Phosphorylation of human replication protein A by the DNA-dependent protein kinase is involved in the modulation of DNA replication

Leigh A Henricksen; Timothy  Carter; Anindya Dutta; Marc S Wold

doi:10.1093/nar/24.15.3107

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Phosphorylation of human replication protein A by the DNA-dependent protein kinase is involved in the modulation of DNA replication

Journal article

Open access

Peer reviewed

Phosphorylation of human replication protein A by the DNA-dependent protein kinase is involved in the modulation of DNA replication

Leigh A Henricksen, Timothy Carter, Anindya Dutta and Marc S Wold

Nucleic acids research, Vol.24(15), pp.3107-3112

08/01/1996

DOI: 10.1093/nar/24.15.3107

PMCID: PMC146026

PMID: 8760901

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Abstract

The single-stranded DNA-binding protein, Replication Protein A (RPA), is a heterotrimeric complex with subunits of 70, 32 and 14 kDa involved in DNA metabolism. RPA may be a target for cellular regulation; the 32 kDa subunit (RPA32) is phosphorylated by several cellular kinases including the DNA-dependent protein kinase (DNA-PK). We have purified a mutant hRPA complex lacking amino acids 1-33 of RPA32 (rhRPA x 32delta1-33). This mutant bound ssDNA and supported DNA replication; however, rhRPA x 32delta1-33 was not phosphorylated under replication conditions or directly by DNA-PK. Proteolytic mapping revealed that all the sites phosphorylated by DNA-PK are contained on residues 1-33 of RPA32. When wild-type RPA was treated with DNA-PK and the mixture added to SV40 replication assays, DNA replication was supported. In contrast, when rhRPA x 32delta1-33 was treated with DNA-PK, DNA replication was strongly inhibited. Because untreated rhRPA x 32delta1-33 is fully functional, this suggests that the N-terminus of RPA is needed to overcome inhibitory effects of DNA-PK on other components of the DNA replication system. Thus, phosphorylation of RPA may modulate DNA replication indirectly, through interactions with other proteins whose activity is modulated by phosphorylation.

Phosphorylation

DNA Replication

Mutation

Sequence Deletion

DNA, Viral - biosynthesis

DNA, Single-Stranded - metabolism

Humans

Protein-Serine-Threonine Kinases - genetics

Structure-Activity Relationship

Simian virus 40 - genetics

Nuclear Proteins

DNA-Binding Proteins - metabolism

DNA-Activated Protein Kinase

Protein Binding

Protein Conformation

Replication Protein A

Protein-Serine-Threonine Kinases - metabolism

Details

Title: Subtitle: Phosphorylation of human replication protein A by the DNA-dependent protein kinase is involved in the modulation of DNA replication
Creators: Leigh A Henricksen - Department of Biochemistry, University of Iowa, Iowa City, 52242, USA
Timothy Carter
Anindya Dutta
Marc S Wold
Resource Type: Journal article
Publication Details: Nucleic acids research, Vol.24(15), pp.3107-3112
Publisher: England
DOI: 10.1093/nar/24.15.3107
PMID: 8760901
PMCID: PMC146026
ISSN: 0305-1048
eISSN: 1362-4962
Grant note: GM44721 / NIGMS NIH HHS GM64499 / NIGMS NIH HHS
Language: English
Date published: 08/01/1996
Academic Unit: Radiation Oncology; Biochemistry and Molecular Biology
Record Identifier: 9984025257002771

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