The replicative DNA polymerase of herpes simplex virus 1 exhibits apurinic/apyrimidinic and 5′-deoxyribose phosphate lyase activities

Abstract

Base excision repair (BER) is essential for maintaining genome stability both to counter the accumulation of unusual bases and to protect from base loss in the DNA. Herpes simplex virus 1 (HSV-1) is a large dsDNA virus that encodes its own DNA replication machinery, including enzymes involved in nucleotide metabolism. We report on a replicative family B and a herpesvirus-encoded DNA Pol that possesses DNA lyase activity. We have discovered that the catalytic subunit of the HSV-1 DNA polymerase (Pol) (UL30) exhibits apurinic/apyrimidinic (AP) and 5 -deoxyribose phosphate (dRP) lyase activities. These activities are integral to BER and lead to DNA cleavage on the 3 side of abasic sites and 5 -dRP residues that remain after cleavage by 5 -AP endonuclease. The UL30-catalyzed reaction occurs independently of divalent cation and proceeds via a Schiff base intermediate, indicating that it occurs via a lyase mechanism. Partial proteolysis of the Schiff base shows that the DNA lyase activity resides in the Pol domain of UL30. These observations together with the presence of a virus-encoded uracil DNA glycosylase indicates that HSV-1 has the capacity to perform critical steps in BER. These findings have implications on the role of BER in viral genome maintenance during lytic replication and reactivation from latency.base excision repair ͉ abasic DNA ͉ uracil DNA glycosylase H erpes simplex virus 1 (HSV-1) is a dsDNA virus with a genome of Ϸ152 kbp (1). HSV-1 switches between lytic replication in epithelial cells and a state of latency in sensory neurons during which there is no detectable DNA replication (1). Replication of the genome is mediated by seven essential virus-encoded factors with the following functions: a DNA polymerase (Pol) catalytic subunit (UL30), its associated processivity factor (UL42), a ssDNA binding protein (UL29), a heterotrimeric helicase-primase (UL5, UL8, and UL52) and an initiator protein (UL9) that binds to and unwinds the viral replication origins (1-4). HSV-1 also encodes several other enzymes that are dispensable for replication in cell culture, which are involved in nucleotide metabolism and perform other important roles in maintaining the viral genome including a thymidine kinase (UL23), a ribonucleotide reductase (UL39 and UL40), a dUTPase (UL50), an exonuclease (UL12), and notably a uracil DNA glycosylase (UDG) (UL2) (4).The presence of a virus-encoded UDG suggests that excision of uracil may be important during viral replication. Hence, it has been shown that uracil substitutions in the viral origins of replication alters their recognition by the viral initiator protein (5). Moreover, whereas UL2 may be dispensable for viral replication in fibroblast (6), UL2 mutants exhibit reduced neurovirulence and a decreased frequency of reactivation from latency (7). Thus, UDG action in HSV-1 may be important for viral reactivation after quiescence in neuronal cells during which the genome may accumulate uracil as a result of spontaneous deamination of cytosine. In cytomegalovirus, the viral UDG ...

Reconstitution of Uracil DNA Glycosylase-initiated Base Excision Repair in Herpes Simplex Virus-1

“…Hence, HSV-1 encodes a uracil DNA glycosylase (UDG) (UL2) as well as a dUTPase to reduce the pool of dUTP and prevent misincorporation by the viral Pol (24,25). Moreover, we recently showed that the catalytic subunit of the viral Pol (UL30) exhibits AP and 5Ј-deoxyribose phosphate (dRP) lyase activities (26). The presence of a virus-encoded UDG and DNA lyase indicates that HSV-1 has the capacity to perform integral steps of BER, specifically for the removal of uracil.…”

Replication and Recombination of Herpes Simplex Virus DNA

“…The antiviral compound acyclovir is incorporated less efficiently than dGTP but causes chain termination, and it is removed with a half-life of 1 h (36). Interestingly, the polymerase can cleave an apurinic/apyrimidinic site in duplex DNA as well as remove the product 5Ј-deoxyribose phosphate by a lyase activity (37). The presence of a separate UL2 uracil glycosylase as well as apurinic/apyrimidinic and 5Ј-deoxyribose phosphate lyase activities in the virus polymerase indicates important roles in enhancing replication fidelity (17).…”

“…The enzyme forms a complex with the viral DNA polymerase and collaborates to perform DNA repair (37). The presence of a dUTPase and a ribonucleotide reductase lacking allosteric control mechanisms may indicate an enhanced risk for misincorporation of dNTPs during replication (35).…”

The Pre-NH 2 -Terminal Domain of the Herpes Simplex Virus 1 DNA Polymerase Catalytic Subunit Is Required for Efficient Viral Replication

“…The extreme C terminus of HSV-1 Pol is crucial for an interaction with processivity factor UL42 that is necessary for long-chain DNA synthesis and indispensable for viral replication (11). Most recently, HSV-1 Pol was found to possess apurinic/apyrimidinic and 5=-deoxyribose phosphate lyase activities consistent with base excision repair processes, which are typically functions of the repair polymerase family X (3,35). Although the active site has yet to be mapped, DNA lyase activity has been localized to a 63-kDa C-terminal fragment of HSV-1 Pol (3).…”