In Vitro Strand Exchange Promoted by the Herpes Simplex Virus Type-1 Single Strand DNA-binding Protein (ICP8) and DNA Helicase-Primase

Abstract

The genome of herpes simplex virus type-1 undergoes a high frequency of homologous recombination in the absence of a virus-encoded RecA-type protein. We hypothesized that viral homologous recombination is mediated by the combined action of the viral single strand DNA-binding protein (ICP8) and helicase-primase. Our results show that ICP8 catalyzes the formation of recombination intermediates (joint molecules) between circular single-stranded acceptor and linear duplex donor DNA. Joint molecules formed by invasion of a 3-terminal strand displaces the non-complementary 5-terminal strand, thereby creating a loading site for the helicaseprimase. Helicase-primase acts on these joint molecules to promote ATP-dependent branch migration. Finally, we have reconstituted strand exchange by the synchronous action of ICP8 and helicase-primase. Based on these data, we present a recombination mechanism for a eukaryotic DNA virus in which a single strand DNAbinding protein and helicase cooperate to promote homologous pairing and branch migration.Homologous recombination is a fundamental biological process. It serves, among other purposes, to repair DNA damage such as double strand DNA breaks or to reinitiate DNA replication at stalled replication forks. Herpes simplex virus (HSV) 1 undergoes a high frequency of homologous recombination during its replicative cycle. One of the earliest reports on HSV recombination dates to 1955; co-infection of chorioallantois with HSV strains of varying virulence led to recombinant progeny with altered virulence (1). Additional proof for recombination of the HSV genome comes from the observation that the subgenomic elements, U L and U S , undergo inversion with respect to each other, generating four possible isomeric forms (2-4). This genome isomerization appears to be a consequence of homologous recombination rather than of a site-specific event involving cleavage at the a sequences that reside in inverted orientations at the termini of U L and U S (5). Nevertheless, the a sequences may act as recombination hotspots. In this context, the a sequences are the sites of double-stranded (ds) DNA breaks created either by a structure-specific endonuclease 2 or during cleavage and packaging of the viral genome (6, 7). The terminal a sequences have also been shown to be involved in the circularization of the viral genome by homologous recombination (8). This process occurs immediately after infection by the virus and appears to be mediated by host factors (9, 10). The frequency of recombination has been estimated to be on the order of 0.5-0.7%/kilobase of the viral genome (11). Furthermore, a screen for replication-competent HSV-1 origins from a library in which particular origin elements were substituted with random sequences resulted in the isolation of wild-type origins at a frequency of 25% as a consequence of homologous recombination (12).Insight into the mechanism by which homologous recombination proceeds during HSV-1 replication is provided by several lines of evidence; it was shown that plasmi...

Replication and Recombination of Herpes Simplex Virus DNA

“…ICP8 has also been demonstrated to promote strand invasion in vitro, and together with helicaseprimase, it can promote strand exchange (67,68). A possible role could be to promote recombination-dependent DNA synthesis during repair of double-strand breaks (69,70). It has, however, not yet been possible to assess the significance of the recombinase properties of ICP8 during the course of a virus infection.…”

The Herpes Simplex Virus Type 1 Alkaline Nuclease and Single-Stranded DNA Binding Protein Mediate Strand Exchange In Vitro

“…Analogous to the interaction of lambda Red␣ and the ssDNA binding protein (SSB) lambda Red␤, UL12 interacts with the SSB of HSV-1, ICP8 (53,55). Furthermore, ICP8 possesses strand-melting (4) and strand-annealing activities (12) and has been reported to mediate limited strand exchange (6,38). A recent study has also shown that ICP8 can promote strand invasion (37).…”

Replication, recombination and packaging of amplicon DNA in cells infected with the herpes simplex virus type 1 alkaline nuclease null mutant ambUL12

“…It is likely that host enzymes, perhaps in conjunction with the HSV-1 single-stranded DNA-binding protein ICP8, mediate the observed recombination. In this context, it is interesting to note that ICP8 has been demonstrated to mediate strand-invasion and -exchange in the absence of AN, and also to interact with host proteins known to be involved in DNA replication and repair (Nimonkar & Boehmer, 2002Taylor & Knipe, 2004). …”