Base-pair substitutions in avian sarcoma virus U5 and U3 long terminal repeat sequences alter the process of DNA integration in vitro

Abstract

We have described a reconstituted avian sarcoma virus (ASV) concerted DNA integration system with specially designed mini-donor DNA containing a supF transcription unit, a supercoiled plasmid acceptor, purified bacterially expressed ASV integrase (IN), and human high-mobility-group protein I(Y). Integration in this system is dependent upon the mini-donor DNA having IN recognition sequences at both ends and upon both ends of the same donor integrating into the acceptor DNA. The integrated DNA product exhibits all of the features associated with integration of viral DNA in vivo (P. Hindmarsh et al., J. Virol., 73:2994-3003, 1999). Individual integrants are isolated from bacteria containing drug-resistant markers with amber mutations. This system was used to evaluate the importance of sequences in the terminal U5 and U3 long terminal repeats at positions 5 and/or 6, adjacent to the conserved CA dinucleotide. Base-pair substitutions introduced at these positions in U5 result in significant reductions in recovered integrants from bacteria, due to increases in one-ended insertion events. Among the recovered integrants from reactions with mutated U5 but not U3 IN recognition sequences were products that contain large deletions in the acceptor DNA. Base-pair substitutions at positions 5 and 6 in U3 mostly reduce the efficiency of integration of the modified donor. Together, these results indicate that sequences directly 5' to the conserved CA dinucleotide are very important for the process of concerted DNA integration. Furthermore, IN interacts with U3 and U5 termini differently, and aberrant end-processing events leading to nonconcerted DNA integration are more common in U5 than in U3.

FIG. 1

Reconstitution of ASV IN-dependent DNA integration with wild-type donor DNA. (A) A diagram of donor DNA showing the U3 (left) and (U5) LTR sequences is shown at the top. The highly conserved CA dinucleotide is underlined; the closed rectangle represents a supF tRNA transcription unit. The donor and acceptor DNAs were incubated on ice with IN and HMG proteins as described in Materials and Methods, and the integration reaction was initiated at 37°C with the addition of MgCl₂. Below are diagrammatic representations of concerted DNA integration products resulting from use of both LTR termini from a single donor (product a) and from use of different LTR termini from two donors (product b) and of nonconcerted integration products resulting from one-ended integration from a single donor (product c), using both ends from a single donor with insertion at different sites on the acceptor DNA (product d), and using one-ended integration from two donors at different sites on the acceptor DNA (product e). (B) Modified integration reaction conditions where the acceptor DNA is introduced into the assay after preincubation overnight at 4°C. (C) Gel electrophoresis analysis of integration products formed with a wild-type donor DNA. Positions of RF II and RF III forms of the acceptor DNA, the RF II and RF III products from integration of the wild-type donor into the acceptor DNA, and possible intermediates as shown in panel A are indicated. The radiolabel is in the donor DNA whose migration position is at the bottom of the gel.

FIG. 2

Gel electrophoresis analysis of integration products formed with wild-type and U5-substituted donor DNA. (A) Integration reactions under the standard conditions as described in the legend to Fig. 1A were carried out with 6 pmol of IN and 4 pmol of HMG-I(Y) with wild-type (WT) donor DNA (lane 1) or donor DNAs containing U5-5A6A (lane 2), U5-5A (lane 3), or U5-6A (lane 4) base-pair substitutions. Integration reactions were also carried out with 6 pmol of IN and 4 of pmol HMG-1 with wild-type donor DNA (lane 5) or donor DNAs containing U5-5AS6A (lane 6), U5-5A (lane 7), or U5-6A (lane 8) base-pair substitutions. (B) Integration reactions with (lanes 2, 4, and 6) or without (lanes 1, 3, and 5) HMG-I(Y) using a wild-type (WT) donor (lanes 1 and 2) or a donor DNA lacking the U3 IN recognition sequence but maintaining a wild-type U5 (lanes 3 and 4) or U5-5A6A (lanes 5 and 6) IN recognition sequence.

FIG. 3

Deletions introduced into the acceptor DNA by nonconcerted DNA integration of mutated donors. The solid horizontal lines represent deletions from individual integrants as depicted above or below the plasmid map. The plasmid acceptor DNA is as described in the legend to Fig. 4.

FIG. 4

Sites of concerted integration of wild-type and mutant donor ASV DNA. The locations and lengths of flanking duplications of plasmid DNA for integrants using wild-type (A) and mutant ASV donor DNAs with base pair substitutions U5-5A6A (B), U5-5A (C), U5-6A (D), U3-5T6A (E), and U3-6A (F) as described in Tables 2 to 5 are presented. Data for wild-type ASV are from Aiyar et al. (1) and Hindmarsh et al. (10). The plasmid acceptor data are drawn in a linear representation to scale. The genes of the plasmid and origin of replication (ori) are indicated by open boxes. MCS, multiple cloning site. The thick vertical lines represent numbers of nucleotides in the plasmid DNA. Each thin vertical line represents a separate sequenced integration event. Lengths of the thin vertical lines represent 4-, 5-, 6-, or 7-bp duplications of the acceptor DNA as indicated.

FIG. 5

Effects of U3 LTR base-pair substitutions on integration into acceptor DNAs. Integration reactions as described in the legend to Fig. 2 were stimulated by HMG-I(Y) (lanes 1 to 3 and 7 to 9) or HMG-1 (lanes 4 to 6). Lanes 1, 4, and 7, wild-type (WT) donor DNA. Donor DNAs contained U3-5T6A (lanes 2 and 5), U3-4G5T6A7G (lanes 3 and 6), U3-5T (lane 7), or U3-6A (lane 8) base pair substitutions.