Residues critical for retroviral integrative recombination in a region that is highly conserved among retroviral/retrotransposon integrases and bacterial insertion sequence transposases.

摘要: pombe, reveals strong conservation ofaconstellation ofaminoacids characterized bytwoinvariant aspartate (D)residues anda glutamate (E)residue, which we refer toastheD,D(35)E region. Thesame constellation isfoundinthetransposases of anumberofbacterial insertion sequences.Theconservation ofthis region suggests that thecomponent residues areinvolved inDNA recognition, cutting, andjoining, since these properties areshared among proteins ofdivergent origin. We introduced aminoacidsubstitutions ininvariant andselected conserved and nonconserved throughout theD,D(35)E ofRoussarcoma virus INandinhumanimmunodeficiency INandassessed their effect upon theactivities ofthepurified, mutantproteins invitro. Changes oftheinvariant andconserved typically produce similar impairment ofbothviral longterminal repeat (LTR)oligonucleotide cleavage referred toas theprocessing reaction andthesubsequent joining ofthe processed LTR-based oligonucleotides toDNA targets. Theseverity ofthedefects depended thesite thenatureoftheaminoacidsubstitution(s). Allsubstitutions acidic D andEresidues inboth Roussarcoma virusandhumanimmunodeficiency virusINdramatically reduced LTR oligonucleotide processing andjoining toa fewpercent orless ofwildtype,suggesting thatthey areessential components theactive site forbothreactions. Onthebasis ofsimilarities withenzymes thatcatalyze analogous reactions, proposethattheinvariant andE may participate incoordination ofthemetalcofactor (Mn2+ orMg2')required forthecatalytic activities ofIN.We further speculate a metal-DNA complex be necessarytoposition bothLTR andtarget DNA substrates fornucleophilic attack during thecleavage reactions.