Repair of gaps in retroviral DNA integration intermediates

Abstract

Diverse mobile DNA elements are believed to pirate host cell enzymes to complete DNA transfer. Prominent examples are provided by retroviral cDNA integration and transposon insertion. These reactions initially involve the attachment of each element 3' DNA end to staggered sites in the host DNA by element-encoded integrase or transposase enzymes. Unfolding of such intermediates yields DNA gaps at each junction. It has been widely assumed that host DNA repair enzymes complete attachment of the remaining DNA ends, but the enzymes involved have not been identified for any system. We have synthesized DNA substrates containing the expected gap and 5' two-base flap structure present in retroviral integration intermediates and tested candidate enzymes for the ability to support repair in vitro. We find three required activities, two of which can be satisfied by multiple enzymes. These are a polymerase (polymerase beta, polymerase delta and its cofactor PCNA, or reverse transcriptase), a nuclease (flap endonuclease), and a ligase (ligase I, III, or IV and its cofactor XRCC4). A proposed pathway involving retroviral integrase and reverse transcriptase did not carry out repair under the conditions tested. In addition, prebinding of integrase protein to gapped DNA inhibited repair reactions, indicating that gap repair in vivo may require active disassembly of the integrase complex.

FIG. 1

Candidate pathways for repair of integration intermediates. The action of integrase protein joins one DNA strand of the viral cDNA to target DNA at each cDNA end. Unfolding of this intermediate yields DNA gaps at each host-virus DNA junction, as shown at the top of each panel. The viral cDNA is shown by the darker line. 5′ DNA ends are shown by filled circles; hash marks indicate unpaired DNA bases. (A) Potential pathway for repair employing a polymerase, a 5′-to-3′ nuclease, and a ligase; (B) potential pathway for repair employing RT and integrase (12).

FIG. 2

Repair of model gapped integration intermediates by Pol beta, FEN, and ligase I. The model gapped integration intermediate substrate is composed of three DNA oligonucleotides annealed to form a five-base gap and two-base 5′ flap (see Table 1 for sequences). The sequence to the right of the gap as drawn matches 27 nucleotides of HIV-1 cDNA sequence; sequence to the left of the HIV sequence is chosen to model target DNA. Addition of proteins or nucleotides is indicated by +. Reaction conditions are as described in Materials and Methods. Reaction products were denatured, separated by SDS-PAGE and visualized by phosphorimaging. ³²P labels were attached to the substrates on the 5′ or 3′ end as indicated by the asterisks in the diagram beside each gel. (A) Repair of the 5′-labeled gapped substrate. Diagrammed markers indicate the unreacted substrate, the substrate extended by five nucleotides to the end of the gap, and the full-length product. (B) Repair of the 3′-labeled gapped substrate. Diagrammed markers indicate the beginning substrate, the substrate shortened by two nucleotides, and the full-length product.

FIG. 3

Gap repair by Pol delta, PCNA, FEN, and ligase I. Repair reactions were performed with either Pol delta (lanes 2 to 4) or Pol beta (lanes 5 to 7). PCNA was added at 20 ng (lanes 3 and 6) and 800 ng (lanes 4 and 7). Labeling is as in Fig. 2. (A) Repair of 5′-labeled gapped substrate; (B) repair of 3′-labeled gapped substrate.

FIG. 4

Gap repair by RT, FEN, and ligase I. Repair reactions were performed as described for Fig. 2 except using HIV RT to support polymerization. Labeling is as in Fig. 2. (A) Repair of 5′-labeled gapped substrate; (B) repair of 3′-labeled gapped substrate.

FIG. 5

Ligases I, III, and IV/XRCC4 all support gap repair. Repair reactions were performed with either Pol beta (lanes 2 to 4) or HIV RT (lanes 5 to 7) and ligase I (lanes 2 and 5), ligase III (lanes 3 and 6), and ligase IV/XRCC4 (lanes 4 and 7). Labeling is as in Fig. 2. (A) Repair of 5′-labeled gapped substrate; (B) repair of 3′-labeled gapped substrate. Percent conversion values for lanes 2 to 7 were, respectively, 25, 36, 53, 2, 2, and 3%.

FIG. 6

Inhibition of gap repair by Ku70/80. Repair reactions were conducted in the presence of either ligase I (lanes 2 to 5) or ligase IV (lanes 6 to 9). The Ku70/80 heterodimer was added in increasing concentrations of 100 pg (lanes 3 and 7), 1 ng (lanes 4 and 8), and 10 ng (lanes 5 and 9). Labeling is as in Fig. 2; each panel contains lanes from the same autoradiogram. (A) Repair of 5′-labeled gapped substrate; (B) repair of 3′-labeled gapped substrate. Percent conversion values for lanes 2 to 9 were, respectively, 44, 40, 36, 2, 48, 57, 53, and 18%.

FIG. 7

Lack of gap repair by HIV-1 RT and integrase. Reactions were carried out in the presence of HIV RT only (lanes 2 to 6) or in the presence of both HIV RT and HIV integrase (lanes 7 to 11). Labeling is as in Fig. 2. (A) Repair of 5′-labeled gapped substrate. (B) repair of 3′-labeled gapped substrate.

FIG. 8

Blocking of polymerase access to DNA gaps by added integrase. (A) A four-arm substrate was synthesized with two arms matching RSV cDNA sequences (bold lines) and two arms of mixed sequence recapitulating target DNA (thin lines). Two separate molecules with complementary gap sequences were studied to permit assembly of the four-armed structure. Addition of polymerase and a labeled nucleotide (asterisks) results in incorporation of radioactive nucleotides in the gap. Addition of integrase, in contrast, can potentially block access. (B) Polymerization on the RSV gapped dumbbell substrate. Labeled DNA products indicates incorporation of ³²P-labeled dCTPs in the dumbbell substrate by Pol beta. RSV integrase was preincubated for 10 min with the substrate in increasing amounts; lanes 2 to 6 contained 0.1, 1, 10, 50, and 100 ng, respectively. BSA was similarly preincubated with the substrate in lanes 7 to 11 in increasing amounts (0.1, 1, 10, 50, and 100 ng, respectively). The preincubation was followed by the addition of Pol beta and further incubation for 10 min. The amounts of relative incorporation in lanes 2 to 6, respectively, were 100, 108, 34, 15, and 2%. The amounts of relative incorporation in lanes 7 to 11, respectively, were 100, 107, 91, 321, and 108%. (C) Polymerization on a 5′-labeled nicked substrate. The diagrams at the left indicate the unreacted nicked substrate and a strand displacement synthesis product. Reaction compositions were the same as for panel B except for the DNA substrate. The relative percent conversion values for lanes 2 to 6 were, respectively, 100, 113, 115, 87, and 58%. The relative percent conversion values for lanes 7 to 11 were, respectively, 100, 88, 152, 127, and 126%.