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

Berquist BR, Singh DK, Fan J, Kim D, Gillenwater E, Kulkarni A, Bohr VA, Ackerman EJ, Tomkinson AE, Wilson DM - Nucleic Acids Res. (2010)

Bottom Line: 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 POLbeta, PARP-1, LIG3alpha, PCNA or DNA.One common population variant (R280H) had no pronounced effect on the interactions with POLbeta, PARP-1, LIG3alpha 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.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Molecular Gerontology, National Institute on Aging, NIH, Baltimore, MD 21224, USA.

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 POLbeta, but did not disrupt the interactions with PARP-1, LIG3alpha and PCNA; and an E98K substitution, identified in EM-C12, reduced protein integrity, marginally destabilized the POLbeta 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 POLbeta, PARP-1, LIG3alpha, PCNA or DNA. One common population variant (R280H) had no pronounced effect on the interactions with POLbeta, PARP-1, LIG3alpha 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 POLbeta-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.

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Relevant amino acid positions and regions of human XRCC1, and purification of XRCC1 proteins. (A) Linear schematic of the XRCC1 protein. Indicated are the four major conserved elements of XRCC1: the N-terminal domain (NTD), the nuclear localization signals (NLS), the BRCT1 and the BRCT2 domains. Numbers represent the amino acid positions within the XRCC1 polypeptide. The locations of the amino acid substitutions are indicated above the diagram. The regions of XRCC1 that interact with specified protein partners are denoted. (B) Nickel column affinity purified XRCC1 proteins. C-terminal His-tagged, N-terminal S-peptide-tagged XRCC1 recombinant proteins expressed in bacteria were purified using nickel chromatography. Shown is an image of the purified proteins separated in a 12% SDS–polyacrylamide gel and stained with coomassie blue. (C) Nickel and S-protein affinity purified XRCC1 proteins. Following nickel purification, dual-tagged XRCC1 recombinant proteins were affinity captured using an S-protein matrix. Shown is an image of silver stained purified proteins after separation in a 12% SDS–polyacrylamide gel. Arrows indicate XRCC1. STD = protein standards (in kDa).
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Figure 1: Relevant amino acid positions and regions of human XRCC1, and purification of XRCC1 proteins. (A) Linear schematic of the XRCC1 protein. Indicated are the four major conserved elements of XRCC1: the N-terminal domain (NTD), the nuclear localization signals (NLS), the BRCT1 and the BRCT2 domains. Numbers represent the amino acid positions within the XRCC1 polypeptide. The locations of the amino acid substitutions are indicated above the diagram. The regions of XRCC1 that interact with specified protein partners are denoted. (B) Nickel column affinity purified XRCC1 proteins. C-terminal His-tagged, N-terminal S-peptide-tagged XRCC1 recombinant proteins expressed in bacteria were purified using nickel chromatography. Shown is an image of the purified proteins separated in a 12% SDS–polyacrylamide gel and stained with coomassie blue. (C) Nickel and S-protein affinity purified XRCC1 proteins. Following nickel purification, dual-tagged XRCC1 recombinant proteins were affinity captured using an S-protein matrix. Shown is an image of silver stained purified proteins after separation in a 12% SDS–polyacrylamide gel. Arrows indicate XRCC1. STD = protein standards (in kDa).

Mentions: The various site-specific XRCC1 variant proteins generated for this study were characterized for one of the following reasons (summarized in Table 2): (i) they had been previously shown or argued as disruptive of a specific function and therefore would serve as a control (V86R, in the POLβ interaction; R109A, in the POLβ interaction and/or in DNA binding) (8,9,26); (ii) they were identified as the principal defect responsible for the repair-deficiencies associated with the CHO cell lines EM-C12 and EM-C11 (e.g. E102K and C390Y, respectively, or E98K and C389Y in humans; see protein alignment in Supplementary Figure S1) (24); or (iii) they were found among the normal healthy human population yet may represent impaired-function, disease susceptibility factors (P161L, R194W, R280H, R399Q and Y576S; Table 3) (27). Since these amino acid substitutions spanned the length of the XRCC1 protein (Figure 1A), they might represent separation-of-function mutants defective in only a single (or a limited number of) interaction(s).Table 2.


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

Berquist BR, Singh DK, Fan J, Kim D, Gillenwater E, Kulkarni A, Bohr VA, Ackerman EJ, Tomkinson AE, Wilson DM - Nucleic Acids Res. (2010)

Relevant amino acid positions and regions of human XRCC1, and purification of XRCC1 proteins. (A) Linear schematic of the XRCC1 protein. Indicated are the four major conserved elements of XRCC1: the N-terminal domain (NTD), the nuclear localization signals (NLS), the BRCT1 and the BRCT2 domains. Numbers represent the amino acid positions within the XRCC1 polypeptide. The locations of the amino acid substitutions are indicated above the diagram. The regions of XRCC1 that interact with specified protein partners are denoted. (B) Nickel column affinity purified XRCC1 proteins. C-terminal His-tagged, N-terminal S-peptide-tagged XRCC1 recombinant proteins expressed in bacteria were purified using nickel chromatography. Shown is an image of the purified proteins separated in a 12% SDS–polyacrylamide gel and stained with coomassie blue. (C) Nickel and S-protein affinity purified XRCC1 proteins. Following nickel purification, dual-tagged XRCC1 recombinant proteins were affinity captured using an S-protein matrix. Shown is an image of silver stained purified proteins after separation in a 12% SDS–polyacrylamide gel. Arrows indicate XRCC1. STD = protein standards (in kDa).
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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Figure 1: Relevant amino acid positions and regions of human XRCC1, and purification of XRCC1 proteins. (A) Linear schematic of the XRCC1 protein. Indicated are the four major conserved elements of XRCC1: the N-terminal domain (NTD), the nuclear localization signals (NLS), the BRCT1 and the BRCT2 domains. Numbers represent the amino acid positions within the XRCC1 polypeptide. The locations of the amino acid substitutions are indicated above the diagram. The regions of XRCC1 that interact with specified protein partners are denoted. (B) Nickel column affinity purified XRCC1 proteins. C-terminal His-tagged, N-terminal S-peptide-tagged XRCC1 recombinant proteins expressed in bacteria were purified using nickel chromatography. Shown is an image of the purified proteins separated in a 12% SDS–polyacrylamide gel and stained with coomassie blue. (C) Nickel and S-protein affinity purified XRCC1 proteins. Following nickel purification, dual-tagged XRCC1 recombinant proteins were affinity captured using an S-protein matrix. Shown is an image of silver stained purified proteins after separation in a 12% SDS–polyacrylamide gel. Arrows indicate XRCC1. STD = protein standards (in kDa).
Mentions: The various site-specific XRCC1 variant proteins generated for this study were characterized for one of the following reasons (summarized in Table 2): (i) they had been previously shown or argued as disruptive of a specific function and therefore would serve as a control (V86R, in the POLβ interaction; R109A, in the POLβ interaction and/or in DNA binding) (8,9,26); (ii) they were identified as the principal defect responsible for the repair-deficiencies associated with the CHO cell lines EM-C12 and EM-C11 (e.g. E102K and C390Y, respectively, or E98K and C389Y in humans; see protein alignment in Supplementary Figure S1) (24); or (iii) they were found among the normal healthy human population yet may represent impaired-function, disease susceptibility factors (P161L, R194W, R280H, R399Q and Y576S; Table 3) (27). Since these amino acid substitutions spanned the length of the XRCC1 protein (Figure 1A), they might represent separation-of-function mutants defective in only a single (or a limited number of) interaction(s).Table 2.

Bottom Line: 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 POLbeta, PARP-1, LIG3alpha, PCNA or DNA.One common population variant (R280H) had no pronounced effect on the interactions with POLbeta, PARP-1, LIG3alpha 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.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Molecular Gerontology, National Institute on Aging, NIH, Baltimore, MD 21224, USA.

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 POLbeta, but did not disrupt the interactions with PARP-1, LIG3alpha and PCNA; and an E98K substitution, identified in EM-C12, reduced protein integrity, marginally destabilized the POLbeta 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 POLbeta, PARP-1, LIG3alpha, PCNA or DNA. One common population variant (R280H) had no pronounced effect on the interactions with POLbeta, PARP-1, LIG3alpha 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 POLbeta-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.

Show MeSH
Related in: MedlinePlus