Repair-specific functions of replication protein A

Cathy S Hass; Koonyee Lam; Marc S Wold

doi:10.1074/jbc.M111.287441

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Repair-specific functions of replication protein A

Journal article

Open access

Peer reviewed

Repair-specific functions of replication protein A

Cathy S Hass, Koonyee Lam and Marc S Wold

The Journal of biological chemistry, Vol.287(6), pp.3908-3918

02/03/2012

DOI: 10.1074/jbc.M111.287441

PMCID: PMC3281679

PMID: 22179778

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Abstract

Replication protein A (RPA), the major eukaryotic single-strand DNA (ssDNA)-binding protein, is essential for replication, repair, recombination, and checkpoint activation. Defects in RPA-associated cellular activities lead to genomic instability, a major factor in the pathogenesis of cancer and other diseases. ssDNA binding activity is primarily mediated by two domains in the 70-kDa subunit of the RPA complex. These ssDNA interactions are mediated by a combination of polar residues and four conserved aromatic residues. Mutation of the aromatic residues causes a modest decrease in binding to long (30-nucleotide) ssDNA fragments but results in checkpoint activation and cell cycle arrest in cells. We have used a combination of biochemical analysis and knockdown replacement studies in cells to determine the contribution of these aromatic residues to RPA function. Cells containing the aromatic residue mutants were able to progress normally through S-phase but were defective in DNA repair. Biochemical characterization revealed that mutation of the aromatic residues severely decreased binding to short ssDNA fragments less than 20 nucleotides long. These data indicate that altered binding of RPA to short ssDNA intermediates causes a defect in DNA repair but not in DNA replication. These studies show that cells require different RPA functions in DNA replication and DNA repair.

Protein Structure, Tertiary

DNA, Single-Stranded - metabolism

Humans

DNA Repair - physiology

Cell Cycle Checkpoints - physiology

Replication Protein A - metabolism

Mutation, Missense

DNA, Single-Stranded - genetics

Animals

DNA Replication - physiology

Replication Protein A - genetics

S Phase - physiology

Mice

HeLa Cells

Drosophila melanogaster

Saccharomyces cerevisiae

Amino Acid Substitution

Details

Title: Subtitle: Repair-specific functions of replication protein A
Creators: Cathy S Hass - Department of Biochemistry, Carver College of Medicine, University of Iowa, Iowa City, Iowa 52242, USA
Koonyee Lam
Marc S Wold
Resource Type: Journal article
Publication Details: The Journal of biological chemistry, Vol.287(6), pp.3908-3918
DOI: 10.1074/jbc.M111.287441
PMID: 22179778
PMCID: PMC3281679
NLM abbreviation: J Biol Chem
ISSN: 0021-9258
eISSN: 1083-351X
Publisher: United States
Grant note: GM44721 / NIGMS NIH HHS P30 CA086862 / NCI NIH HHS R01 GM044721 / NIGMS NIH HHS
Language: English
Date published: 02/03/2012
Academic Unit: Radiation Oncology; Biochemistry and Molecular Biology
Record Identifier: 9984024562202771

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