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Binding and repair of mismatched DNA mediated by Rhp14, the fission yeast homologue of human XPA


Hohl, M; Christensen, O; Kunz, C; Naegeli, H; Fleck, O (2001). Binding and repair of mismatched DNA mediated by Rhp14, the fission yeast homologue of human XPA. Journal of Biological Chemistry, 276(33):30766-30772.

Abstract

Rhp14 of Schizosaccharomyces pombe is homologous to human XPA and Saccharomyces cerevisiae Rad14, which act in nucleotide excision repair of DNA damages induced by ultraviolet light and chemical agents. Cells with disrupted rhp14 were highly sensitive to ultraviolet light, and epistasis analysis with swi10 (nucleotide excision repair) and rad2 (Uve1-dependent ultraviolet light damage repair pathway) revealed that Rhp14 is an important component of nucleotide excision repair for ultraviolet light-induced damages. Moreover, defective rhp14 caused instability of a GT repeat, similar to swi10 and synergistically with msh2 and exo1. Recombinant Rhp14 with an N-terminal hexahistidine tag was purified from Escherichia coli. Complementation studies with a rhp14 mutant demonstrated that the tagged Rhp14 is functional in repair of ultraviolet radiation-induced damages and in mitotic mutation avoidance. In bandshift assays, Rhp14 showed a preference to substrates with mismatched and unpaired nucleotides. Similarly, XPA bound more efficiently to C/C, A/C, and T/C mismatches than to homoduplex DNA. Our data show that mismatches and loops in DNA are substrates of nucleotide excision repair. Rhp14 is likely part of the recognition complex but alone is not sufficient for the high discrimination of nucleotide excision repair for modified DNA.

Abstract

Rhp14 of Schizosaccharomyces pombe is homologous to human XPA and Saccharomyces cerevisiae Rad14, which act in nucleotide excision repair of DNA damages induced by ultraviolet light and chemical agents. Cells with disrupted rhp14 were highly sensitive to ultraviolet light, and epistasis analysis with swi10 (nucleotide excision repair) and rad2 (Uve1-dependent ultraviolet light damage repair pathway) revealed that Rhp14 is an important component of nucleotide excision repair for ultraviolet light-induced damages. Moreover, defective rhp14 caused instability of a GT repeat, similar to swi10 and synergistically with msh2 and exo1. Recombinant Rhp14 with an N-terminal hexahistidine tag was purified from Escherichia coli. Complementation studies with a rhp14 mutant demonstrated that the tagged Rhp14 is functional in repair of ultraviolet radiation-induced damages and in mitotic mutation avoidance. In bandshift assays, Rhp14 showed a preference to substrates with mismatched and unpaired nucleotides. Similarly, XPA bound more efficiently to C/C, A/C, and T/C mismatches than to homoduplex DNA. Our data show that mismatches and loops in DNA are substrates of nucleotide excision repair. Rhp14 is likely part of the recognition complex but alone is not sufficient for the high discrimination of nucleotide excision repair for modified DNA.

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Item Type:Journal Article, refereed, original work
Communities & Collections:05 Vetsuisse Faculty > Institute of Veterinary Pharmacology and Toxicology
Dewey Decimal Classification:570 Life sciences; biology
Language:English
Date:2001
Deposited On:09 Nov 2010 15:15
Last Modified:07 Dec 2017 03:20
Publisher:American Society for Biochemistry and Molecular Biology
ISSN:0021-9258
Additional Information:This research was originally published in: Hohl, M; Christensen, O; Kunz, C; Naegeli, H; Fleck, O (2001). Binding and repair of mismatched DNA mediated by Rhp14, the fission yeast homologue of human XPA. Journal of Biological Chemistry, 276(33):30766-30772. © the American Society for Biochemistry and Molecular Biology.
Free access at:Publisher DOI. An embargo period may apply.
Publisher DOI:https://doi.org/10.1074/jbc.M104039200
PubMed ID:11408483

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