Extracts of the yeast Saccharomyces cerevisiae support DNA replication on exogenous yeast 2-pm plasmid DNA templates. A crude extract from a S. cerevisae cell division cycle mutant, cdc8-1, expressed the temperature-sensitive phenotype since it could be inactivated at 42°C in vitra This heat-inactivated extract was fully complemented by the addition ofeither wild-type or cdc8-1 single-stranded DNA binding protein (SSB). Restoration by SSB ofthe activity ofthe mutant cell extract allowed replication like that of a wild-type crude extract, as shown by bidirectional DNA synthesis from the in vivo origin. The DNA binding protein specifically stimulates the reaction catalyzed by yeast DNA polymerase I, a true DNA replicase, using the hybrid of 4X174 singlestranded DNA and a restriction endonuclease fragment as a template. It also increases processivity of DNA polymerase I at least 10-fold. Escherichia coli SSB, but not T4 gene 32 protein, can substitute for yeast SSB. Both restoration of DNA synthesis in the heated mutant cell extract and stimulation ofthe DNA polymerase I reaction by SSB from cdc8-1 cells are inactivated at nonpermissive temperatures, suggesting that yeast SSB is the CDC8 gene product.We have recently shown that crude extracts of Saccharomyces cerevisiae catalyze DNA replication of exogenous yeast 2-,um plasmid DNA and a chimeric plasmid containing a yeast nuclear DNA replicon (autonomously replicating segment) (1). In this in vitro system, DNA replication initiates at or near the in vivo origin and proceeds bidirectionally as in vivo. This system should permit the identification and characterization of the components required for both plasmid and yeast nuclear DNA replication, since each requires the same gene products (2-4).In studies in prokaryotes, conditional lethal mutations in the genes needed by DNA replication have been used to assay for required proteins and establish physiological relevance. Analogous mutants have been isolated and mapped in yeast (5) (8). Phosphocellulose (P-li) and DEAE-Sephacel were from Whatman; Sephacryl S200 was from Pharmacia; and acrylamide, bisacrylamide, and hydroxylapatite (Bio-Gel HT) were from Bio-Rad. Other chemicals were as used previously (1).DNAs. Yeast 2-gm plasmid DNA and the chimeric plasmid pJDB36 DNA (which consists ofpMB9 and 2-gm A-form DNAs)were prepared as described (1). 4X174 viral ss DNA and replicative form (RF) DNA were purchased from Bethesda Research Laboratories. Hae III restriction endonuclease fragments of cX174 RF DNA were purified by electrophoresis on a 5% polyacrylamide gel and eluted by a published procedure (9). 4X174 viral ss DNA was hybridized with the complementary strand of a Hae III restriction endonuclease fragment as described (10)

The activities of the three known catabolic and the one anabolic polyadenylate enzymes have been determined in synchronized L5178y cells : endoribonuclease, exoribonuclease, 5'-nucleotidase and poly(A) polymerase (Mg' +-dependent). These four enzymes were found primarily in the nuclear fraction. The activity of poly(A) polymerase remains essentially constant during the transition from GI to S phase. However, the poly(A) catabolic enzyme activities increase parallel with DNA synthesis; the endoribonuclease activity increases 4-fold during GI to S phase, the exoribonuclease and the nucleotidase activities increasing 30-fold and 16-fold. During the S phase the poly(A)-degrading enzymes are far more active than the poly(A)-synthesizing activity of poly(A) polymerase. We conclude that in L5178y cells the poly(A)-degrading enzymes probably function in regulation of the post-transcriptional net-polyadenylation of heterogeneous nuclear RNA during the phase of DNA synthesis.

SUMMARYThe phosphorylation of arabinofuranosylthymine (araThd) has been studied both in non-infected cells and in those infected with herpes simplex virus (HSV-I, Lennette; HSV-I, IES and HSV-2, D-316 ). In these experiments, HSV strains were used which either contain (Lennette, TK + and D-316 TK +) or lack (IES, TK-) the capacity to induce pyrimidine deoxyribonucleoside kinase. It was found that extracellularly administered araThd is phosphorylated to araTTP via araTMP and araTDP in both non-infected and in HSV-infected ceils. The phosphorylating capacity is more than tenfold lower in non-infected cells than in infected cells. Interestingly, cells infected with the TK-strain have a tenfold higher phosphorylating capacity than normal, uninfected cells, a fact which might indicate that host cell deoxythymidine kinase is induced during HSV infection. AraTMP is incorporated into cellular DNA but not into HSV DNA. This finding is in contrast to observations with arabinofuranosyladenine, which is incorporated into both cellular and HSV DNA. In vitro experiments with HSV-induced DNA polymerase show that araTTP strongly inhibits the enzyme activity. Therefore we conclude that the inhibition of HSV DNA polymerase by araTTP (formed intracellularly from araThd) is the explanation for the observed antiviral activity of araThd.

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