Reconstitution of recombination-dependent DNA synthesis in herpes simplex virus 1

Abstract

The repair of double-strand DNA breaks by homologous recombination is essential for the maintenance of genome stability. In herpes simplex virus 1, double-strand DNA breaks may arise as a consequence of replication fork collapse at sites of oxidative damage, which is known to be induced upon viral infection. Double-strand DNA breaks are also generated by cleavage of viral a sequences by endonuclease G during genome isomerization. We have reconstituted a system using purified proteins in which strand invasion is coupled with DNA synthesis. In this system, the viral single-strand DNA-binding protein promotes assimilation of single-stranded DNA into a homologous supercoiled plasmid, resulting in the formation of a displacement loop. The 3' terminus of the invading DNA serves as a primer for long-chain DNA synthesis promoted by the viral DNA replication proteins, including the polymerase and helicase-primase. Efficient extension of the invading primer also requires a DNA-relaxing enzyme (eukaryotic topoisomerase I or DNA gyrase). The viral polymerase by itself is insufficient for DNA synthesis, and a DNA-relaxing enzyme cannot substitute for the viral helicase-primase. The viral single-strand DNA-binding protein, in addition to its role in the invasion process, is also required for long-chain DNA synthesis. Form X, a topologically distinct, positively supercoiled form of displacement-loop, does not serve as a template for DNA synthesis. These observations support a model in which recombination and replication contribute toward maintaining viral genomic stability by repairing double-strand breaks. They also account for the extensive branching observed during viral replication in vivo.

Fig. 1.

Recombination-dependent DNA synthesis. D-loops were formed, and the ability of the invading strand to prime DNA synthesis was examined as described in Materials and Methods. (A) Schematic depiction of the band pattern obtained by resolving the CR by 2D native-denaturing agarose gel electrophoresis. The arrows indicate the direction of migration in each dimension. The species are shaded to distinguish between primer extension originating from D-loops (black) and nicked D-loops (gray). Shown are storage phosphor images of first (Upper) and second (Lower) dimensions for each. (B) CR with gyrase. (C) CR without dNTP. (D) CR without UL30/UL42 (Pol). (E) Primer extension with UL30/UL42 (Pol) alone. (F) CR without UL5/UL52/UL8 (H/P). (G) CR without gyrase/topoisomerase I. (H) CR with topoisomerase I instead of gyrase. The positions of D-loops (D), products (P), nicked D-loops (N), 100-mer, full-length extension products (2,686 nt), intermediates, and markers (HindIII-digested λ DNA) are as indicated.

Fig. 2.

ICP8 is required for full-length DNA synthesis. Reactions were performed as a function of ICP8 concentration with purified D-loops as described in Materials and Methods, followed by 1D denaturing agarose gel electrophoresis. (A) Storage phosphor image. Lane 1, mock-treated D-loops; lanes 2-7, extension of D-loops with 0, 150, 300, 600, 900, and 1,300 nM ICP8, respectively. (B) Quantitation of full-length synthesis products. The positions of 100-mer, full-length extension products (2,686 nt), intermediates, and markers (HindIII-digested λ DNA) are as indicated.

Fig. 3.

Kinetics of recombination-dependent DNA synthesis. Reactions were performed as a function of time with purified D-loops as described in Materials and Methods, followed by 1D denaturing agarose gel electrophoresis. (A) Storage phosphor image. Lanes 1-7: 0, 2, 5, 10, 15, 30, and 60 min, respectively. (B) Quantitation of full-length synthesis products (open circles) and total extension products (filled circles). The positions of 100-mer, full-length extension products (2,686 nt), intermediates, and markers (HindIII-digested λ DNA) are as indicated.

Fig. 4.

Form X is not a substrate for DNA synthesis. D-loops were formed, and the ability of the invading strand to prime DNA synthesis was examined as described in Materials and Methods. Reaction products were resolved by 2D native chloroquine-denaturing agarose gel electrophoresis. Storage phosphor images of first (Upper) and second (Lower) dimensions. The positions of D-loops (D), form X (X), products (P), nicked D-loops (N), 100-mer, full-length extension products (2,686 nt), intermediates, and markers (HindIII-digested λ DNA) are as indicated.

Fig. 5.

Model for recombination-dependent replication in HSV-1. (Upper) The assimilation of a ssDNA (red arrow) into a homologous duplex (black circle) by ICP8. (Lower) The detailed events that culminate in the assembly of a replication fork after ICP8 (yellow circles)-mediated strand assimilation. The three ovals (orange, green, and blue) represent the heterotrimeric helicaseprimase (UL5/UL52/UL8). The cylinder and attached semicircle represent the polymerase (UL30) and its processivity factor (UL42), respectively. The pair of scissors represents the action of a relaxing enzyme. The solid and dashed lines represent DNA and RNA synthesis, respectively. The 3′ primer end is depicted by an arrowhead.