Retroviral Integrases Promote Fraying of Viral DNA Ends

Abstract

In the initial step of integration, retroviral integrase (IN) introduces precise nicks in the degenerate, short inverted repeats at the ends of linear viral DNA. The scissile phosphodiester bond is located immediately 3 of a highly conserved CA/GT dinucleotide, usually 2 bp from the ends. These nicks create new recessed 3 -OH viral DNA ends that are required for joining to host cell DNA. Previous studies have indicated that unpairing, "fraying," of the viral DNA ends by IN contributes to end recognition or catalysis. Here, we report that end fraying can be detected independently of catalysis with both avian sarcoma virus ( Within hours after infection of a host cell, a colinear DNA copy of the retroviral RNA genome becomes integrated into the host chromosome, through a highly coordinated series of enzymatic events. The steps are carried out by the virus-encoded reverse transcriptase, which makes a double-stranded copy of the viral RNA genome, and the viral integrase (IN), 2 which catalyzes the integration of this newly synthesized DNA into the DNA of the host cell. Following integration, an essential step for virus replication, the viral DNA is permanently associated with the genome of the host cell and all daughters and serves as a template for viral gene transcription and the production of progeny virus.The underlying chemical steps in retroviral DNA integration are generally understood (1). The reaction can be divided into two steps, which can be reproduced in vitro using purified IN and model DNA substrates. (i) The nuclease activity of IN introduces nicks, 3Ј of the highly conserved CA dinucleotide, usually 2 bp from the DNA terminus, in a "processing" step that releases a dinucleotide and generates new, recessed 3Ј-OH ends. (ii) In the second step, these newly exposed 3Ј-oxygen atoms are joined to staggered backbone phosphates on opposite strands of the host DNA, via concerted cleavage and ligation. Because the target phosphates are staggered, this "joining" step leaves gaps in the opposite strands of host DNA, and host mechanisms are required to repair the gaps and complete the covalent joining (for review, see Ref.2). These combined viral and host-catalyzed enzymatic steps produce the characteristic sequence signatures of retroviral integration: loss of the terminal 2 bp from the linear viral DNA ends and the duplication, depending on the virus, of 4 -6 bp of host DNA flanking the integration site.Substrate Recognition by IN Does Not Depend on Sequence Alone-Sequences at the tips of the blunt-ended retroviral DNA (10 -20 bp) are sufficient for IN recognition, processing, and joining. However, the mechanism by which such substrates are recognized is not completely understood, and it has been difficult to detect sequence-specific binding to viral DNA in vitro (3). Sequence identity of the generally imperfect terminal inverted repeats that are embedded in the U3 and U5 regions of the long terminal repeat (LTR) at the ends of each viral DNA can range from 16/18 for murine leukemia virus, to 12/18 for avian s...

Retroviral Integrase: Then and Now

“…Such an association would be consistent with the observation that end fraying by ASLV IN occurs in cis with the active site and, though detectable with isolated CCDs, is most efficient with fragments that contain both the CCD and CTD (85). As a dimer appears to be the minimal form able to process vDNA ends (73), full fraying and end processing may require formation of an inner dimer-like structure in which each NTD makes contact with the DNA held by the other inner protomer in trans .…”

“…There are no clues to the function of the outer protomers, of which only the CCDs were resolved. As predicted from earlier studies (84, 85), the last three base pairs of the vDNA ends do not remain double stranded in the active sites of the complex; rather, each is distorted and partially unwound. The frayed 5′-PO 4 end of the noncleaved vDNA strand is threaded between the CCD and CTD, and the strand with the 3′-OH end is positioned in the active site for cleavage.…”

Retroviral DNA Transposition: Themes and Variations

“…9A), appear to participate in catalysis. As predicted from earlier studies (96) there is a reciprocal ( trans ) arrangement in the inner dimer of the crystal structure: Each viral DNA end is bound by the two terminal domains of one monomer (with tips frayed (97, 98) via interaction of the 5'-ends with the CTD), but processed in the CCD of the second monomer. Removal of the two nucleotides at the 3'-ends in the processing reaction allows binding of a bent target DNA, and the concerted joining of the 3'-ends of viral DNA to both strands of the target, five base pairs apart.…”

DNA-Based Sensor for Real-Time Measurement of the Enzymatic Activity of Human Topoisomerase I

“…These include measurements of helicase-, endonuclease- or repair activities as well as protein-DNA interactions and were achieved by ensemble or single-molecule fluorescence resonance energy transfer (FRET) between two fluorophores or by various fluorophore-quenching strategies [1–7]. Optical sensor systems allow investigation of enzymatic steps otherwise difficult to address using conventional methods as exemplified by the measurement of unpairing of viral DNA ends by retroviral integrases [2] or gate-DNA bending by human topoisomerase IIα [3]. Furthermore, sensors have been designed to allow easy real-time measurement of enzyme activity useful for prognostic, diagnostic or drug testing purposes [4,7].…”