Herpes simplex virus-1 (HSV-1) is a large dsDNA virus that encodes its own DNA replication machinery and other enzymes involved in DNA transactions. We recently reported that the HSV-1 DNA polymerase catalytic subunit (UL30) exhibits apurinic/apyrimidinic and 5 -deoxyribose phosphate lyase activities. Moreover, UL30, in conjunction with the viral uracil DNA glycosylase (UL2), cellular apurinic/apyrimidinic endonuclease, and DNA ligase III␣-XRCC1, performs uracil-initiated base excision repair. Base excision repair is required to maintain genome stability as a means to counter the accumulation of unusual bases and to protect from the loss of DNA bases. Here we show that the HSV-1 UL2 associates with the viral replisome. We identified UL2 as a protein that co-purifies with the DNA polymerase through numerous chromatographic steps, an interaction that was verified by co-immunoprecipitation and direct binding studies. The interaction between UL2 and the DNA polymerase is mediated through the UL30 subunit. Moreover, UL2 co-localizes with UL30 to nuclear viral prereplicative sites. The functional consequence of this interaction is that replication of uracil-containing templates stalls at positions ؊1 and ؊2 relative to the template uracil because of the fact that these are converted into non-instructional abasic sites. These findings support the existence of a viral repair complex that may be capable of replication-coupled base excision repair and further highlight the role of DNA repair in the maintenance of the HSV-1 genome.Herpes simplex virus-1 (HSV-1) is a large double-stranded DNA virus with a genome of ϳ152 kbp (reviewed in Refs. 1 and 2). HSV-1 switches between a lytic replication cycle in epithelial cells that is tightly regulated by a temporal order of viral gene expression and a state of latency in sensory neurons during which the genome is proposed to be maintained in heterochromatin form with limited gene expression and no detectable DNA replication (3). Viral DNA replication is dependent on seven viral genes (4, 5). Six of these genes encode components of a typical DNA replication fork. The heterodimeric replicase (reviewed in Refs. 6 and 7) consists of a catalytic subunit encoded by UL30, with DNA polymerase (pol), 2 3Ј-5Ј-proofreading exonuclease, and RNase H activities (8 -11), and a tightly associated DNA-binding protein that confers a high degree of processivity on the pol, encoded by UL42 (11-17). The viral single-strand DNA-binding protein (ICP8) is encoded by UL29 (9), whereas the three subunits of the DNA helicaseprimase are encoded by the UL5, UL8, and UL52 genes (18). Finally, UL9 encodes an origin-binding protein with associated helicase activity that targets the viral replication origins (oriS and oriL) to promote unwinding and thereby initiate DNA replication at those sites (19 -24). A key step in HSV-1 DNA replication is the intricately orchestrated assembly of the essential viral replication proteins into prereplicative sites adjacent to nuclear domain 10 (ND10) structures that mature int...

We are interested in studying events at the HSV‐1 replication fork, processes that are dependent on six core viral gene products: the heterodimeric DNA polymerase‐clamp assembly (UL30/UL42), the DNA helicase‐primase (UL5/UL8/UL52) and the single‐strand DNA binding protein (ICP8/UL29). In an effort to identify additional factors that are active at the viral DNA replication fork, we searched for polymerase interacting proteins. We identified the viral uracil DNA glycosylase (UL2) as a protein that co‐purifies with the polymerase through numerous chromatographic steps, an interaction that was verified by co‐immunoprecipitation. We found that the interaction with UL2 is mediated through the UL30 subunit. Moreover, UL2 co‐localizes with UL30 to nuclear viral replication compartments. In addition, we have observed that UL30 specifically cleaves abasic DNA in a manner independent of divalent cation, resembling the activity of a lyase. This property would make the HSV‐1 DNA polymerase functionally analogous to the mitochondrial DNA polymerase gamma in as much as it possesses DNA synthesis, proofreading and lyase activities. These findings raise the interesting notion that the viral replisome promotes replication‐coupled base excision repair.