HIV-1 integrase can process a 3'-end crosslinked substrate.
Integrase of the human immunodeficiency virus type-1 (HIV-1) recognizes specific sequences located in the U3 and U5 regions at the ends of viral DNA. We synthesized DNA duplexes mimicking the U5 region and containing either 2'-aminonucleosides or non-nucleoside 1,3-propanediol insertions at the third and terminal positions and studied their interactions with HIV-1 integrase. Both modifications introduced a local structural distortion in the DNA double helix. Replacement of the terminal nucleosides by corresponding 2'-aminonucleosides had no significant effect on integrase activity. We used an integrase substrate bearing terminal 2'-aminonucleosides in both strands to synthesize a duplex with cross-linked strands. This duplex was then used to determine whether terminal base pair disruption is an obligatory step of retroviral DNA 3'-processing. Processing of the cross-linked analog of the integrase substrate yielded a product of the same length as 3'-processing of the wild-type substrate but the reaction efficiency was lower. Replacement of the third adenosine in the processed strand by a corresponding 2'-aminonucleoside did not affect integrase activity, whereas, its replacement by 1,3-propanediol completely inhibited 3'-processing. Both modifications of the complementary thymidine in the nonprocessed strand increased the initial rate of 3'-processing. The same effect was observed when both nucleosides, at the third position, were replaced by corresponding 2'-aminonucleosides. This indicates that the local duplex distortion facilitated the cleavage of the phosphodiester bond. Thus, a localized destabilization of the third A-T base pair is necessary for efficient 3'-processing, whereas 3'-end-fraying is important but not absolutely required.
Agapkina
Julia
J
Institute of the Chemical Physics Problems, Russian Academy of Sciences, Chernogolovka, Russia.
Smolov
Maksim
M
Zubin
Evgeni
E
Mouscadet
Jean-François
JF
Gottikh
Marina
M
D020029:Base Pairing; D001483:Base Sequence; D003432:Cross-Linking Reagents; D019427:HIV Integrase / Q000378:metabolism*; D007700:Kinetics; D009838:Oligodeoxyribonucleotides / Q000737:chemistry / Q000378:metabolism; D013379:Substrate Specificity
D003432:Cross-Linking Reagents; D009838:Oligodeoxyribonucleotides; D019427:HIV Integrase