Site-directed deletion mutagenesis within the T4 endonuclease V gene: dispensable sequences within putative loop regions

M. L. Dodson, Melissa A. Prince, Wayne F. Anderson, R. Stephen Lloyd*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

4 Scopus citations

Abstract

Endonuclease V from bacteriophage T4 may be one of the first DNA-repair enzymes to have its three-dimensional structure determined by X-ray crystallography (Morikawa et al., 1988). However, since this structure is not yet available, analyses of the sequence of the protein were performed in order to guide site-directed mutational studies of enzyme structure-function relationships. The enzyme is predominantly α-helical, so that an algorithm which finds the locations of turns or loops in the structure would be expected to approximately locate the helices along the sequence. Two loop sites were identified which might be adjacent in the tertiary structure according to a model developed from the loop predictions and the derived secondary structure. Deletion of three residues at each loop site produced protein molecules which retained considerable in vitro enzyme activity and in vivo repair function. However, the mutant proteins did not accumulate as well within the cell as the wild-type enzyme, suggesting that the nascent molecules folded inefficiently. Combination of the two deletions yielded a molecule with activity enhanced over one of the individual mutants, a result which can be interpreted as a classic second-site mutational reversion. This result supports the hypothesis that these regions are adjacent in the enzyme tertiary structure.

Original languageEnglish (US)
Pages (from-to)19-29
Number of pages11
JournalMutation Research-DNA Repair
Volume255
Issue number1
DOIs
StatePublished - Jul 1991

Funding

We wish to express our genuine thanks to Dr. Robert Abarbanel for furnishing the implementation of the Cohen et al. algorithm used in this investigation, to the Howard Hughes Medical Institute, Vanderbilt University, for computer time, and to Dr. C.M. Kay of the University of Alberta for the use of his spectropolarimeter. We are also very grateful to Ms. Doris Harris for her careful preparation of this manuscript. This work was supported by ES 04091, ES 00267 and the Canadian Medical Research Council through the Group in Protein Structure and Function. RSL is the recipient of an ACS Faculty Research Award, FRA No. 381.

Keywords

  • DNA-repair enzymes
  • DenV gene
  • Endonuclease V
  • Pyrimidine dimers
  • Structure predictions

ASJC Scopus subject areas

  • Molecular Biology
  • Toxicology
  • Genetics

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