DNA hydrolytic activity associated with the Ustilago maydis REC1 gene product analyzed on hairpin oligonucleotide substrates.
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.
Naureckiene
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Hearst Microbiology Research Center, Department of Microbiology and Immunology, Cornell University Weill Medical College, New York 10021, USA.
Holloman
W K
WK
D001080:Apurinic Acid / Q000378:metabolism; D020137:Base Pair Mismatch; D001483:Base Sequence; D004270:DNA, Circular / Q000378:metabolism; D004271:DNA, Fungal / Q000378:metabolism*; D043211:Exodeoxyribonuclease V; D005090:Exodeoxyribonucleases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006868:Hydrolysis; D007700:Kinetics; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D009691:Nucleic Acid Denaturation; D009841:Oligonucleotides / Q000378:metabolism*; D013329:Structure-Activity Relationship; D013379:Substrate Specificity; D014588:Ustilago / Q000201:enzymology* / Q000235:genetics
D004270:DNA, Circular; D004271:DNA, Fungal; D009841:Oligonucleotides; D001080:Apurinic Acid; D005090:Exodeoxyribonucleases; D043211:Exodeoxyribonuclease V