The roles of arginine 41 and tyrosine 76 in the coupling of DNA recognition to phosphodiester bond cleavage by DNase I: a study using site-directed mutagenesis.
Bovine pancreatic deoxyribonuclease I is an endonuclease of low specificity that interacts with the minor groove of DNA. Two amino acids, R41 and Y76, completely fill this groove, with R41 hydrogen bonding to the O2/N3 positions of pyrimidines and purines, and Y76 contacting a deoxyribose via an unusual hydrophobic "stacking" interaction. The roles of these amino acids in phosphodiester bond cleavage and in DNA hydrolysis selectivity have been studied by site-directed mutagenesis. Alterations have been made that are either conservative (R41K, Y76F) or more drastic (R41A, R41G, Y76A, Y76G). The surface loop (residues 73 to 76) that contains Y76 has also been deleted. Several double mutants in which both R41 and Y76 have been altered have also been prepared. The integrity of the catalytic site of the mutants has been investigated using the small, non-DNA, chromophoric substrate deoxythymidine-3',5'-di-(p-nitrophenyl)-phosphate. Hydrolysis of this compound was hardly changed, even by the most extreme alterations to R41 and Y76. In contrast, all the mutants bound DNA about ten times more weakly than the wild-type and, with the exception of R41K and Y76F, hydrolysed DNA much more slowly. This suggests that changes to R41 and Y76 have little effect on catalytic amino acids at the hydrolysis site, but are required to bind DNA and, more importantly, to correctly position the scissile phosphate for efficient hydrolysis. The selectivity of DNA hydrolysis for all the mutants has been tested using the 160 base-pair Escherichia coli Tyr T promoter DNA fragment. Very small differences were seen in global hydrolysis selectivity when either amino acid was altered. However, changes to R41 resulted in some differences to local cutting specificity that could be explained by the role of this amino acid in hydrogen bonding to particular bases relative to the scissile phosphate.
Doherty
A J
AJ
Department of Biochemistry, University of Southampton, UK.
Worrall
A F
AF
Connolly
B A
BA
D000818:Animals; D001120:Arginine / Q000378:metabolism*; D001483:Base Sequence; D002417:Cattle; D004247:DNA / Q000378:metabolism*; D003850:Deoxyribonuclease I / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics; D006860:Hydrogen Bonding; D006868:Hydrolysis; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides / Q000378:metabolism; D010727:Phosphoric Diester Hydrolases / Q000378:metabolism; D011401:Promoter Regions, Genetic / Q000235:genetics; D013379:Substrate Specificity; D013938:Thymidine Monophosphate / Q000031:analogs & derivatives / Q000378:metabolism; D014443:Tyrosine / Q000378:metabolism*
D009838:Oligodeoxyribonucleotides; D013938:Thymidine Monophosphate; D014443:Tyrosine; D004247:DNA; D001120:Arginine; D003850:Deoxyribonuclease I; D010727:Phosphoric Diester Hydrolases