Mutational analysis of 26 residues of vaccinia DNA topoisomerase identifies Ser-204 as important for DNA binding and cleavage Journal Article
| Authors: | Wang, L. K.; Wittschieben, J.; Shuman, S. |
| Article Title: | 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 down 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 thr 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. |
| Keywords: | sequence; gene; camptothecin; resistance; sites; duplex dna; covalent; sv40 dna; strand cleavage; i cleavage |
| Journal Title: | Biochemistry |
| Volume: | 36 |
| Issue: | 26 |
| ISSN: | 0006-2960 |
| Publisher: | American Chemical Society |
| Date Published: | 1997-07-01 |
| Start Page: | 7944 |
| End Page: | 7950 |
| Language: | English |
| ACCESSION: | WOS:A1997XH66900002 |
| DOI: | 10.1021/bi970498q |
| PROVIDER: | wos |
| PUBMED: | 9201940 |
| Notes: | Article -- Source: Wos |
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