Document Detail

The DNA scanning mechanism of T4 endonuclease V. Effect of NaCl concentration on processive nicking activity.
MedLine Citation:
PMID:  3525529     Owner:  NLM     Status:  MEDLINE    
T4 endonuclease V is a pyrimidine dimer-specific endonuclease which generates incisions in DNA at the sites of pyrimidine dimers by a processive reaction mechanism. A model is presented in which the degree of processivity is directly related to the efficacy of the one-dimensional diffusion of endonuclease V on DNA by which the enzyme locates pyrimidine dimers. The modulation of the processive nicking activity of T4 endonuclease V on superhelical covalently closed circular DNA (form I) which contains pyrimidine dimers has been investigated as a function of the ionic strength of the reaction. Agarose gel electrophoresis was used to separate the three topological forms of the DNA which were generated in time course reactions of endonuclease V with dimer-containing form I DNA in the absence of NaCl, and in 25, 50, and 100 mM NaCl. The degree of processivity was evaluated in terms of the mass fraction of form III (linear) DNA which was produced as a function of the fraction of form I DNA remaining. Processivity is maximal in the absence of NaCl and decreases as the NaCl concentration is increased. At 100 mM NaCl, processivity is abolished and endonuclease V generates incisions in DNA at the site of dimers by a distributive reaction mechanism. The change from the distributive to a processive reaction mechanism occurs at NaCl concentrations slightly below 50 mM. The high degree of processivity which is observed in the absence of NaCl is reversible to the distributive mechanism, as demonstrated by experiments in which the NaCl concentration was increased during the time course reaction. In addition, unirradiated DNA inhibited the incision of irradiated DNA only at NaCl concentrations at which processivity was observed.
E A Gruskin; R S Lloyd
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Publication Detail:
Type:  Journal Article; Research Support, U.S. Gov't, P.H.S.    
Journal Detail:
Title:  The Journal of biological chemistry     Volume:  261     ISSN:  0021-9258     ISO Abbreviation:  J. Biol. Chem.     Publication Date:  1986 Jul 
Date Detail:
Created Date:  1986-09-17     Completed Date:  1986-09-17     Revised Date:  2012-07-04    
Medline Journal Info:
Nlm Unique ID:  2985121R     Medline TA:  J Biol Chem     Country:  UNITED STATES    
Other Details:
Languages:  eng     Pagination:  9607-13     Citation Subset:  IM    
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MeSH Terms
DNA / analysis*,  radiation effects
Endodeoxyribonucleases / metabolism*
Escherichia coli / enzymology
Osmolar Concentration
Sodium Chloride / pharmacology*
Time Factors
Ultraviolet Rays
Grant Support
Reg. No./Substance:
0/Polymers; 7647-14-5/Sodium Chloride; 9007-49-2/DNA; EC 3.1.-/Endodeoxyribonucleases; EC V

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