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Mutational analysis of 26 residues of vaccinia DNA topoisomerase identifies Ser-204 as important for DNA binding and cleavage.

Abstract：

:Vaccinia DNA topoisomerase, a 314 amino acid type I enzyme, catalyzes the cleavage and rejoining of DNA strands through a DNA-(3'-phosphotyrosyl)-enzyme intermediate formed at a specific target sequence, 5'-(C/T)CCTT downward arrow. To identify amino acids that participate in the DNA binding and transesterification steps, we introduced alanine substitutions at 18 positions within a centrally located 27 amino acid segment (181-RLYKPLLKLTDDSSPEEFLFNKLSERK-207) and at 8 positions near the N-terminus (1-MRALFYKDGK-10). All mutant proteins except two displayed wild-type activity in relaxing supercoiled DNA. F200A and S204A exhibited reduced rates of relaxation and were subjected to a kinetic analysis of the strand cleavage reaction under single-turnover and equilibrium conditions. The F200A and S204A mutations reduced the rate of single-turnover DNA cleavage by factors of 5 and 70, respectively. Both mutations shifted the cleavage-religation equilibrium in favor of the noncovalently bound state. The S204A mutation reduced the affinity of topoisomerase for CCCTT-containing DNA, but did not alter the site-specificity of DNA cleavage. Vaccinia residue Ser-204, which is conserved in all poxvirus topoisomerases, but not in the cellular homologues, may contribute to the unique cleavage site specificity of the poxvirus enzymes. Phe-200 is conserved in all members of the type IB topoisomerase family.

journal_name

Biochemistry

journal_title

Biochemistry

authors

Wang LK,Wittschieben J,Shuman S

doi

10.1021/bi970498q

subject

Has Abstract

pub_date

1997-07-01 00:00:00

pages

7944-50

issue

26

eissn

0006-2960

issn

1520-4995

pii

bi970498q

journal_volume

36

pub_type

杂志文章

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