CHARACTERIZATION OF RECOMBINANT MURINE LEUKEMIA-VIRUS INTEGRASE

Retroviral integration involves two DNA substrates that play different roles. The viral DNA substrate is recognized by virtue of specific nu cleotide sequences near the end of a double-stranded DNA molecule. The target DNA substrate is recognized at internal sites with little sequ ence preference; nucleosomal DNA appears to be preferred for this role . Despite this apparent asymmetry in the sequence, structure, and role s of the DNA substrates in the integration reaction, the existence of distinct binding sites for viral and target DNA substrates has been co ntroversial. In this report, we describe the expression in Escherichia coli and purification of Moloney murine leukemia virus integrase as a fusion protein with glutathione S-transferase, characterization of it s activity by using several model DNA substrates, and the initial kine tic characterization of its interactions with a model viral DNA substr ate. We provide evidence for functionally and kinetically distinct bin ding sites for viral and target DNA substrates and describe a cross-li nking assay for DNA binding at a site whose specificity is consistent with the target DNA binding site.