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Since replication of DNA is a fundamental biological phenomenon, an understanding of the mechanism of DNA synthesis and the complex levels of control of this process is central to our understanding of how cells work. Although the mechanisms and some aspects of control of DNA synthesis in procaryotes have been elucidated (22,23,34), a similar understanding of DNA synthesis in eucaryotic cells is lacking. As with early work on procaryote DNA replication, initial studies of replication in eucaryotes have concentrated on small, autonomously replicating DNA molecules such as the DNA viruses. Some of these DNAs, such as papillomavirus (32) and Epstein-Barr virus DNAs (49), can exist in cells as stable extrachromosomal plasmids. Other studies have exploited the lytic DNA viruses of mammalian cells of which the best understood are the adenoviruses, primarily as a result of in vitro studies (6, 12, 37a). Although adenovirus has an atypical mechanism for initiation and elongation of DNA synthesis, these studies have led to the identification of some cellular proteins that are required for the replication of DNA in vitro.The monkey virus simian virus 40 (SV40) contains a small (5,243-bp), double-stranded, circular genome with a single origin of DNA replication (reviewed in references 1 and 9). The virus genome encodes one protein that is required for replication of virus DNA, the SV40 large tumor (T) antigen (41). This protein is a multifunctional phosphoprotein that is required for DNA synthesis, control of RNA synthesis, and cell transformation. Four biochemical properties of the purified protein have been observed: an ATPase activity (7, 14, 44), a nucleotide-binding activity distinct from the ATPase-binding site (8), adenylation of the protein (5), and site-specific DNA binding to three sites that lie at the origin of DNA replication and early promoter region (reviewed in reference 43). The general characteristics of the mechanism of SV40 DNA replication have been elucidated in vivo (reviewed in reference 9), and these studies suggest that SV40 may replicate its DNA in a way similar to that of the chromosomes of the host cell. This may be true not only for * Corresponding author.initiation and elongation of DNA synthesis but also for the segregation of daughter DNA molecules after replication (39, 40). However, a detailed understanding of the mechanism and control of SV40 DNA synthesis must derive from studies of DNA replicated in vitro.Two reports of cell-free systems for the replication of SV40 DNA have appeared (3,26). Recently, Li and Kelly (26) demonstrated that cytoplasmic extracts prepared from SV40-infected or -uninfected monkey cells supplemented with purified SV40 T antigen can efficiently replicate DNA templates containing a functional origin of DNA replication. In this report, we have confirmed this finding by using similarly prepared cytoplasmic extracts from human 293 cells grown in suspension. We show that DNA synthesis proceeds bidirectionally from the origin of DNA replication and that multiple rounds ...

Novel suppressor variants of conditionally lethal HSV‐1 gamma34.5 deletion mutants have been isolated which exhibit restored ability to grow on neoplastic neuronal cells. Deletion of the viral gamma34.5 genes, whose products share functional similarity with the cellular GADD34 gene, renders the virus non‐neurovirulent and imposes a block to viral replication in neuronal cells. Protein synthesis ceases at late times post‐infection and the translation initiation factor eIF2alpha is phosphorylated by the cellular PKR kinase [Chou et al. (1990) Science, 252, 1262–1266; (1995) Proc. Natl Acad. Sci. USA, 92, 10516–10520]. The suppressor mutants have overcome the translational block imposed by PKR. Multiple, independent isolates all contain rearrangements within a 595 bp element in the HSV‐1 genome where the unique short component joins the terminal repeats. This alteration, which affects the production of the viral mRNA and protein from the Us11 and Us12 genes, is both necessary and sufficient to confer the suppressor phenotype on gamma34.5 mutant viruses. HSV‐1 thus encodes a specific element which inhibits ongoing protein synthesis in the absence of the viral GADD34‐like function. Since this inhibition involves the accumulation of phosphorylated eIF2alpha, the element identified by the suppressor mutations may be a discrete PKR activator. Activation of the PKR kinase thus does not proceed through a general, cellular ‘antiviral’ sensing mechanism. Instead, the virus deliberately activates PKR and encodes a separate function which selectively prevents the phosphorylation of at least one PKR target, eIF2alpha. The nature of this potential activator element, and how analogous cellular elements could affect PKR pathways which affect growth arrest and differentiation are discussed.

Simian virus 40 large tumour antigen (T) is a replication origin binding protein required for viral DNA synthesis. Unphosphorylated T antigen is deficient in promoting DNA replication in vitro but can be activated by phosphorylation at residue threonine 124 by the cdc2 protein kinase. This observation demonstrates that T is regulated by phosphorylation and provides a model for cdc2 function in the control of DNA replication.

A cell‐free system for replication of SV40 DNA was used to assess the effect of mutations altering either the SV40 origin of DNA replication or the virus‐encoded large tumor (T) antigen. Plasmid DNAs containing various portions of the SV40 genome that surround the origin of DNA replication support efficient DNA synthesis in vitro and in vivo. Deletion of DNA sequences adjacent to the binding sites for T antigen either reduce or prevent DNA synthesis. This analysis shows that sequences that had been previously defined by studies in vivo to constitute the minimal core origin sequences are also necessary for DNA synthesis in vitro. Five mutant T antigens containing amino acid substitutions that affect SV40 replication have been purified and their in vitro properties compared with the purified wild‐type protein. One protein is completely defective in the ATPase activity of T antigen, but still binds to the origin sequences. Three altered proteins are defective in their ability to bind to origin DNA, but retain ATPase activity. Finally, one of the altered T antigens binds to origin sequences and contains ATPase activity and thus appears like wild‐type for these functions. All five proteins fail to support SV40 DNA replication in vitro. Interestingly, in mixing experiments, all five proteins efficiently compete with the wild‐type protein and reduce the amount of DNA replication. These data suggest that an additional function of T antigen other than origin binding or ATPase activity, is required for initiation of DNA replication.

Bacteroides fragilis TAL3636 produces a class B, Zn2+-requiring 13-lactamase. The gene, ccrA, was cloned and expressed in Escherichia coli. The gene was sequenced and shown to share greater than 33% identity with the metalloenzyme from Bacillus cereus 569/H.The unique class B 1-lactamases require a metal cofactor,

Enhancers are cis-acting control elements which can stimulate at a distance the activity of a variety of eukaryotic promoters. First identified as a repeated 72 base pair (bp) sequence upstream of the simian virus 40 (SV40) early gene promoter, enhancers have since been shown to be associated with numerous other viral and cellular genes. Although there are no strong homologies between the sequences of different enhancers, a number of short and degenerate consensus sequences have been identified, including the 'core' element GTGGA/TA/TA/TG and stretches of alternating purines and pyrimidines which may have the potential to form left-handed Z DNA. To study the functional significance of two alternating purine and pyrimidine sequences in the SV40 enhancer, we have introduced various combinations of point mutations into a modified SV40 enhancer which contained only one copy of the 72 bp element (W.H., Y.G., A. Nordheim and A. Rich, unpublished results); one of these combinations impaired both the activity of the enhancer and growth of SV40. We describe here the structure of 18 revertants of this mutant and suggest that in each of the 18 revertants, the defects of the original mutant have been overcome by simple tandem duplications in the enhancer region, all of which include the 'core' element.