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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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Relative affinities of XRCC1 proteins for PARP-1 (A), LIG3α (B) and PCNA (C). Shown are the average and standard deviation of three independent affinity capture experiments. See Figure 2, as well as text, for further details. Values represent the resulting signal ratio, with WT being arbitrarily set to 100. Standard deviation of the values for the WT-binding reactions is shown as well.
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Figure 3: Relative affinities of XRCC1 proteins for PARP-1 (A), LIG3α (B) and PCNA (C). Shown are the average and standard deviation of three independent affinity capture experiments. See Figure 2, as well as text, for further details. Values represent the resulting signal ratio, with WT being arbitrarily set to 100. Standard deviation of the values for the WT-binding reactions is shown as well.

Mentions: As the above experiments with DNA POLβ validated the use of the affinity capture technique to assess relative binding affinity [see for instance, ref. (26)], similar interaction studies using the purified protein partners PARP-1, LIG3α and PCNA, which span the various functional regions of the XRCC1 polypeptide, were performed (Figure 1A). As shown in Figure 3A, ADP-ribosylated PARP-1 interacted with all of the XRCC1 variant proteins with similar affinity as WT. We note that ADP-ribosylation of PARP-1 enhanced the interaction with WT XRCC1 (Supplementary Figure S2), as described previously (7,33). As with PARP-1, no interaction deficiency for any of the XRCC1 variants was observed with DNA LIG3α (Figure 3B). Given that the association of XRCC1 with PCNA appears 5-fold weaker than its association with the other proteins examined herein, we used WB analysis to improve the detection of captured PCNA. These studies revealed that none of the variant XRCC1 proteins exhibited a statistically significant defect in their affinity for PCNA (Figure 3C).Figure 3.


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)

Relative affinities of XRCC1 proteins for PARP-1 (A), LIG3α (B) and PCNA (C). Shown are the average and standard deviation of three independent affinity capture experiments. See Figure 2, as well as text, for further details. Values represent the resulting signal ratio, with WT being arbitrarily set to 100. Standard deviation of the values for the WT-binding reactions is shown as well.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2926592&req=5

Figure 3: Relative affinities of XRCC1 proteins for PARP-1 (A), LIG3α (B) and PCNA (C). Shown are the average and standard deviation of three independent affinity capture experiments. See Figure 2, as well as text, for further details. Values represent the resulting signal ratio, with WT being arbitrarily set to 100. Standard deviation of the values for the WT-binding reactions is shown as well.
Mentions: As the above experiments with DNA POLβ validated the use of the affinity capture technique to assess relative binding affinity [see for instance, ref. (26)], similar interaction studies using the purified protein partners PARP-1, LIG3α and PCNA, which span the various functional regions of the XRCC1 polypeptide, were performed (Figure 1A). As shown in Figure 3A, ADP-ribosylated PARP-1 interacted with all of the XRCC1 variant proteins with similar affinity as WT. We note that ADP-ribosylation of PARP-1 enhanced the interaction with WT XRCC1 (Supplementary Figure S2), as described previously (7,33). As with PARP-1, no interaction deficiency for any of the XRCC1 variants was observed with DNA LIG3α (Figure 3B). Given that the association of XRCC1 with PCNA appears 5-fold weaker than its association with the other proteins examined herein, we used WB analysis to improve the detection of captured PCNA. These studies revealed that none of the variant XRCC1 proteins exhibited a statistically significant defect in their affinity for PCNA (Figure 3C).Figure 3.

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