis, Site-Directed Oncogene Proteins, Viral/genetics Peptide Mapping Phosphopeptides/isolation & purification Phosphorylation Replication Protein A S Phase/physiology Simian virus 40/genetics
Chemicals
Adenovirus Early Proteins Cyclins DNA-Binding Proteins Oncogene Proteins, Viral Phosphopeptides RPA1 protein, human Replication Protein A CDC2 Protein Kinase
Authors & Affiliations
2 authors, click to expand affiliations / ORCID
Dutta A
Cold Spring Harbor Laboratory, NY 11724.
Stillman B
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Article Info
Journal
The EMBO journal
Abbr.
EMBO J
ISSN
0261-4189
Published
1992-06-00
Pages
2189-99
Language
English
Region
England
NLM ID
8208664
PMCID
PMC556686
Subset
IM
Grants
NCI NIH HHS · CA 13106 · United States
NIGMS NIH HHS · GM 45436 · United States
External Links
PubMed source PMC Full Text View journal details
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