Functional capacity of XRCC1 protein variants identified in DNA repair-deficient Chinese hamster ovary cell lines and the human population

Abstract

XRCC1 operates as a scaffold protein in base excision repair, a pathway that copes with base and sugar damage in DNA. Studies using recombinant XRCC1 proteins revealed that: a C389Y substitution, responsible for the repair defects of the EM-C11 CHO cell line, caused protein instability; a V86R mutation abolished the interaction with POLβ, but did not disrupt the interactions with PARP-1, LIG3α and PCNA; and an E98K substitution, identified in EM-C12, reduced protein integrity, marginally destabilized the POLβ interaction, and slightly enhanced DNA binding. Two rare (P161L and Y576S) and two frequent (R194W and R399Q) amino acid population variants had little or no effect on XRCC1 protein stability or the interactions with POLβ, PARP-1, LIG3α, PCNA or DNA. One common population variant (R280H) had no pronounced effect on the interactions with POLβ, PARP-1, LIG3α and PCNA, but did reduce DNA-binding ability. When expressed in HeLa cells, the XRCC1 variants—excluding E98K, which was largely nucleolar, and C389Y, which exhibited reduced expression—exhibited normal nuclear distribution. Most of the protein variants, including the V86R POLβ-interaction mutant, displayed normal relocalization kinetics to/from sites of laser-induced DNA damage: except for E98K and C389Y, and the polymorphic variant R280H, which exhibited a slightly shorter retention time at DNA breaks.

Interaction with OGG1 Is Required for Efficient Recruitment of XRCC1 to Base Excision Repair and Maintenance of Genetic Stability after Exposure to Oxidative Stress

“…Comet assays were performed on the EM9 populations to measure the repair kinetics of DNA strand breaks induced by ionizing radiation. In agreement with previous reports (27,38), cells expressing XRCC1(R194W) did not show a defect in their capacity to repair strand breaks (data not shown). Moreover, both variants were equally efficient in reversing the sensitivity of EM9 cells to the alkylating agent MMS (Fig.…”

“…6A and B, while LIG3 was almost undetectable in EM9 cells, both XRCC1 variants allowed the recovery of the levels of LIG3 found in the XRCC1-proficient CHO cells (AA8). This is consistent with in vitro interaction experiments that showed that the R194W substitution does not affect XRCC1 interactions with PARP1, POL␤, and LIG3, involving the BRCT1, NTD, and BRCT2 domains of XRCC1, respectively (27). Furthermore, it has been shown that the BRCT2 domain of XRCC1 is required for the recruitment of LIG3 to the sites of repair in order to perform the last step of BER (43).…”

“…We thus decided to evaluate the impact of this amino acid substitution on XRCC1 interaction with OGG1 and its recruitment to BER after induction of oxidative base damage. We show here that the XRCC1(R194W) variant, while able to repair SSBs, as suggested by previous studies (27), is defective in the interaction with the DNA glycosylase OGG1 and is not correctly recruited to BER of 8-oxoG. Furthermore, we observed an increase in genetic instability in cells expressing the XRCC1(R194W) variant, suggesting an accumulation of BER intermediates.…”

Targeting XRCC1 Deficiency in Breast Cancer for Personalized Therapy

“…Zdzienicka, Department of Molecular Cell Genetics, Collegium Medicum in Bydgoszcz, Nicolaus-Copernicus University in Torun (Bydgoszcz, Poland; ref. 25,26). Cells were grown in Ham F-10 media (supplemented with 10% FBS and 1% penicillin/streptomycin; PAA).…”

The Interaction between Polynucleotide Kinase Phosphatase and the DNA Repair Protein XRCC1 Is Critical for Repair of DNA Alkylation Damage and Stable Association at DNA Damage Sites

“…Following site-directed laser damage, fluorescence recovery after photobleaching was recorded at various intervals until fluorescence returned to background levels. Data were analyzed using the Velocity software described above and "region of interest fluorescent intensity" was measured at various time intervals (35,36).…”