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J. Biol. Chem., Vol. 275, Issue 50, 39287-39295, December 15, 2000
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From the Model oligodeoxyribonucleotide substrates
representing viral DNA integration intermediates with a gap and a
two-nucleotide 5' overhang were used to examine late steps in
human immunodeficiency virus, type 1 (HIV-1) retroviral
integrase (IN)-catalyzed DNA integration in vitro. HIV-1 or
avian myeloblastosis virus reverse transcriptase (RT) were capable of
quantitatively filling in the gap to create a nicked substrate but did
not remove the 5' overhang. HIV-1 IN also failed to remove the 5'
overhang with the gapped substrate. However, with a nicked substrate
formed by RT, HIV-1 IN removed the overhang and covalently closed the
nick in a disintegration-like reaction. The efficiency of this closure
reaction was very low. Such closure was not stimulated by the addition
of HMG-(I/Y), suggesting that this protein only acts during the early
processing and joining reactions. Addition of Flap endonuclease-1, a
nuclease known to remove 5' overhangs, abolished the closure reaction
catalyzed by IN. A series of base pair inversions, introduced into the
HIV-1 U5 long terminal repeat sequence adjacent to and/or including the
conserved CA dinucleotide, produced no or only a small decrease in the
HIV-1 IN-dependent strand closure reaction. These same mutations caused a significant decrease in the efficiency of concerted DNA integration by a modified donor DNA in vitro,
suggesting that recognition of the ends of the long terminal repeat
sequence is required only in the early steps of DNA integration.
Finally, a combination of HIV-1 RT, Flap endonuclease-1, and DNA ligase is capable of quantitatively forming covalently closed DNA with these
model substrates. These results support the hypothesis that cellular
enzyme(s) may catalyze the late steps of retroviral DNA integration.
Modeling the Late Steps in HIV-1 Retroviral
Integrase-catalyzed DNA Integration*
,¶
Department of Microbiology and Immunology,
Northwestern University School of Medicine, Chicago, Illinois 60611 and
the § Fox Chase Cancer Center,
Philadelphia, Pennsylvania 19111
*
This work was supported in part by United States Public
Health Service Grants CA38046 (to J. L.) and CA49042, CA06927, and RR05539 (to A. M. S.).The costs of publication of this
article were defrayed in part by the
payment of page charges. The article must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
Copyright © 2000 by The American Society for Biochemistry and Molecular Biology, Inc.
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