Abstract: We have determined by use of DNA sequencing techniques the exact location of the deletion in d1 892, a viable deletion mutant of Simian virus 40 (SV40) reported to map very near the unique replication origin or SV40. With the help of this localization we have narrowed down the boundaries of the replication origin to 85 nucleotides within the sequence of SV40.

Abstract: Normally, bacteria cease DNA replication in the absence of protein synthesis. A variety of treatments, such as thymine starvation or a shift-up to rich medium, lead to continued DNA replication in the absence of protein synthesis. Mutants are described which always terminate replication under these conditions. These conditional lethal mutants, dnaT1 and dnaT2, contransduce with serB and dnaC. The mutation also affects cell division. All aspects of the mutant phenotype (obligatory termination of replication, temperature sensitivity of DNA replication and growth, and aberrant cell division at permissive growth temperatures) were transdominant to the wild-type phenotype. Episomes carrying the dnaT mutation appeared to be unstable. The existence of such a dominant mutation was predicted by a model of chromosome termination proposed by Kogoma and Lark (J. Mol. Biol. 94:243-256, 1975).

Abstract: The synthesis of the bacteriophage G4 negative strand is an example of the de novo initiation of a polynucleotide chain. This initiation is performed by the Escherichia coli replication protein dna G which selects a unique site on 5400-base positive-strand template. In this paper we present the nucleotide sequence of the G4 negative-strand initiation site. This is the template element recognized by the dna G priming protein. In conjunction with the sequence of the nascent negative strand, obtained by Bouche, Rowen, and Kornberg [Bouche, J.-P., Rowen, L. & Kornberg, A. (1978) J. Biol. Chem. 253, 765-769], the present data provide a description of a dna G-dependent origin of replication in which one knows the place at which polymerization starts at the nucleotide level.

Abstract: Ring-to-ring (early) replication of bacteriophage lambda DNA was blocked after heat inactivation of the P protein. Rolling circle (late) replication continued for several rounds at the rate reached when the temperature shift was carried out. The same differential effect was observed after inhibition of RNA or protein synthesis during the two different phases of replication. In contrast, inactivation of the O protein resulted in a fast stop of lambda DNA synthesis at early and late times after infection. The results were consistent with the following interpretations. (i) The lambda P gene product plays a role in the initiation of the ring-to-ring replication. (ii) Ring-to-ring replication continues parallel to rolling circle replication, possibly diminishing with time after infection. (iii) The O function is stable in and necessary for the structural integrity of an elongation complex. It is unstable in free form and probably released from such a replication complex after each round of replication at the ring-to-ring stage.

Abstract: The genome of Bacillus subtilis bacteriophage SPP1, a linear, 28.5-megadalton DNA duplex, was mapped by analysis with the restriction endonucleases endo R.Sal I, Sma I, Xba I, Bgl I, Bgl II, and EcoRI. The SPP1 genome, like that of the Salmonella typhimurium phage, P22, was found to be a terminally repetitious, circularly permuted molecule. 6-(p-Hydroxyphenylazo)uracil, a selective, reversible inhibitor of SPP1 DNA synthesis, was exploited to synchronize the initiation of genome replication and to selectively label the site of its initiation with radioactive thymidine. Restriction endonuclease analysis of the distribution of the label located the origin of replicative synthesis at an area approximately 0.2 genome length from one molecular terminus.

Abstract: DNA replication in isolated nuclei is highly dependent on the availability of soluble proteins from the cell cytoplasm. Activity is distributed nonrandomly among the different proteins, and the range of proteins that are required for optimal DNA replication varies with the fractions of DNA being replicated. Support of DNA replication has been correlated with the uptake of these proteins by nuclei and their integration into an immature form of the newly replicated chromatin; the latter has been shown by density analysis to be richer in protein content than the bulk of nonreplicating chromatin. Pulse labeling of DNA in living cells has revealed that a similar protein-rich chromatin is formed as an intermediate in chromatin replication in vivo ; however, this form rapidly matures by the exclusion of proteins. The dependency of DNA replication on the presence of soluble cytoplasmic proteins and the physical association of these entities with newly replicated chromatin prompt the proposal that availability of specific proteins may play an important role in determining the ultimate genetic expressability of the matured chromatin and thus the cell phenotype. The finding that dexamethasone, a steroid that regulates the expression of several genes and directs the differentiation of certain cells, can modify the uptake of proteins in isolated nuclei is in accord with this hypothesis.

Abstract: It is just twenty years since Robert Sinsheimer reported the results of his initial studies on a small and previously uncharacterized virus, ϕ X174. It is one of the ironies of research that a virus initially so different and odd should gradually emerge as a system offering some of the deepest insights into a major area of molecular biology, DNA replication. In contrast to the disruptive entrance into the host cell of larger viruses such as T4 and T7, bacteriophage ϕ X and the related isometric phage G4 set to work quietly, not disturbing the host cell except for an eventual cessation of bacterial DNA synthesis (Tessman 1966; Stone 1970). Even in the midst of this seeming order, however, the virus diverts the host sufficiently to generate a thousandfold replication of itself—a process which is readily observed if one studies a mutant virus that does not lyse the host prematurely. The host cell supplies almost all of the enzymatic machinery required for the viral DNA replication cycle. It is precisely for this reason that the single-stranded (SS) DNA phages are able to serve as a window into the DNA replication apparatus of Escherichia coli. The way in which the viral chromosome is processed during its replication reveals the reaction mechanisms of many host enzymes that normally play a role in bacterial DNA synthesis but which are commandeered by the virus after infection. In this article we will consider the various steps in the ϕ X and G4 replication cycles and discuss the...

Abstract: The initiation of DNA replication in a mammalian cell is a complex series of processes of which we have limited knowledge. We are aware of three parameters which must be regulated. The first and most stringent of these is the necessity to replicate every segment of each chromosome once and only once each cell cycle. Repair replication, which is a continuing process whether the cell is cycling or not, can occur, in the absence of this highly controlled semi-conservative replication. Since a chromosome consists of a single segment of DNA which is too long to replicate from a single origin in the time available for most cell cycles, each chromosome must have many initiation sites. Yet, each must function no more than once each cell cycle. If a potential site fails to function it must be modified as the replication fork passes over it so that it cannot function until the next cycle. It appears likely that this type of modification or regulation will be unique for chromosomal DNA. The DNA of animal viruses, and perhaps organelle DNA, may be designed to initiate several to many rounds of replication during an interval when the chromosomes complete one round or fail to replicate at all. The basis for this type of regulation is unknown and probably cannot be determined until we know more about the distribution and structure of potential origins for replication.

Abstract: Data presently available support the hypothesis that inhibition of DNA replication in ultraviolet-irradiated mammalian cells is due to long pauses when replication forks encounter pyrimidine dimers in the template DNA.

Abstract: A group of small plasmids from enterobacteria including the ColEl plasmid and the ampicillin resistance plasmid RSF103O is capable of extensive replication in the presence of chloramphenicol (Clewell, 1972; Crosa et al., 1976). This replicative behaviour has been termed “relaxed replication” and is defined operationally by an increase in the ratio of plasmid to chromosomal DNA upon inhibition of protein synthesis. Plasmid replication in the presence of chloramphenicol is still sensitive to inhibition by rifampicin indicating a direct role of E.coli RNA polymerase in the replication process (Clewell et al., 1972).

Abstract: The first evidence that DNA replication proceeds in an organized, nonrandom manner at the level of the chromosome was obtained by Taylor (1960) using autoradiographic techniques to examine labeled metaphase chromosomes. His work, and that of Hsu (1964), demonstrated that DNA replication is initiated at multiple sites along the chromosome and that each chromosome shows a reproducible, characteristic pattern of replication in different stages of S phase. It has also been shown that hetero-chromatin tends to be late replicating (see Lima-de-Faria and Jaworska, 1968). Even better resolution is now possible with the fluoresence labeling technique developed by Latt (1973). Both Latt (1975) and Stubblefield (1975) have suggested that chromosome bands which have been visualized as structural units by classical staining procedures are actually units of replication as observed by fluoresence labeling.

Abstract: For a long time it has been thought that the only energy-requiring event occurring in DNA replication was the breakage of the dNTP phosphodiester bond during the process of polymerization. In 1968, however, Cairns and Denhardt noticed that carbon monoxide or cyanide inhibited the replication of the chromosome E.coli and that the inhibition could not be explained by depletion of the intracellular pool of dNTP (1). This result suggested the existence of another energy-consuming step in DNA replication and lead the authors to postulate the existence of an energy-dependent unwinding of the DNA duplex. The discovery of in vitro replication systems soon confirmed the requirement of ATP for DNA replication; for example, toluene-treated bacteria require ATP in addition to the four dNTP for continuation of chromosome replication (2). Since then, many investigators have concentrated their efforts on discovering the mechanism of this energy dependence. It is now clear that not only one, but several, ATP-dependent reactions may take place in the process which leads to the duplication of the chromosome. In this article, we will focus on some enzymes potentially capable of unwinding the chromosome in the presence of ATP, as well as on the role of ATP in chromosome replication in situ.

Abstract: DNA replication in eukaryotes begins at multiple sites along the length of the chromosome (1) and proceeds bidirectionally from each point of origin (2). The factors which govern the temporal order of replication initiation remain unknown. These unresolved determinants may endow early embryonic cells with replication characteristics which distinguish them from adult cells. Among these are an altered temporal sequence of DNA replication and an extremely brief S phase. In this report I will provide evidence supporting the former contention and indirectly address the latter. It has been argued that the brevity of the early embryonic S phase is due to the activation of a number of replication initiation sites that are not available in adult cells with longer S phases (3,4). The argument proposes that the increase in the number of available replication initiation sites is accompanied by a decrease in replicon size. I wish to interject a cautionary note with respect to the second part of the argument by demonstrating that replicons as small as any seen in embryonic cells can be detected in cultured mammalian cells.

Abstract: The synthesis of DNA in prokaryotic and eukaryotic cells is complex, involving numerous enzymes and other proteins that may function in multi-enzyme systems A molecular description of DNA replication in Escherichia coli, is only beginning to be realized, after fifteen years of intensive studies using small bacteriophage DNA molecules as probes Full details, however, will no doubt require many years of additional study Several of the approximately fifteen proteins that are involved in DNA replication in Ecoli (excluding DNA precursor enzymes), as indicated by biochemical and/ or genetic studies, have not yet been identified Moreover, functions have been established for only some of these proteins, and their complex interactions have only recently begun to be appreciated