Subunits of human replication protein A are crosslinked by photoreactive primers synthesized by DNA polymerases

Olga I. Lavrik; Heinz-Peter Nasheuer; Klaus Weisshart; Marc S. Wold; Rajendra Prasad; William A. Beard; Samuel H. Wilson; Alain Favre

doi:10.1093/nar/26.2.602

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Subunits of human replication protein A are crosslinked by photoreactive primers synthesized by DNA polymerases

Journal article

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Subunits of human replication protein A are crosslinked by photoreactive primers synthesized by DNA polymerases

Olga I. Lavrik, Heinz-Peter Nasheuer, Klaus Weisshart, Marc S. Wold, Rajendra Prasad, William A. Beard, Samuel H. Wilson and Alain Favre

Nucleic acids research, Vol.26(2), pp.602-607

01/1998

DOI: 10.1093/nar/26.2.602

PMCID: PMC147271

PMID: 9421522

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Abstract

Human replication protein A (huRPA) is a multisubunit protein which is involved in DNA replication, repair and recombination processes. It exists as a stable heterotrimer consisting of p70, p32 and p14 subunits. To understand the contribution of huRPA subunits to DNA binding we applied the photoaffinity labeling technique. The photoreactive oligonucleotide was synthesized in situ by DNA polymerases. 5-[N-(2-nitro-5-azidobenzoyl)-trans-3-aminopropenyl-1]deoxyuridine-5-triphosphate (NABdUTP) was used as substrate for elongation of a radiolabeled primerortemplate either by human DNA polymerase α primase (polα), human DNA polymerase β (polβ) or Klenow fragment of Escherichia coli DNA polymerase I (KF). The polymerase was incubated with NABdUTP and radiolabeled primer-template in the presence or absence of huRPA. The reaction mixtures were then irradiated with monochromatic UV light (315 nm) and the crosslinked products were separated by SDS-PAGE. The results clearly demonstrate crosslinking of the huRPA p70 and p32 subunits with DNA. The p70 subunit appears to bind to the single-stranded part of the DNA duplex, the p32 subunit locates near the 3′-end of the primer, while the p14 subunit locates relatively far from the 3β-end of the primer. This approach opens new possibilities for analysis of huRPA loading on DNA in the course of DNA replication and DNA repair.

Details

Title: Subtitle: Subunits of human replication protein A are crosslinked by photoreactive primers synthesized by DNA polymerases
Creators: Olga I. Lavrik - Novosibirsk Institute of Bioorganic Chemistry, Siberian Division of Russian Academy of Sciences, 630090 Novosibirsk, Russia.
Heinz-Peter Nasheuer - Institut für Molekulare Biotechnologie Jena, D-07708 Jena, Germany
Klaus Weisshart - Institut für Molekulare Biotechnologie Jena, D-07708 Jena, Germany
Marc S. Wold - University of Iowa
Rajendra Prasad - National Institute of Environmental Health Sciences
William A. Beard - National Institute of Environmental Health Sciences
Samuel H. Wilson - National Institute of Environmental Health Sciences
Alain Favre - Institut Jacques Monod
Resource Type: Journal article
Publication Details: Nucleic acids research, Vol.26(2), pp.602-607
DOI: 10.1093/nar/26.2.602
PMID: 9421522
PMCID: PMC147271
NLM abbreviation: Nucleic Acids Res
ISSN: 0305-1048
eISSN: 1362-4962
Publisher: Oxford University Press
Language: English
Date published: 01/1998
Academic Unit: Radiation Oncology; Biochemistry and Molecular Biology
Record Identifier: 9984288726502771

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