EFFICIENT GAP REPAIR CATALYZED IN-VITRO BY AN INTRINSIC DNA-POLYMERASE-ACTIVITY OF HUMAN-IMMUNODEFICIENCY-VIRUS TYPE-1 INTEGRASE

Cleavage and DNA joining reactions, carried out by human immunodeficie ncy virus type 1 (HIV-1) integrase, are necessary to effect the covale nt insertion of HIV-1 DNA into the host genome. For the integration of HIV-1 DNA into the cellular genome to be completed, short gaps flanki ng the integrated proviral DNA must be repaired, It has been widely as sumed that host cell DNA repair enzymes are involved, Here we report t hat HIV-1 integrase multimers possess an intrinsic DNA-dependent DNA p olymerase activity. The activity was characterized by its dependence o n Mg2+, resistance to N-ethylmaleimide, and inhibition by 3'-azido-2', 3'-dideoxythymidine-5'-triphosphate. coumermycin A(1), and pyridoxal 5 '-phosphate. The enzyme efficiently utilized poly(dA)-oligo(dT) or sel f-annealing oligonucleotides as a template primer but displayed relati vely low activity with gapped calf thymus DIVA and no activity with po ly(dA) or poly(rA)-oligo(dT). A monoclonal antibody binding specifical ly to an epitope comprised of amino acids 264 to 273 near the C termin us of HIV-1 integrase severely inhibited the DNA polymerase activity. A deletion of 50 amino acids at the C terminus of integrase drasticall y altered the gel filtration properties of the DNA polymerase, althoug h the level of activity was unaffected by this mutation, The DNA polym erase efficiently extended a hail,pin DNA primer up to Ib nucleotides on a T-20, DNA template, although addition of the last nucleotide occu rred infrequently or not at all, The ability of integrase to repair ga ps in DNA was also investigated. We designed a series of gapped molecu les containing a single-stranded region flanked by a duplex U5 viral a rm on one side and by a duplex nonviral arm on the other side, Molecul es varied structurally depending on the size of the Sap (one, two, fiv e, or seven nucleotides), their content of T's or C's in the single-st randed region, whether the CA dinucleotide in the vir al arm had been replaced with a nonviral sequence, or whether they contained 5' AC din ucleotides as unpaired tails, The results indicated that the integrase DNA polymerase is specifically designed to repair gaps efficiently an d completely, regardless of gap size, base composition, or structural features such as the internal CA dinucleotide or unpaired 5'-terminal AC dinucleotides. When the U5 arm of the gapped DNA substrate was remo ved, leaving a nongapped DNA template-primer, the integrase DNA polyme rase failed to repair the last nucleotide in the DNA template effectiv ely. A post-gap repair reaction did depend on the CA dinucleotide, Thi s secondary reaction was highly regulated, Only two nucleotides beyond the gap were synthesized, and these were complementary to and depende nt for their synthesis on the CA dinucleotide. We were also able to id entify a specific requirement for the C tel minus of integrase in the post gap repair reaction, The results are consistent with a direct rol e for a heretofore unsuspected DNA polymerase function of HIV-1 integr ase in the repair of short gaps flanking proviral DNA integration inte rmediates that arise during virus infection.