HIV-1 integrase catalyzes the insertion of the viral genome into chromosomal DNA. We characterized the structural determinants of the 3'-processing reaction specificity-the first reaction of the integration process-at the DNA-binding level. We found that the integrase N-terminal domain, containing a pseudo zinc-finger motif, plays a key role, at least indirectly, in the formation of specific integrase-DNA contacts. This motif mediates a cooperative DNA binding of integrase that occurs only with the cognate/viral DNA sequence and the physiologically relevant Mg(2+) cofactor. The DNA-binding was essentially non-cooperative with Mn(2+) or using non-specific/random sequences, regardless of the metallic cofactor. 2,2'-Dithiobisbenzamide-1 induced zinc ejection from integrase by covalently targeting the zinc-finger motif, and significantly decreased the Hill coefficient of the Mg(2+)-mediated integrase-DNA interaction, without affecting the overall affinity. Concomitantly, 2,2'-dithiobisbenzamide-1 severely impaired 3'-processing (IC(50) = 11-15 nM), suggesting that zinc ejection primarily perturbs the nature of the active integrase oligomer. A less specific and weaker catalytic effect of 2,2'-dithiobisbenzamide-1 is mediated by Cys 56 in the catalytic core and, notably, accounts for the weaker inhibition of the non-cooperative Mn(2+)-dependent 3'-processing. Our data show that the cooperative DNA-binding mode is strongly related to the sequence-specific DNA-binding, and depends on the simultaneous presence of the Mg(2+) cofactor and the zinc effector.