Proteolytic footprinting of vaccinia topoisomerase bound to DNA Journal Article
| Authors: | Sekiguchi, J.; Shuman, S. |
| Article Title: | Proteolytic footprinting of vaccinia topoisomerase bound to DNA |
| Abstract: | Vaccinia DNA topoisomerase, a member of the eukaryotic type I enzyme family, binds duplex DNA and forms a covalent protein. DNA complex at sites containing a conserved sequence element 5'-CCCTT ↓ . The structure of the enzyme in the free and DNA-bound states was probed by limited proteolysis. The free topoisomerase (a 314-amino acid polypeptide) consists of protease- resistant amino- and carboxyl-terminal structural domains flanking a protease-sensitive 'hinge.' The hinge region, located between residues 135 and 142, is defined by accessibility to three different proteases. The amino- terminal region is punctuated by a trypsin-sensitive 'bridge' at Arg-80, suggesting at least a tripartite domain structure overall. A specific subset of residues accessible to proteases in the free enzyme becomes resistant to proteolysis in the DNA-bound state. The trypsin-sensitive site at Arg-80 is protected almost completely in the covalent complex. Within the hinge region, Lys-135, Tyr-136, and Glu-139 are protected from trypsin, chymotrypsin, and V8, respectively. Acquisition of altered protease sensitivity upon DNA binding occurs prior to covalent adduct formation. The 20-kDa carboxyl domain by itself binds noncovalently to duplex DNA, albeit without the sequence specificity characteristic of the full-sized topoisomerase. |
| Keywords: | sequence analysis; nonhuman; comparative study; polymerase chain reaction; protein conformation; animal; carboxy terminal sequence; protein degradation; cloning, molecular; dna; double stranded dna; amino acid sequence; conserved sequence; molecular sequence data; amino terminal sequence; kinetics; peptide fragments; recombinant proteins; vaccinia virus; base sequence; dna flanking region; binding sites; conformational transition; sequence homology, nucleic acid; dna binding; enzyme structure; chymotrypsin; autoradiography; dna topoisomerase; dna topoisomerases, type i; oligodeoxyribonucleotides; vaccinia; dna protein complex; restriction mapping; human; priority journal; article; support, non-u.s. gov't; support, u.s. gov't, p.h.s. |
| Journal Title: | Journal of Biological Chemistry |
| Volume: | 270 |
| Issue: | 19 |
| ISSN: | 0021-9258 |
| Publisher: | American Society for Biochemistry and Molecular Biology |
| Date Published: | 1995-05-01 |
| Start Page: | 11636 |
| End Page: | 11645 |
| Language: | English |
| DOI: | 10.1074/jbc.270.19.11636 |
| PUBMED: | 7744804 |
| PROVIDER: | scopus |
| DOI/URL: | |
| Notes: | Article -- Export Date: 28 August 2018 -- Source: Scopus |
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