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 specif icity that interacts with the minor groove of DNA. Two amino acids, R4 1 and Y76, completely fill this groove, with R41 hydrogen bonding to t he O-2/N-3 positions of pyrimidines and purines, and Y76 contacting a deoxyribose via an unusual hydrophobic ''stacking'' interaction. The r oles of these amino acids in phosphodiester bond cleavage and in DNA h ydrolysis selectivity have been studied by site-directed mutagenesis. Alterations have been made that are either conservative (R41K, Y76F) o r more drastic (R41A, R41G, Y76A, Y76G). The surface loop (residues 73 to 76) that contains Y76 has also been deleted. Several double mutant s in which both R41 and Y76 have been altered have also been prepared. The integrity of the catalytic site of the mutants has been investiga ted using the small, non-DNA, chromophoric substrate deoxythymidine-3' ,5'-di-(p-nitrophenyl)-phosphate. Hydrolysis of this compound was hard ly changed, even by the most extreme alterations to R41 and Y76. In co ntrast, all the mutants bound DNA about ten times more weakly than the wild-type and, with the exception of R41K and Y76F, hydrolysed DNA mu ch more slowly. This suggests that changes to R41 and Y76 have little effect on catalytic amino acids at the hydrolysis site, but are requir ed to bind DNA and, more importantly, to correctly position the scissi le phosphate for efficient hydrolysis. The selectivity of DNA hydrolys is for all the mutants has been tested using the 160 base-pair Escheri chia coli Tyr T promoter DNA fragment. Very small differences were see n 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 phospha te.