Abstract
Several DNA-interactive proteins, including the DNA repair enzyme T4 endonuclease V, have been shown to locate their target recognition sites utilizing an electrostatically mediated facilitated diffusion mechanism. Previous work indicates that a decrease in the affinity of endonuclease V for nontarget DNA results in an increased nontarget dissociation rate. This study was designed to investigate the effect of an increase in the affinity of endonuclease V for nontarget DNA. Using a working structural model of the enzyme as a guide, the electrostatic character of endonuclease V was altered. Substitution of Thr-7 with Lys-7 resulted in an enzyme with wild type in vitro characteristics. Mutations which increased the positive charge along a proposed solvent-exposed alpha-helical face had significant effects. The mutants Ala-30, Val-31----Lys-30, Leu-31 and Asn-37----Lys-37 displayed wild type in vitro apurinic-specific and dimer-specific nicking activities. Although the processive dimer-specific nicking rate of the Lys-37 mutant resembled that of wild type, the rate of the Lys-30, Leu-31 mutant was reduced by 60%. In addition, the salt concentration range over which these mutants processively nick dimer-containing DNA has been greatly expanded. Both mutants are shown to have an increased affinity for nontarget DNA.
Highlights
The protein scans or slides along DNAin a nonspecifically bound state
The protein is more tightly associated with nonspecific DNA, has areduced dissociation rate, and, is able to diffuse a great distance along the DNA strand
The apyrimidinic endonuclease activity cleaves the phosphodiester backbone between the two pyrimidines (Gordon and Haseltine, 1980; Radany and Friedberg, 1980; Seawell et al, 1980; McMillan et al, 1981; Nakabeppu and Sekiguchi, 1981; Warner et al, 1981;Nakabeppu et al, 1982).At monovalent salt concentrations less than 40mM, the enzyme slides on nontarget sequences, generating incisions in DNA at the sites of pyrimidine dimers by a processive nicking mechanism
Summary
Ated by 254 nm UV light at 100 pW/cmYfor 245 s in order to generate 20-25 pyrimidinedimers/plasmid molecule (Gruskinand Lloyd, Model BuildingProcedures-Comparative model building pro- 1986). The reaction sequence of endonuclease V was singly and simult~aneouslyaligned was stopped by the addition of an equal volume of electrophoresis with the amino acid sequences of a number of four-a-helix bundle loading buffer This analysis identified a-helical seg- HCl, pH 8.0, 20 mM EDTA, 0.01% bromphenol blue). The reaction ments of endonuclease V which are analogous to a-helical regions of products were subjected to electrophoresis througah1%(w/v) agarose several four-a-helix bundle proteins Utilizing this predictive infor- gel, stained with ethidium bromide in electrophoresis buffer consistmation, four structurally ideal endonuclease V a-helices were gener- ing of 40 mM sodium acetate and 2 mM EDTA, pH 8.0, and the ated using Biosym Technology's Insight molecular modeling software topological forms of DNA visualized by exposure to long-wave UV on a Silicon Graphics 4D/70GT system.
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