MUTATIONAL ANALYSIS OF HUMAN DNASE-I AT THE DNA-BINDING INTERFACE - IMPLICATIONS FOR DNA RECOGNITION, CATALYSIS, AND METAL-ION DEPENDENCE

Human deoxyribonuclease I(DNase I), an enzyme used to treat cystic fib rosis patients, has been systematically analyzed by site-directed muta genesis of residues at the DNA binding interface. Crystal structures o f bovine DNase I complexed with two different oligonucleotides have im plicated the participation of over 20 amino acids in catalysis or DNA recognition. These residues have been classified into four groups base d on the characterization of over 80 human DNase I variants. Mutations at any of the four catalytic amino acids His 134, His 252, Glu 78, an d Asp 212 drastically reduced the hydrolytic activity of DNase I. Repl acing the three putative divalent metal ion-coordinating residues Glu 39, Asp 168, or Asp 251 led to inactive variants. Amino acids Gln 9, A rg 41, Tyr 76, Arg 111, Asn 170, Tyr 175, and Tyr 211 were also critic al for activity, presumably because of their close proximity to the ac tive site, while more peripheral DNA interactions stemming from 13 oth er positions were of minimal significance. The relative importance of these 27 positions is consistent with evolutionary relationships among DNase I across different species, DNase I-like proteins, and bacteria l sphingomyelinases, suggesting a fingerprint for a family of DNase I- like proteins. Furthermore, we found no evidence for a second active s ite that had been previously implicated in Mn2+-dependent DNA degradat ion. Finally, we correlated our mutational analysis of human DNase I t o that of bovine DNase I with respect to their specific activity and d ependence on divalent metal ions.