TY1 IN-VITRO INTEGRATION - EFFECTS OF MUTATIONS IN CIS AND IN TRANS

Mutations within the TYB gene of Ty1 encoding integrase (IN) as,well a s alterations in its substrate, a linear DNA molecule, were examined f or their effects on in vitro IN activity, using a recently developed p hysical assay. Five different codon-insertion mutations, two frameshif t mutations, and one missense mutation, previously identified as trans position-deficient mutations, were tested. Virus-like particles, the s ource of IN, from two different protease mutants and a reverse transcr iptase mutant exhibited near-normal to normal IN activity. Two framesh ift mutations mapping within the phylogenetically variable C-terminal domain of IN resulted in significant in vitro IN activity. In contrast , three mutations within the amino-terminal conserved domain of IN com pletely abolished IN activity. When the substrate termini were mutated , we found that substrates with as few as 4 bp of Ty1 termini were cap able of efficiently generating integration products. Surprisingly, cer tain substrates that lacked obvious similarity to Ty1 termini mere als o readily integrated into both linear and circular targets, whereas ot hers were not used as substrates at all. Termini rich in adenosine res idues were among the more active substrates; however, certain substrat es lacking terminal adenosine residues can form small quantities of in tegration products, including complete integration reactions.