Document Detail


DNA binding and cleavage by the fowlpox virus resolvase.
MedLine Citation:
PMID:  19004818     Owner:  NLM     Status:  MEDLINE    
Abstract/OtherAbstract:
The first steps of poxvirus DNA synthesis yield concatemeric arrays of covalently linked genomes. The virus-encoded Holliday junction resolvase is required to process concatemers into unit-length genomes for packaging. Previous studies of the vaccinia virus resolvase have been problematic due to poor protein solubility. We found that fowlpox virus resolvase was much more tractable. Fowlpox resolvase formed complexes with a variety of branched DNA substrates, but not linear DNA, and had the highest affinity for a Holliday junction substrate, illustrating a previously unappreciated affinity for Holliday junctions over other substrates. The cleavage activity was monitored in fixed time assays, showing that, as with vaccinia resolvase, the fowlpox enzyme could cleave a wide array of branched DNA substrates. Single turnover kinetic analysis revealed the Holliday junction substrate was cleaved 90-fold faster than a splayed duplex substrate containing a single to double strand transition. Multiple turnover kinetic analysis, however, showed that the cleavage step was not limiting for the full reaction cycle. Cleavage by resolvase was also tightly coupled at symmetrical positions across the junction, and coupling required the complete Holliday junction structure. Last, we found that cleavage of an extruded cruciform yielded a product, which after treatment with ligase, had the properties expected for covalently closed DNA hairpin ends, as is seen for poxvirus genome monomers. These findings provide a tractable poxvirus resolvase usable for the development of small molecule inhibitors.
Authors:
Matthew J Culyba; Young Hwang; Nana Minkah; Frederic D Bushman
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Publication Detail:
Type:  Journal Article; Research Support, N.I.H., Extramural     Date:  2008-11-12
Journal Detail:
Title:  The Journal of biological chemistry     Volume:  284     ISSN:  0021-9258     ISO Abbreviation:  J. Biol. Chem.     Publication Date:  2009 Jan 
Date Detail:
Created Date:  2009-01-05     Completed Date:  2009-03-06     Revised Date:  2014-04-03    
Medline Journal Info:
Nlm Unique ID:  2985121R     Medline TA:  J Biol Chem     Country:  United States    
Other Details:
Languages:  eng     Pagination:  1190-201     Citation Subset:  IM    
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MeSH Terms
Descriptor/Qualifier:
DNA, Viral / chemistry,  metabolism*
Fowlpox virus / enzymology*
Kinetics
Nucleic Acid Conformation
Plasmids / metabolism
Protein Binding
Recombinases / metabolism*
Substrate Specificity
Virus Replication
Grant Support
ID/Acronym/Agency:
T32 AI 07324/AI/NIAID NIH HHS; T32 AI007324/AI/NIAID NIH HHS; U54 AI057168/AI/NIAID NIH HHS
Chemical
Reg. No./Substance:
0/DNA, Viral; 0/Recombinases
Comments/Corrections

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