CHARACTERIZATION OF THE DNA-BINDING ACTIVITY OF HIV-1 INTEGRASE USINGA FILTER BINDING ASSAY

Based on the selective binding of proteins and DNA to distinct filter materials a double-layered dot blot radio assay was developed to evalu ate the binding of DNA to HIV-1 integrase. In this assay the DNA-bindi ng was found to be independent of Mn2+ concentration, inhibited by con centrations of Mg2+ above 5 mM, abolished by zinc chelation and inhibi ted by monoclonal antibodies reacting with either the N-terminal or C- terminal regions of integrase. Atomic absorption spectroscopy revealed the molar ratio between integrase and zinc to be close to 1. It is co ncluded that both the N-terminal and the C-terminal regions of integra se are involved in DNA-binding and that the reported double-layered do t blot radio assay is well suited for further characterization of the integrase. (C) 1995 Academic Press, Inc.