The UL8 subunit of the helicase–primase complex of herpes simplex virus promotes DNA annealing and has a high affinity for replication forks

Abstract

During lytic infection, herpes simplex virus (HSV) DNA is replicated by a mechanism involving DNA recombination. For instance, replication of the HSV-1 genome produces X- and Y-branched structures, reminiscent of recombination intermediates. HSV-1's replication machinery includes a trimeric helicase-primase composed of helicase (UL5) and primase (UL52) subunits and a third subunit, UL8. UL8 has been reported to stimulate the helicase and primase activities of the complex in the presence of ICP8, an HSV-1 protein that functions as an annealase, a protein that binds complementary single-stranded DNA (ssDNA) and facilitates its annealing to duplex DNA. UL8 also influences the intracellular localization of the UL5/UL52 subunits, but UL8's catalytic activities are not known. In this study we used a combination of biochemical techniques and transmission electron microscopy. First, we report that UL8 alone forms protein filaments in solution. Moreover, we also found that UL8 binds to ssDNAs >50-nucletides long and promotes the annealing of complementary ssDNA to generate highly branched duplex DNA structures. Finally, UL8 has a very high affinity for replication fork structures containing a gap in the lagging strand as short as 15 nucleotides, suggesting that UL8 may aid in directing or loading the trimeric complex onto a replication fork. The properties of UL8 uncovered here suggest that UL8 may be involved in the generation of the X- and Y-branched structures that are the hallmarks of HSV replication.

The three-component helicase/primase complex of herpes simplex virus-1

“…Unless all three subunits are co-expressed, they are mostly cytoplasmic as examined by immunofluorescence and do not localize to the cell nucleus [96,98]. Biochemical analyses of UL8 DNA binding showed that UL8 does not bind oligonucleotides smaller than 50 nucleotides [99], but it can bind to 90-nucleotide-long oligonucleotides [100]. Intriguingly, UL8 does stably associate with long ssM13 DNA, and forms protein-ssDNA filamentous structures [100].…”

“…The nucleoprotein filament formation is a property shared by many ssDNA-binding proteins involved in recombination including archaeal RadA [101], bacterial RecA [102], HSV-1 ICP8 [103], KHSV ORF6 [24] and human Rad51 [104]. Compared to these proteins, UL8 filaments are distinct in that they are not helical filaments [100]. The significance of UL8 nucleofilament formation in an infected cell is currently unknown.…”

“…Biochemical analyses of UL8 DNA binding showed that UL8 does not bind oligonucleotides smaller than 50 nucleotides [99], but it can bind to 90-nucleotide-long oligonucleotides [100]. Intriguingly, UL8 does stably associate with long ssM13 DNA, and forms protein-ssDNA filamentous structures [100]. The nucleoprotein filament formation is a property shared by many ssDNA-binding proteins involved in recombination including archaeal RadA [101], bacterial RecA [102], HSV-1 ICP8 [103], KHSV ORF6 [24] and human Rad51 [104].…”