Deletions at the carboxyl terminus of vaccinia DNA topoisomerase affect DNA binding and enhance distributivity in DNA relaxation Journal Article
| Authors: | Wang, L. K.; Shuman, S. |
| Article Title: | Deletions at the carboxyl terminus of vaccinia DNA topoisomerase affect DNA binding and enhance distributivity in DNA relaxation |
| Abstract: | Vaccinia topoisomerase relaxes DNA through the formation of a covalent DNA-(3'-phosphotyrosyl)protein intermediate at sites containing the sequence 5'-(T/C)CCTT↓. The active site, Tyr-274, is situated near the carboxyl terminus of the 314 amino acid enzyme. Here, we report the effects of serial C-terminal deletions. Removal of five amino acids had no effect on topoisomerase activity. However, deletion of 10, 15, or 20 amino acids rendered the enzyme distributive in DNA relaxation, incrementally slowed the rate of single-turnover DNA cleavage, and progressively diminished DNA binding affinity, without altering the sequence specificity of DNA cleavage. These effects lead us to speculate that the region downstream of the active site, which is not well-conserved among the poxvirus-encoded topoisomerases, is a component of the proposed circumferential interface between the enzyme and duplex DNA. |
| Keywords: | protein expression; mutation; dna-binding proteins; sequence deletion; nonhuman; polymerase chain reaction; carboxy terminal sequence; enzyme activity; dna; amino acid sequence; molecular sequence data; kinetics; escherichia coli; substrate specificity; vaccinia virus; mutagenesis, site-directed; binding sites; nucleic acid conformation; dna binding; enzyme specificity; sodium chloride; deletion mutant; dna cleavage; electrophoresis, polyacrylamide gel; dna topoisomerase; dna topoisomerases, type i; enzyme active site; dna denaturation; magnesium; priority journal; article |
| Journal Title: | Biochemistry |
| Volume: | 36 |
| Issue: | 13 |
| ISSN: | 0006-2960 |
| Publisher: | American Chemical Society |
| Date Published: | 1997-04-01 |
| Start Page: | 3909 |
| End Page: | 3916 |
| Language: | English |
| DOI: | 10.1021/bi962754p |
| PUBMED: | 9092821 |
| PROVIDER: | scopus |
| DOI/URL: | |
| Notes: | Article -- Export Date: 17 March 2017 -- Source: Scopus |
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