Functional Characterization of a Cancer Causing Mutation in Human Replication Protein A

Abstract

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.

DNA Damage Follows Repair Factor Depletion and Portends Genome Variation in Cancer Cells after Pore Migration

“…Knockdown of BRCA2 notably increases γH2AX foci (Figure S2), despite BRCA2 being functionally low in U2OS cells [15]. The si4 is intended to approximate nuclear depletion of multiple factors after constricted migration (Figure 2A,B,S1), and the increased DNA damage is consistent with past studies of individual factors in other cells [27-30]. Control transfections with siCtrl/lipofectamine also increase DNA damage (by ~40% when averaged across all control assays) and might reflect cell stress in transfection [31].…”

Repair-specific Functions of Replication Protein A

“…Aro1 binds ssDNA with an affinity ϳ0.1% of wild-type RPA (1% of the other Aro mutants) and does not support DNA replication in vitro (37). We have observed other RPA1 mutants with severe ssDNA-binding defects that are non-functional (16,45). We conclude that Aro1 does not support RPA function in cells.…”

“…Loss of heterozygosity of the RPA locus has been linked to cancer development (12,13) and abnormal checkpoint signaling (14). Loss of heterozygosity of RPA has also been demonstrated to cause genome instability in mice, suggesting a role for RPA in cancer progression (15,16).…”

Chemical inhibitor targeting the replication protein A–DNA interaction increases the efficacy of Pt-based chemotherapy in lung and ovarian cancer

“…These data demonstrate that to achieve in vivo activity a balance between potency and bioavailability can lean towards lower affinity as long as PK parameters allow clinically effective concentrations to be maintained. This balance is especially important in targeting RPA an essential protein with homozygous mutations being embryonically lethal in mice, while heterozygous mutants having an early predisposition to cancer [34]. No loss of function mutation for RPA has been reported in humans and genetic knockdown of RPA affects cellular viability [32].…”