Rate Of Replication Fork Progression Research Articles

Identification of a Region of Escherichia coli DnaB Required for Functional Interaction with DnaG at the Replication Fork

The fundamental activities of the replicative primosomes of Escherichia coli are provided by DnaB, the replication fork DNA helicase, and DnaG, the Okazaki fragment primase. As we have demonstrated previously, DnaG is recruited to the replication fork via a transient protein-protein interaction with DnaB. Here, using site-directed amino acid mutagenesis, we have defined the region on DnaB required for this protein-protein interaction. Mutations in this region of DnaB affect the DnaB-DnaG interaction during both general priming-directed and phiX174 complementary strand DNA synthesis, as well as at replication forks reconstituted in rolling circle DNA replication reactions. The behavior of the purified mutant DnaB proteins in the various replication systems suggests that access to the DnaG binding pocket on DnaB may be restricted at the replication fork.

Resolution of head-on collisions between the transcription machinery and bacteriophage phi29 DNA polymerase is dependent on RNA polymerase translocation.

The outcome of collisions between Bacillus subtilis phage Phi29 DNA polymerase and oppositely oriented transcription complexes has been studied in vitro. We found that the replication fork was unable to go past a transcription ternary complex stalled head-on. However, head-on collisions did not lead to a deadlock. Both DNA and RNA polymerase remained bound to the template and, when the halted transcription complex was allowed to move, the replication machinery resumed normal elongation. These results suggested that a replication fork that encounters an RNA polymerase head-on whose movement is not impeded would bypass the transcription machinery. Our results for head-on collisions between concurrently moving replication and transcription complexes are indeed consistent with the existence of a resolving mechanism. The ability of Phi29 DNA polymerase to resolve head-on collisions with itself during symmetrical replication of Phi29 DNA in vivo is likely to be related to its ability to pass a head-on oriented RNA polymerase.

Replicon size and rate of DNA replication fork movement are correlated in grasses.

In eukaryotes, most nuclear DNA replication proceeds bidirectionally from multiple origins of replication. A unit of DNA, replicated by two replication forks from a single origin, is termed a replicon. Using results from DNA fiber autoradiography we show a novel positive correlation between replicon size and the rate of replication fork movement in root meristem nuclei of 13 grass species. Although there is interspecific variation in replicon size, it is balanced by similar variation in the rate of replication fork progression.

DNA replication fork progression rate and temporal organization of S phase in normal epidermis and in basal cell carcinoma.

The double-pulse labeling technique for DNA fiber autoradiography was applied to epidermal cells from normal human skin and from human basal cell carcinoma (BCC). We aimed to measure the size and replication rate of the replication unit (RU) for both types of cell and to account, from these results, for our previous observation of a near doubling of S-phase duration in BCC, compared with normal skin. The mean RU size was 76 +/- 4 micron in BCC, not significantly different from the 68 +/- 6 micron value found in normal skin, so the mean of those two values (i.e., 72 micron), was used in further calculations. The rate of replication fork progression was 0.59 +/- 0.005 micron/min in the normal epidermis and 0.33 +/- 0.03 micron/min in BCC, corresponding to a replication time of the average RU equal to 61 min and 109 min, respectively. Thus, with an unchanged RU size in BCC, the observed 1.8-fold decrease in the rate of fork progression in the tumor can account entirely for our previous observation of a 1.8-fold increase in S-phase duration in this tumor, without requiring the assumption of any change in the temporal organization of DNA synthesis in the malignant cells. Considering S phase as an ordered process in which a major part, if not all, of the genome replicates at genetically determined times, we suggest that the clusters of replication units are, in turn, organized into temporally defined "sets". These sets are composed of all the clusters (whatever their chromosomal location) that are programmed to initiate replication during the same fraction of the S period. This hypothesis implies that DNA synthesis in a given set is triggered by some event coupled to progression of replication in the immediately preceding set. Based on a S-phase duration of 10.2 hours in normal skin and of 19.2 hours in BCC (our previous data), and assuming perfect synchrony and homogeneity of the clusters within each set and of each cluster's constitutive RUs, the minimum number of sequentially replicating sets, in both instances, can be estimated as roughly equal to 10.

Effects of reovirus infection on the spatial and temporal organization of DNA replication in L cells.

DNA fiber autoradiography was used to analyze the spatial and temporal organization of activated initiation sites for DNA replication in mouse L929 cells infected with reovirus type 3 (Dearing strain) and in uninfected control cells. Cells were labeled for 10 min with 3H-thymidine at high specific activity followed by 3 h of low specific activity labeling. Reovirus infection causes no change in the rate of replication fork progression, but increases both the mean distance between activated initiation sites by approximately 30% and the nonrandomness in the spatial distribution of the sites along the DNA fibers. Significant synchronization of initiation in adjacent activated sites was detected on DNA fibers from uninfected cells and from reovirus-infected cells. The mean relative initiation time for pairs of initiation events which had occurred prior to high specific activity labeling did not differ significantly between the infected and uninfected cells. The data are consistent with the interpretation that reovirus infection shuts off initiation sites in a coordinated fashion, possibly by preventing activation of entire clusters of potential initiation sites.

Decreased rate of DNA-chain growth in human basal cell carcinoma

The DNA synthetic (S) phase of cell division is about twice as long in human basal cell carcinoma (BCC) as the normal epidermis and several non-malignant skin diseases. A similar increase in S phase duration seems to occur in tumours of the human oesophagus, larynx and recto-colon. At the molecular level, the overall rate of replication in a nucleus is determined by two factors--the frequency of initiation of replication and the rate of replication fork progression along the units of replication (for review see refs 7,8). Here we present evidence (based on DNA fibre autoradiography) that the observed increase in S phase duration may be due to a marked reduction in the rate of DNA replication along replication units, the density of simultaneously operating replication units remaining unchanged. This indicates an alteration in some biological component(s) of DNA synthesis in BCC that is of potential interest for characterizing the malignant transformation of the epidermal cell and that may perhaps be exploited therapeutically.

The rate of fork movement during DNA replication in mammalian cells.

DNA fiber autoradiography was used to measure the rate of replication fork progression along replication units in human diploid cells. The rate in different replication units differs very significantly and lies within the range 0.1 to 1.2 micrograms/min. However, no significant changes were found in the rate of fork movement along single replication units operating during long intervals of S phase. Moreover, the fork progression rate is constant in many replication units of human cells.

Chromosomal DNA replication: Retarded fork progression and altered initiation in cells infected with mengovirus or Newcastle disease virus

Infection of mouse L cells with mengovirus or Newcastle disease virus inhibits DNA synthesis. The effects of this inhibition on chromosome replication have now been investigated using DNA fiber autoradiography, a technique permitting analysis of events on clusters of active replication units. At 5 hr after infection at high multiplicity with either virus, DNA replication is normal despite a marked inhibition of [ 3H]thymidine incorporation into DNA. By 6 hr, virus infection slows the rate of replication fork progression. In addition, virus infection alters the pattern of initiation of DNA replication in that the proportion of units with bidirectional replication is reduced and the usual synchrony of initiation events on clusters of units is decreased.

Deoxyribonucleic acid fiber autoradiography as a technique for studying the replication of the mammalian chromosome.

Deoxyribonucleic acid (DNA) fiber autoradiography is a technique that allows analysis of replication events on mammalian chromosomal DNA. 3H-thymidine is used to pulse-label DNA, which is subsequently released from lysed cells and extended linearly over a glass slide. Autoradiograms are produced by exposing the labeled DNA to light-sensitive emulsion. The effects of inhibition of protein synthesis on DNA replication were examined using this technique. Analysis of the autoradiographic patterns has shown that inhibition of protein synthesis with either cycloheximide or puromycin retards the rate of replication fork progression. In addition, these antibiotics after the normal pattern of multifocal initiation on replication units. The spatial interval between active initiation sites is decreased, there is a decay in synchrony of initiation events on subchromosomal clusters of replication units, the decay in synchrony of initiation events on subchromosomal clusters of replication units, the frequency of initiation is inhibited and the frequency of units showing the normal bidirectional mode of replication is decreased. The retarded fork progression and shorter initiation intervals may result either from the continued operation of a subset of replication units resistant to the inhibition of protein synthesis, or be manifestations of the inhibition of protein synthesis on all active sites. The other alterations, decreased synchrony and frequency of initiation and increased unidirectional replication probably occur on all active units when protein synthesis is inhibited.