The small size of endonuclease V from bacteriophage T4, combined with the relative complexity of the reactions it catalyzes, makes it an interesting and important DNA repair enzyme to be structurally characterized. Among the T-even bacteriophages, T4 has been shown to be approximately twice as resistant to ultraviolet light inactivation as T2 and T6. Luria, S. E. (1947) Reactivation of a Radiated Bacteriophage by Transfer of Self-reproducing Units, Proc. Natl. Acad. Sci. 33, 253-264. Endonuclease V is coded for by the den V gene. Endonuclease V has been shown by biochemical and genetic analysis to possess four distinctly separable activities: (1) a pyrimidine dimer-specific DNA binding activity; (2) a pyrimidine dimer N-glycosylase activity; (3) an apyrimidinic/apurinic (AP)-endonuclease activity which may be manifest at the site of the N-glycosylase event or which may incise DNA at the site of any missing base; and (4) a salt sensitive linear diffusion along double-stranded DNA.
Site-directed mutational studies of the carboxyl terminal region of the enzyme support the concept that this part of the enzyme is intimately involved in binding to DNA at the site of the pyrimidine dimer. This carboxy terminal region of endonuclease V is essential for pyrimidine dimer binding.
The endonuclease V enzyme has commercial potential for in vivo as well as in vitro studies and uses. Especially for in vitro studies, it is desirable to possess an altered endonuclease V enzyme having increased specific activity thereby requiring less enzyme to provide a predetermined specific activity.
The present invention provides a T4 endonuclease V repair enzyme having increased specific activity at low salt concentrations.