The Herpes Simplex virus replisomeIn Herpes Simplex virus type 1, HSV-1, six viral proteins form a multiprotein machine, replisome, which is responsible for the bulk of viral DNA replication. Our understanding of the molecular mechanisms of HSV-1 replisome function has been hampered by the lack of an in vitro DNA replication system. We believed that careful investigations of the individual components of the HSV-1 replisome would allow us to identify proper conditions for an in vitro system capable of performing rolling circle DNA replication.Unwinding of DNA and primer synthesis at the viral replication fork require the viral helicase-primase complex. This complex contains a helicase (UL5), a primase (UL52), and protein of unknown function (UL8). We first wanted to understand the role of the UL8 protein. We could in a series of experiments demonstrate that the UL8 subunit of the helicase-primase complex was required for the unwinding of single-strand DNA binding protein (ICP8)-coated DNA substrates. The rate of unwinding was however low, only 2 bp/s, much lower than the 50-60 bp/s reported for a related herpesvirus in vivo. We therefore tried to optimize the experimental conditions. We found that the low helicase rate could be explained by an extraordinary sensitivity of ICP8 to reaction conditions. At the appropriate Mg2+ concentration and ionic strength, the rate of the helicase approached the rate of replication fork movement in vivo. The HSV-1 DNA polymerase does not enhance the rate of unwinding by the helicase-primase. Instead, DNA synthesis by the HSV-1 DNA polymerase appears to be rate-limiting during leading strand synthesis.Finally we established an in vitro system for rolling circle DNA replication. The synthesis of double-stranded DNA on a mini-circular template by a reconstituted replisome, consisting of DNA polymerase-UL42, trimeric helicase-primase and ICP8, was measured. Our experiments demonstrated synthesis of Okazaki fragments of about 3 kb in length and leading strands of approximately 10 kb. Lagging strand synthesis was stimulated by ribonucleoside triphosphates, and DNA replication appeared to be coordinated and processive since it resists competition with an excess of (dT)150/(dA)20. From our experiments we conclude that only HSV-1 encoded proteins are needed for leading and lagging strand synthesis and that HSV-1 rolling circle DNA replication is a highly coordinated process.