Background:The single-strand DNA-binding protein, replication protein A (RPA), is essential for replication, repair, and recombination. Results: This study elucidates the molecular defects of RPA mutants that cause cell cycle arrest. Conclusion: These mutants are active in DNA replication but not in DNA repair because of altered binding to short ssDNA intermediates. Significance: This study indicates that DNA repair and replication require different RPA-DNA interactions.

Replication protein A (RPA) is the primary ssDNA-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 lethal when homozygous and to cause high rates of cancer when heterozygous. 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 nonfunctional protein complex. This suggests that haploinsufficiency of RPA causes an increase in the levels of DNA damage and in the incidence of cancer.

Summary Replication Protein A (RPA) is a single-strand DNA-binding protein that is found in all eukaryotes. RPA is subjected to multiple post-translational modifications including serine- and threonine-phosphorylation, poly-ADP ribosylation and SUMOylation. These modifications are believed to regulate RPA activity through modulating interactions with DNA and partner proteins. This article describes two methods used to detect post-translational modified RPA: immunofluorescence and immmuoblotting.

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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. The ssDNA-binding activity of RPA is primarily mediated by two domains in the RPA1 subunit. I characterized mutant forms v 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. The ssDNA-binding activity of RPA is primarily mediated by two domains in the RPA1 subunit. I characterized mutant forms of RPA to elucidate the contribution of specific residues in the high affinity DNA binding domains to the cellular function of RPA. These studies enhance the understanding of the properties of RPA that contribute to DNA repair and cellular checkpoints. 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 a high rate of chromosomal rearrangements in yeast and mice. I characterized the equivalent mutation in human RPA. My studies show that the mutation causes a defect in ssDNA binding and a nonfunctional protein. Combined with the mice studies, the data suggest that haploinsufficiency of RPA causes an increase in DNA damage and in the incidence of cancer. The ssDNA-interactions of the high affinity binding domains in RPA1 are mediated by several residues including four highly conserved aromatic residues. Mutation of these residues had no effect on DNA replication but caused defects in DNA repair pathways. I conclude that DNA intermediates in different DNA metabolic pathways require different RPA binding function and that the aromatic residues are indispensable for binding in DNA repair. These studies illustrate that different DNA metabolic pathways have distinct requirements for RPA function. A decrease in binding to ssDNA of any length has specific consequences in vivo. These data also demonstrate that a single mutation in RPA in a residue that does not even contact ssDNA can result in a non-functional RPA complex. I conclude that even a modest decrease in RPA protein levels is not compatible vi with long term cell survival. Taken together, these studies highlight the importance of proper regulation of RPA protein levels and its ssDNA binding affinity to proper maintenance of the integrity of the genome. vii TABLE OF CONTENTS LIST OF TABLES .