DNA Replication Catalyzed by Herpes Simplex Virus Type 1 Proteins Reveals Trombone Loops at the Fork*

Bermek, Oya; Willcox, Smaranda; Griffith, Jack D.

doi:10.1074/jbc.m114.623009

Cited by 9 publications

(9 citation statements)

References 25 publications

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“…The QF mutant can form filaments and binds to ssDNA with a similar affinity to WT ICP8; therefore, we asked whether it retained other functions associated with replicative SSBs. ICP8 has been reported to stimulate the activities of other core HSV-1 replication proteins involved in leading-and laggingstrand DNA synthesis: HSV polymerases (UL30/UL42) and helicase-primase (UL5/8/52) (34,36,37,39,(45)(46)(47). To assess the ability of WT and QF mutant proteins to stimulate these activities, we have employed an in vitro minicircle replication assay using a DNA substrate consisting of a 90-mer linear oligo annealed to a 70-bp minicircle (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…To assess the ability of WT and QF mutant proteins to stimulate these activities, we have employed an in vitro minicircle replication assay using a DNA substrate consisting of a 90-mer linear oligo annealed to a 70-bp minicircle (Fig. 4A) (46,47). In this assay, the minicircle is the template for leading-strand synthesis and lacks thymidine residues.…”

Section: Resultsmentioning

confidence: 99%

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Herpes simplex virus 1 ICP8 mutant lacking annealing activity is deficient for viral DNA replication

Weerasooriya

DiScipio

Darwish

et al. 2018

Proc. Natl. Acad. Sci. U.S.A.

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Most DNA viruses that use recombination-dependent mechanisms to replicate their DNA encode a single-strand annealing protein (SSAP). The herpes simplex virus (HSV) single-strand DNA binding protein (SSB), ICP8, is the central player in all stages of DNA replication. ICP8 is a classical replicative SSB and interacts physically and/or functionally with the other viral replication proteins. Additionally, ICP8 can promote efficient annealing of complementary ssDNA and is thus considered to be a member of the SSAP family. The role of annealing during HSV infection has been difficult to assess in part, because it has not been possible to distinguish between the role of ICP8 as an SSAP from its role as a replicative SSB during viral replication. In this paper, we have characterized an ICP8 mutant, Q706A/F707A (QF), that lacks annealing activity but retains many other functions characteristic of replicative SSBs. Like WT ICP8, the QF mutant protein forms filaments in vitro, binds ssDNA cooperatively, and stimulates the activities of other replication proteins including the viral polymerase, helicase–primase complex, and the origin binding protein. Interestingly, the QF mutant does not complement an ICP8-null virus for viral growth, replication compartment formation, or DNA replication. Thus, we have been able to separate the activities of ICP8 as a replicative SSB from its annealing activity. Taken together, our data indicate that the annealing activity of ICP8 is essential for viral DNA replication in the context of infection and support the notion that HSV-1 uses recombination-dependent mechanisms during DNA replication.

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Herpes simplex virus 1 ICP8 mutant lacking annealing activity is deficient for viral DNA replication

Weerasooriya

DiScipio

Darwish

et al. 2018

Proc. Natl. Acad. Sci. U.S.A.

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“…In addition, ICP8 the major ssDNA binding protein from herpes simplex virus-1 is also distantly related to gp2.5, sharing a common ancestor (14). Like gp2.5, ICP8 can anneal ssDNA and it is essential for replication of leading-and lagging-strands using minicircle DNA in vitro (109). However, only the middle segment of ICP8 shares any significant homology to gp2.5 and the two differ in their oligomerization state.…”

Section: Homologs Of Gp25mentioning

confidence: 99%

Gp2.5, the multifunctional bacteriophage T7 single-stranded DNA binding protein

Hernandez

Richardson

2019

Seminars in Cell & Developmental Biology

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The essential bacteriophage T7-encoded single-stranded DNA binding protein is the nexus of T7 DNA metabolism. Multiple layers of macromolecular interactions mediate its function in replication, recombination, repair, and the maturation of viral genomes. In addition to binding ssDNA, the protein binds to DNA polymerase and DNA helicase, regulating their activities. The protein displays potent homologous DNA annealing activity, underscoring its role in recombination.

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“…Seven virus-encoded proteins are required for viral DNA synthesis including an origin-binding protein (UL9) and six core replication proteins: a two subunit DNA polymerase (UL30 and UL42), a three subunit helicase-primase complex (UL5, UL8, and UL52), and a multifunctional ssDNA-binding protein (ICP8) (for review, see Ref.1). The six purified core replication proteins can function to produce linear concatemers in vitro if provided a primed template (2). On the other hand, in vivo, late replicating viral DNA has been reported to adopt a mixture of complex structures such as X-and Y-shaped branches and tangled masses, suggesting that recombination may play a role in the replication of HSV-1 DNA (3)(4)(5)(6)(7).…”

mentioning

confidence: 99%

“…ICP8, the ssDNA-binding protein (SSB), 2 is a multifunctional protein essential for viral DNA replication including ssDNA binding and stimulation of HSV polymerase and helicaseprimase activities and has a major role in the formation of replication compartments in which DNA replication takes place (9 -11). ICP8 forms helical protein filaments in the absence of DNA (12), and filament formation appears to be required for pre-replicative site and replication compartment formation (13).…”

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confidence: 99%

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

Bermek

Weller

Griffith

2017

Journal of Biological Chemistry

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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.

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DNA Replication Catalyzed by Herpes Simplex Virus Type 1 Proteins Reveals Trombone Loops at the Fork*

Cited by 9 publications

References 25 publications

Herpes simplex virus 1 ICP8 mutant lacking annealing activity is deficient for viral DNA replication

Herpes simplex virus 1 ICP8 mutant lacking annealing activity is deficient for viral DNA replication

Gp2.5, the multifunctional bacteriophage T7 single-stranded DNA binding protein

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

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