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DNA hydrolytic activity associated with the Ustilago maydis REC1 gene product analyzed on hairpin oligonucleotide substrates.

TitleDNA hydrolytic activity associated with the Ustilago maydis REC1 gene product analyzed on hairpin oligonucleotide substrates.
Publication TypeJournal Article
Year of Publication1999
AuthorsNaureckiene S, Holloman WK
JournalBiochemistry
Volume38
Issue43
Pagination14379-86
Date Published1999 Oct 26
ISSN0006-2960
KeywordsApurinic Acid, Base Pair Mismatch, Base Sequence, DNA, Circular, DNA, Fungal, Exodeoxyribonuclease V, Exodeoxyribonucleases, Hydrolysis, Kinetics, Molecular Sequence Data, Nucleic Acid Conformation, Nucleic Acid Denaturation, Oligonucleotides, Structure-Activity Relationship, Substrate Specificity, Ustilago
Abstract

The REC1 gene of Ustilago maydis functions in the maintenance of genome stability as evidenced by the mutator phenotype resulting from inactivation of the gene. The biochemical function of the Rec1 protein was previously identified as a 3'-5'-directed DNA exonuclease. Here studies on the mechanism of action of Rec1 were performed using radiolabeled oligonucleotide DNAs as substrates, enabling detection of single cleavage events after electrophoresis on DNA sequencing gels. The oligonucleotides that were utilized were designed to be self-annealing so that they formed hairpin structures. This simplified interpretation of the data since each molecule contained only one 3'-terminus. Analysis revealed that digestion proceeded by a distributive mode of action and that degradation of DNA was governed by an interplay between sequence context and conformation. The preferential substrate was DNA with a recessed 3'-end. It was discovered that the enzyme had abasic endonuclease activity, was capable of initiating at an internal nick, and had no preference for mismatched bases either internally or terminally. Endonucleolytic cleavage was 5' to the abasic site.

Alternate JournalBiochemistry
PubMed ID10572012
Grant ListGM42482 / GM / NIGMS NIH HHS / United States

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