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

TY - JOUR

T1 - Functional capacity of XRCC1 protein variants identified in DNA repair-deficient Chinese hamster ovary cell lines and the human population

AU - Berquist, Brian R

AU - Singh, Dharmendra Kumar

AU - Fan, Jinshui

AU - Kim, Daemyung

AU - Gillenwater, Elizabeth

AU - Kulkarni, Avanti

AU - Bohr, Vilhelm A

AU - Ackerman, Eric J

AU - Tomkinson, Alan E

AU - Wilson, David M

PY - 2010/4/10

Y1 - 2010/4/10

N2 - 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 POLb, 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, LIG3a, 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.

AB - 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 POLb, 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, LIG3a, 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.

KW - Amino Acid Substitution

KW - Animals

KW - CHO Cells

KW - Cricetinae

KW - Cricetulus

KW - DNA

KW - DNA Repair

KW - DNA-Binding Proteins

KW - Humans

U2 - 10.1093/nar/gkq193

DO - 10.1093/nar/gkq193

M3 - Journal article

C2 - 20385586

SN - 0261-3166

VL - 38

SP - 5023

EP - 5035

JO - Nucleic Acids Symposium Series

JF - Nucleic Acids Symposium Series

IS - 15

ER -