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. 2003 Jul;77(13):7425-33.
doi: 10.1128/jvi.77.13.7425-7433.2003.

The herpes simplex virus type 1 alkaline nuclease and single-stranded DNA binding protein mediate strand exchange in vitro

Nina Bacher Reuven  1 , Amy E Staire, Richard S Myers, Sandra K Weller
Affiliations

The herpes simplex virus type 1 alkaline nuclease and single-stranded DNA binding protein mediate strand exchange in vitro

Nina Bacher Reuven et al. J Virol. 2003 Jul.

Abstract

The replication of herpes simplex virus type 1 (HSV-1) DNA is associated with a high degree of homologous recombination. While cellular enzymes may take part in mediating this recombination, we present evidence for an HSV-1-encoded recombinase activity. HSV-1 alkaline nuclease, encoded by the UL12 gene, is a 5'-->3' exonuclease that shares homology with Redalpha, commonly known as lambda exonuclease, an exonuclease required for homologous recombination by bacteriophage lambda. The HSV-1 single-stranded DNA binding protein ICP8 is an essential protein for HSV DNA replication and possesses single-stranded DNA annealing activities like the Redbeta synaptase component of the phage lambda recombinase. Here we show that UL12 and ICP8 work together to effect strand exchange much like the Red system of lambda. Purified UL12 protein and ICP8 mediated the complete exchange between a 7.25-kb M13mp18 linear double-stranded DNA molecule and circular single-stranded M13 DNA, forming a gapped circle and a displaced strand as final products. The optimal conditions for strand exchange were 1 mM MgCl(2), 40 mM NaCl, and pH 7.5. Stoichiometric amounts of ICP8 were required, and strand exchange did not depend on the nature of the double-stranded end. Nuclease-defective UL12 could not support this reaction. These data suggest that diverse DNA viruses appear to utilize an evolutionarily conserved recombination mechanism.

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Figures

FIG. 1.
FIG. 1.
Model for strand exchange by UL12 and ICP8. A schematic representation of the strand exchange reaction is presented. Sigma, alpha, and gapped circle forms represent strand exchange products at different stages of the reaction. The asterisk marks the internal 32P label, which is 2 kb from the 5′ end of the displaced strand and 5.25 kb from the 5′ end of the pairing strand.
FIG. 2.
FIG. 2.
Time course of joint molecule formation catalyzed by UL12 and ICP8. Strand exchange reactions were carried out using the 32P-labeled linear M13 dsDNA and unlabeled circular M13 ssDNA substrates as described in Materials and Methods. Left panel: phosphorimager image of dried gel. Right panel: photograph of ethidium bromide-stained gel. Incubations were at 37°C for the times indicated. Lane 1, control reaction, no proteins added; lane 2, strand exchange with ICP8 alone; lane 3, strand exchange with UL12 alone; lanes 4 to 12, strand exchange by UL12 and ICP8. jm, joint molecules; ds, linear M13 dsDNA; ss, circular M13 ssDNA.
FIG. 3.
FIG. 3.
Analysis of strand exchange products. Both panels represent phosphorimager images of dried gels. Left panel, strand exchange was performed as described in Materials and Methods with 32P-labeled dsDNA and unlabeled ssDNA substrates. Lane 1, control reaction, no proteins added, 40-min incubation; lanes 2 and 3, strand exchange with ICP8 and UL12, 20- and 40-min time points, respectively. Lanes 4 to 7 represent various DNA-only controls. DNAs (double stranded only in lanes 4 to 5, both double stranded and single stranded in lanes 6 to 7) were boiled for 2 min in strand exchange buffer and either quickly cooled on ice (lanes 4 and 6) or slowly cooled to allow strands to reanneal (lanes 5 and 7). Right panel, strand exchange reactions were performed as in the left panel (lanes 1 to 3) but were electrophoresed on a 1% low-melting-point agarose gel. Three gel slices were cut from each lane: A, containing joint molecules; B, containing remaining double-stranded substrate; and C, containing low-molecular-weight products. The positions of A, B, and C gel slices are indicated on the left panel. The gel slices were melted at 65°C and divided into two portions. One portion was loaded directly into the well of a second 1% agarose gel (shown in right panel). The other portion was boiled for 2 min prior to loading. Electrophoresis was performed as for the strand exchange assay. jm, joint molecules; nc, nicked circle; ds, dsDNA; ss, ssDNA.
FIG. 4.
FIG. 4.
Southern blot of strand exchange reaction. Unlabeled dsDNA and ssDNA were used in strand exchange reactions as described in Materials and Methods and incubated for the times indicated. The gel was loaded with duplicate samples originating from the same reaction tubes (except for lanes 7 to 8), and the DNA was blotted onto a GeneScreen Plus membrane according to the manufacturer's protocol. The membrane was cut into two, and each half was hybridized with its respective oligonucleotide probe. jm, joint molecules. The arrow marks the position of the putative displaced strand.
FIG. 5.
FIG. 5.
Strand exchange and UL12 nuclease activity at different conditions of [Mg2+], [Na+], and pH. Strand exchange and nuclease assays were performed as described in Materials and Methods under the various conditions shown. Open circles, strand exchange; closed squares, nuclease activity. Unless indicated otherwise, the conditions were pH 7.5, 1 mM MgCl2, and 40 mM NaCl. Strand exchange assay mixtures were incubated for 20 min. Percent strand exchange was calculated as the percentage of radioactivity in joint molecule products out of the total radioactivity in the lane. The buffers used were HEPES-NaOH, pH 6.5 and 7.0; Tris-Cl, pH 7.5, 8.0, and 8.5; and glycine-NaOH, pH 9.0 and 9.5. The nuclease activity of UL12 was assayed by using the [3H]DNA E. coli substrate and is represented as the amount of DNA (in nanograms) digested by 47 ng of UL12 (13.9 nM) in a 10-min assay at 37°C.
FIG. 6.
FIG. 6.
Titration of ssDNA and ICP8 in the strand exchange assay. Strand exchange reactions were performed as described in Materials and Methods, with 20-min incubations. UL12 and dsDNA were added according to standard conditions while the amounts of ssDNA and ICP8 used are indicated on the figure. A photograph of the ethidium bromide-stained gel is shown. Lanes 1 to 2 are no-protein controls. jm, joint molecules; ds, dsDNA; ss, ssDNA.
FIG. 7.
FIG. 7.
Strand exchange by UL12, UL12D340E, and ICP8. A photograph of the ethidium bromide-stained gel is shown. Strand exchange was carried out as described in Materials and Methods, with 4.5 μg of ICP8, 18.8 ng of UL12, and 20 ng of UL12D340E, as indicated. Reaction mixtures were incubated at 37°C for the times indicated.
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