Dynamics of termination during in vitro replication of ultraviolet-irradiated DNA with DNA polymerase III holoenzyme of Escherichia coli (original) (raw)
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Proceedings of the National Academy of Sciences, 1986
Replication of UV-irradiated circular singlestranded phage M13 DNA by Escherichia coli RNA polymerase (EC 2.7.7.6) and DNA polymerase HI holoenzyme (EC 2.7.7.7) in the presence of single-stranded DNA binding protein yielded full-length as well as partially replicated products. A similar result was obtained with phage G4 DNA primed with E. coli DNA primase, and phage 4X174 DNA primed with a synthetic oligonucleotide. The fraction of full-length DNA was several orders of magnitude higher than predicted if pyrimidine photodimers were to constitute absolute blocks to DNA replication. Recent models have suggested that pyrimidine photodimers are absolute blocks to DNA replication and that SOS-induced proteins are required to allow their bypass. Our results demonstrate that, under in vitro replication conditions, E. coli DNA polymerase m holoenzyme can insert nucleotides opposite pyrimidine dimers to a significant extent, even in the absence of SOS-induced proteins.
The Journal of biological chemistry, 1989
The cycling time of DNA polymerase III holoenzyme during replication of UV-irradiated single-stranded (ss) DNA was longer than with unirradiated DNA (8 versus 3 min, respectively), most likely due to slow dissociation from lesion-terminated nascent DNA strands. Initiation of elongation on primed ssDNA was not significantly inhibited by the presence of UV lesions as indicated by the identical distribution of replication products synthesized at early and late reaction times and by the identical duration of the initial synthesis bursts on both unirradiated and UV-irradiated DNA templates. When replication was performed with DNA polymerase III* supplemented with increasing quantities of purified beta 2 subunit, the cycling time on UV-irradiated DNA decreased from 14.8 min at 1.7 nM beta 2 down to 6 min at 170 nM beta 2, a concentration in which beta 2 was in large excess over the polymerase. In parallel to the reduction in cycling time, also the bypass frequency of cyclobutane-photodime...
The Journal of biological chemistry, 1986
Replication of UV-irradiated oligodeoxynucleotide-primed single-stranded phi X174 DNA with Escherichia coli DNA polymerase III holoenzyme in the presence of single-stranded DNA-binding protein was investigated. The extent of initiation of replication on the primed single-stranded DNA was not altered by the presence of UV-induced lesions in the DNA. The elongation step exhibited similar kinetics when either unirradiated or UV-irradiated templates were used. Inhibition of the 3'----5' proofreading exonucleolytic activity of the polymerase by dGMP or by a mutD mutation did not increase bypass of pyrimidine photodimers, and neither did purified RecA protein influence the extent of photodimer bypass as judged by the fraction of full length DNA synthesized. Single-stranded DNA-binding protein stimulated bypass since in its absence the fraction of full length DNA decreased 5-fold. Termination of replication at putative pyrimidine dimers involved dissociation of the polymerase from ...
Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis, 1994
Overproduction of the 13 subunit of DNA polymerase III holoenzyme caused a 5-to 10-fold reduction of UV mutagenesis along with a slight increase in sensitivity to UV light in Escherichia coli. The same effects were observed in excision-deficient cells, excluding the possibility that they were mediated via changes in excision repair. In contrast, overproduction of the a subunit of the polymerase did not influence either UV mutagenesis or UV sensitivity. The presence of the mutagenesis proteins MucA and MucB expressed from a plasmid
Journal of Bacteriology, 1989
Cloning of the phi X174 viral origin of replication into phage M13mp8 produced an M13-phi X174 chimera, the DNA of which directed efficient replicative-form----single-strand rolling-circle replication in vitro. This replication assay was performed with purified phi X174-encoded gene A protein, Escherichia coli rep helicase, single-stranded DNA-binding protein, and DNA polymerase III holoenzyme. The nicking of replicative-form I (RFI) DNA by gene A protein was essentially unaffected by the presence of UV lesions in the DNA. However, unwinding of UV-irradiated DNA by the rep helicase was inhibited twofold as compared with unwinding of the unirradiated substrate. UV irradiation of the substrate DNA caused a strong inhibition in its ability to direct DNA synthesis. However, even DNA preparations that contained as many as 10 photodimers per molecule still supported the synthesis of progeny full-length single-stranded DNA. The appearance of full-length radiolabeled products implied at lea...
Journal of Biological Chemistry, 1994
W e have developed an experimental assay to monitor the rate of dissociation of the T4 DNA polymerase holoenzyme (polymerase plus gene 44/62 and 45 proteins) once it has been stalled by nucleotide omission. Using this assay, we determined that the dissociation of the DNA polymerase holoenzyme follows a first order decay with a half-life of 2.5 min. The long half-life resembles that expected for the holoenzyme processively synthesizing DNA on the leading strand of the replication fork. The holoenzyme dissociation rate is independent of polymerase accessory protein concentration and of ATP hydrolysis. The dissociation rate is increased if the gene 32 protein is omitted or if the primer-template region is shortened from 46 to 28 base pairs. But the rate of holoenzyme dissociation is most strikingly increased when the circular DNA template is converted to a linear form. By analogy with other well studied systems, these results support a model in which ATP hydrolysis by the 44/62 proteins serves to load a ring-like 45 protein onto the DNA. Once loaded, the 45 protein, possibly along with the 44/62 complex, acts as a sliding clamp that tethers the DNA polymerase to the template.