Journal article
Functional characterization of a cancer causing mutation in human Replication Protein A
Molecular cancer research, Vol.8(7), pp.1017-1026
07/2010
DOI: 10.1158/1541-7786.MCR-10-0161
PMCID: PMC2905489
PMID: 20587534
Abstract
Replication protein A (RPA) is the primary single-stranded DNA-binding protein in eukaryotes. RPA is essential for DNA replication, repair and recombination. Mutation of a conserved leucine residue to proline in the high affinity DNA binding site of RPA (residue L221 in human RPA) has been shown to have defects in DNA repair and a high rate of chromosomal rearrangements in yeast. The homologous mutation in mice was found to be homozygous lethal and cause high rates of cancer when heterozygous (Wang et al. (2005)
Nat Genet
37, 750). To understand the molecular defect causing these phenotypes, we created the homologous mutation in the human RPA1 gene (L221P) and analyzed its properties in cells and
in vitro
. RPA1(L221P) does not support cell cycle progression when it is the only form of RPA1 in HeLa cells. This phenotype is caused by defects in DNA replication and repair. No phenotype is observed when cells contain both wild-type and L221P forms of RPA1 indicating that L221P is not dominant. Recombinant L221P polypeptide forms a stable complex with the other subunits of RPA indicating that the mutation does not destabilize the protein; however, the resulting complex has dramatically reduced ssDNA binding activity and cannot support SV40 DNA replication
in vitro
. These findings indicate that in mammals the L221P mutation causes a defect in ssDNA binding and a non-functional protein complex. This suggests that haploinsufficiency of RPA causes an increase in the levels of DNA damage and in the incidence of cancer.
Details
- Title: Subtitle
- Functional characterization of a cancer causing mutation in human Replication Protein A
- Creators
- Cathy S Hass - Department of Biochemistry, Carver College of Medicine, University of IowaLokesh Gakhar - Protein Crystallography Facility, Carver College of Medicine, University of IowaMarc S Wold - Department of Biochemistry, Carver College of Medicine, University of Iowa
- Resource Type
- Journal article
- Publication Details
- Molecular cancer research, Vol.8(7), pp.1017-1026
- DOI
- 10.1158/1541-7786.MCR-10-0161
- PMID
- 20587534
- PMCID
- PMC2905489
- ISSN
- 1541-7786
- eISSN
- 1557-3125
- Language
- English
- Date published
- 07/2010
- Academic Unit
- Radiation Oncology; Biochemistry and Molecular Biology; Medicine Administration
- Record Identifier
- 9984025288902771
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