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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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Relocalization of XRCC1 proteins to and from sites of laser-induced DNA damage. (A) Redistribution kinetics of WT and the XRCC1 C389Y variant. Shown are still images taken at the indicated time point (in seconds) after prebleach. The box denotes the region of laser-induced DNA damage, while the arrow indicates the first point and location at which clear ‘stripe’ formation is observed. (B) Retention kinetics of WT and R280H XRCC1 proteins. After laser-induced DNA damage induction, peak stripe was observed at 5 min. Shown is the loss of signal at 15, 30 and 45 min post-irradiation. Each protein (denoted) was analyzed five times and the data presented are mean intensity values obtained in a given experiment following of substraction of the prebleach background.
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Figure 6: Relocalization of XRCC1 proteins to and from sites of laser-induced DNA damage. (A) Redistribution kinetics of WT and the XRCC1 C389Y variant. Shown are still images taken at the indicated time point (in seconds) after prebleach. The box denotes the region of laser-induced DNA damage, while the arrow indicates the first point and location at which clear ‘stripe’ formation is observed. (B) Retention kinetics of WT and R280H XRCC1 proteins. After laser-induced DNA damage induction, peak stripe was observed at 5 min. Shown is the loss of signal at 15, 30 and 45 min post-irradiation. Each protein (denoted) was analyzed five times and the data presented are mean intensity values obtained in a given experiment following of substraction of the prebleach background.

Mentions: Since XRCC1 interacts with several protein partners during the response to DNA SSBs (6), we reasoned that examination of the relocalization kinetics of the XRCC1 variants would uncover any deleterious effect of a particular amino acid substitution on protein coordination events that take place in vivo. Specifically, the redistribution dynamics of the XRCC1 variants to/from site-specifically, laser-induced DNA damage under parameters that generated DNA single-, but not double-strand breaks, were determined (see ‘Materials and methods’ section). For relocalization to the sites of DNA damage, each of the variant proteins displayed WT kinetics, appearing at the laser-targeted region ∼10–15 s (Supplementary Figure S4), except for E98K, which never formed observable stripes (data not shown), presumably due to its abnormal localization pattern (Figure 5), and C389Y, which also never formed detectable stripes (Figure 6A), likely due to its reduced protein stability and possibly as a consequence of a defective interaction with PARP-1 (see site of substitution in Figure 1A).Figure 6.


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)

Relocalization of XRCC1 proteins to and from sites of laser-induced DNA damage. (A) Redistribution kinetics of WT and the XRCC1 C389Y variant. Shown are still images taken at the indicated time point (in seconds) after prebleach. The box denotes the region of laser-induced DNA damage, while the arrow indicates the first point and location at which clear ‘stripe’ formation is observed. (B) Retention kinetics of WT and R280H XRCC1 proteins. After laser-induced DNA damage induction, peak stripe was observed at 5 min. Shown is the loss of signal at 15, 30 and 45 min post-irradiation. Each protein (denoted) was analyzed five times and the data presented are mean intensity values obtained in a given experiment following of substraction of the prebleach background.
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Figure 6: Relocalization of XRCC1 proteins to and from sites of laser-induced DNA damage. (A) Redistribution kinetics of WT and the XRCC1 C389Y variant. Shown are still images taken at the indicated time point (in seconds) after prebleach. The box denotes the region of laser-induced DNA damage, while the arrow indicates the first point and location at which clear ‘stripe’ formation is observed. (B) Retention kinetics of WT and R280H XRCC1 proteins. After laser-induced DNA damage induction, peak stripe was observed at 5 min. Shown is the loss of signal at 15, 30 and 45 min post-irradiation. Each protein (denoted) was analyzed five times and the data presented are mean intensity values obtained in a given experiment following of substraction of the prebleach background.
Mentions: Since XRCC1 interacts with several protein partners during the response to DNA SSBs (6), we reasoned that examination of the relocalization kinetics of the XRCC1 variants would uncover any deleterious effect of a particular amino acid substitution on protein coordination events that take place in vivo. Specifically, the redistribution dynamics of the XRCC1 variants to/from site-specifically, laser-induced DNA damage under parameters that generated DNA single-, but not double-strand breaks, were determined (see ‘Materials and methods’ section). For relocalization to the sites of DNA damage, each of the variant proteins displayed WT kinetics, appearing at the laser-targeted region ∼10–15 s (Supplementary Figure S4), except for E98K, which never formed observable stripes (data not shown), presumably due to its abnormal localization pattern (Figure 5), and C389Y, which also never formed detectable stripes (Figure 6A), likely due to its reduced protein stability and possibly as a consequence of a defective interaction with PARP-1 (see site of substitution in Figure 1A).Figure 6.

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