RETROVIRAL INTEGRASE - CONSERVED SEQUENCE INFORMATION AS A GUIDE TO UNDERSTANDING STRUCTURE AND FUNCTION OF THE RETROVIRAL AND BACTERIAL IS3 DD(35)E TRANSPOSASES

The retroelement integrases (IN) are one of the three enzymes required for the retroviral life cycle. Much attention has been focused on ret roviruses as a result of the AIDS epidemic and IN became a target for the rational development of anti-AIDS drug therapies. However two thin gs that would help this effort, the mechanism of the chemical reaction catalysed by IN and the structure were initially unknown. This review shows how sequence alignments helped design and interpret experiments which showed that the conserved DDE residues were catalytically requi red, and which showed the catalytic function of three acidic residues in a motif here called the DD(35)E motif, in turn leading to the solut ion of its structure by xray crystallography; and finding the DNA bind ing function of the C-terminus in turn leading to the solution of its structure by NMR. Sequence alignments showed that IN belonged to a gre ater family of transposases found in retroelements and IS3 bacterial i nsertion sequences, whose function was to transpose a piece of dsDNA w ith the consensus sequence CA at the 3'-ends. IN was composed of three separate motifs. The central region of about 110 residues which very similar in sequence and which shared 3 conserved acidic groups (D,D an d E). The 2nd D and the E were always separated by 35 residues, and to gether with the first D, were used to name this family the DD(35)E tra nsposases. It was proposed that the DD(35)E region was an autonomously folding domain; had the catalytic site which recognised the CA 3'-end s; and that these 3 residues were involved in DNA bond breaking/making and divalent metal ion binding. The N-terminal part of IN contained a conserved HHCC motif, presumably a Zn finger motif, which was not fou nd in the other DD(35)E transposases. Aside from the HHCC residues, th is part of the enzymes did not align well suggesting that if the struc ture was conserved in the family, then the binding of the Zn atom woul d have to be the overriding factor. The C-terminus was very variable n ot able to be aligned within the DD(35)E transposase family, except fo r very closely related enzymes. The C-terminus for another transposase of similar size (gamma-delta res, Abdel-Meguid et al., Proc. Nat. Aca d. Sci. USA 81, (1984) 2001-2005) was known to bind DNA. However no DN A binding motifs such as helix-turn-helix were found by computer assis ted searches in the C-terminus of the IN family and no function was ab le to be assigned to it. It seemed likely that IN was a 3 domain prote in and it was reasonable;to explore the function of IN in domains in t he same manner as had be pioneered with res. (C) 1998 Elsevier Science B.V.