Abstract:
:The human endonuclease III (hNTH1) is an important DNA glycosylase with associated abasic lyase activity. We previously demonstrated that the K212Q mutant was totally inactive, while the K212R mutant had reduced DNA glycosylase/lyase activity and could form a covalent complex with the substrate DNA upon reduction. We further characterized the biochemical properties of this K212R mutant protein. NH2- (N-) terminal sequencing in combination with mass spectrometry of the peptide-DNA adduct suggested that "opportunistic" lysine(s) in the lysine-rich N-terminal tail formed a Schiff base which might result in beta-elimination. However, simultaneous substitution of Lys-75 with Gln and deletion of first 72 residues in the N-terminal tail could not cause further alteration in the glycosylase reaction or beta-elimination event. Nonetheless, the time kinetics of K212R and its subsequent mutants showed glycosylase activity without any detectable AP-lyase activity during the first 10 min of the reaction. These results suggest that a single point mutation at the active site (K212R) uncoupled the glycosylase activity from the lyase activity. We propose that the uncoupled reaction carried out by K212R is a result of direct attack either by the nonionized form of the guanidino group of arginine which forms an unstable Schiff base that hydrolyzes prior to the beta-elimination event or by hydroxide ion to cleave the glycosylic bond. In either case this reaction is followed by a secondary beta-elimination event performed by random lysine residues primarily from the N-terminal tail region.
journal_name
Biochemistryjournal_title
Biochemistryauthors
Liu X,Roy Rdoi
10.1021/bi011053bsubject
Has Abstractpub_date
2001-11-13 00:00:00pages
13617-22issue
45eissn
0006-2960issn
1520-4995pii
bi011053bjournal_volume
40pub_type
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