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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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Intracellular distribution of XRCC1 variants. The indicated XRCC1 YFP-tagged fusion protein constructs were transiently transfected into HeLa cells, and fluorescent protein visualized by 491 nm GFP laser. The inset highlights a WT expressing cell with punctated nuclear staining. The arrows indicate cells where E98K is concentrated in the nucleolus, while the open arrow head denotes a cell with broad nuclear staining and strong punctated foci. The closed arrow heads point out cells that express C389Y at low levels.
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Figure 5: Intracellular distribution of XRCC1 variants. The indicated XRCC1 YFP-tagged fusion protein constructs were transiently transfected into HeLa cells, and fluorescent protein visualized by 491 nm GFP laser. The inset highlights a WT expressing cell with punctated nuclear staining. The arrows indicate cells where E98K is concentrated in the nucleolus, while the open arrow head denotes a cell with broad nuclear staining and strong punctated foci. The closed arrow heads point out cells that express C389Y at low levels.

Mentions: YFP-tagged XRCC1 expression constructs, for each of the variants outlined in Table 2, were created to determine protein localization and functional capacity in human cells. Specifically, the intracellular localization and the damage-induced redistribution kinetics of each XRCC1 protein after transient transfection into HeLa cells were evaluated. As shown in Figure 5, under normal cell culture conditions, other than mutant E98K, each of the XRCC1 variants appeared similar to WT in that the protein resided largely in the nucleoplasm, exhibiting in some instances a punctated foci pattern that was found previously to reflect S-phase cells and active replication factories (e.g. see WT, inset) (14), and was excluded from the nucleolus. C389Y, although showing a sub-cellular distribution similar to WT, exhibited a reduced fluorescent signal intensity in many of the cells (Figure 5, solid arrow heads), likely indicative of the protein instability observed in bacteria (Figure 1). As for E98K, this mutant displayed an irregular distribution pattern in which the protein was sequestered primarily in the nucleolus in most cells (Figure 5, arrows), although in some instances, the mutant exhibited sharp punctated staining in the nucleoplasm (Figure 5, open arrow head).Figure 5.


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)

Intracellular distribution of XRCC1 variants. The indicated XRCC1 YFP-tagged fusion protein constructs were transiently transfected into HeLa cells, and fluorescent protein visualized by 491 nm GFP laser. The inset highlights a WT expressing cell with punctated nuclear staining. The arrows indicate cells where E98K is concentrated in the nucleolus, while the open arrow head denotes a cell with broad nuclear staining and strong punctated foci. The closed arrow heads point out cells that express C389Y at low levels.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

Figure 5: Intracellular distribution of XRCC1 variants. The indicated XRCC1 YFP-tagged fusion protein constructs were transiently transfected into HeLa cells, and fluorescent protein visualized by 491 nm GFP laser. The inset highlights a WT expressing cell with punctated nuclear staining. The arrows indicate cells where E98K is concentrated in the nucleolus, while the open arrow head denotes a cell with broad nuclear staining and strong punctated foci. The closed arrow heads point out cells that express C389Y at low levels.
Mentions: YFP-tagged XRCC1 expression constructs, for each of the variants outlined in Table 2, were created to determine protein localization and functional capacity in human cells. Specifically, the intracellular localization and the damage-induced redistribution kinetics of each XRCC1 protein after transient transfection into HeLa cells were evaluated. As shown in Figure 5, under normal cell culture conditions, other than mutant E98K, each of the XRCC1 variants appeared similar to WT in that the protein resided largely in the nucleoplasm, exhibiting in some instances a punctated foci pattern that was found previously to reflect S-phase cells and active replication factories (e.g. see WT, inset) (14), and was excluded from the nucleolus. C389Y, although showing a sub-cellular distribution similar to WT, exhibited a reduced fluorescent signal intensity in many of the cells (Figure 5, solid arrow heads), likely indicative of the protein instability observed in bacteria (Figure 1). As for E98K, this mutant displayed an irregular distribution pattern in which the protein was sequestered primarily in the nucleolus in most cells (Figure 5, arrows), although in some instances, the mutant exhibited sharp punctated staining in the nucleoplasm (Figure 5, open arrow head).Figure 5.

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