Ann K. Ganesan

Persistence of pyrimidine dimers during post-replication repair in ultraviolet light-irradiated Escherichia coli K12

We have used a new assay for pyrimidine dimers to obtain evidence regarding the mechanism of post-replication repair of ultraviolet light-induced damage in excision-deficient (uvr) mutants of Escherichia coli. Our data indicate that dimers are gradually removed from the irradiated DNA under conditions permitting post-replication repair. Concomitantly, dimers appear in daughter strands synthesized after irradiation. The daughter strands initially contain gaps. During post-replication repair the gaps are filled and the originally discontinuous DNA is joined into long molecules resembling those observed in unirradiated control cells. Density transfer experiments reported by other investigators have provided evidence that the gap-filling involves exchanges between irradiated parental DNA and unirradiated daughter strands. The results of our experiments are in accord with this possibility and suggest that some dimers are included in the exchanged regions. Our data imply that intact, dimer-free DNA molecules are not necessarily generated by gap-filling and may not appear in uvr cells until several hours after u.v. irradiation. Instead, dimers may be gradually diluted among successive generations of DNA molecules synthesized after irradiation.

The effect of lexA and recF mutations on post-replication repair and DNA synthesis in Escherichia coli K-12

SummaryWe have examined lexA1 uvr A6 and recF143 uvr Bδ derivatives of Escherichia coli K-12 for post-replication repair and DNA synthesis after UV irradiation. Compared to corresponding lex+ rec+strains, we found that the lexA and recF cells were defective in (1) conveting short DNA segments synthesized after irradiation to DNA of normal size; (2) synthesizing high molecular weight DNA after irradiation; (3) transferring pyrimidine dimers from irradiated DNA into unirradiated daughter strands. Our results support the hypothesis that after UV irradiation the formation of large DNA molecules in excision-deficient cells of E. coli depends directly or indirectly upon joining short DNA segments into longer strands, concomitant with the transfer of DNA from irradiated tamplates into unirradiated daughter strands. This process appears to require the activity of lexA and recF genes.

Transport systems for galactose and galactosides in Escherichia coli: I. Genetic determination and regulation of the methyl-galactoside permease

Four transport systems for galactose and galactosides in Escherichia coli are known. The results presented here show that the gene, P-MG , determining one of these, the methyl-galactoside permease, is linked to the His marker on the bacterial chromosome. In addition, evidence for a regulator gene controlling the inducibility of this sytem is given. This regulator gene, R-MG , is distinct from regulator genes of both the Lac and Gal operons. It can be co-transduced with Gal by the phage λ.

Enhanced transformation of human cells by UV-irradiated pSV2 plasmids.

Irradiating the plasmid pSV2-gpt with UV (254 nm) doses up to 200 J m-2 caused a dose-dependent increase in the yield of Gpt+ transformants when the plasmid was introduced into human cells by calcium phosphate coprecipitation. UV doses greater than 1 kJ m-2 were required to reduce the efficiency of transformation below that obtained with unirradiated DNA.

Transcription-coupled DNA repair in prokaryotes.

Transcription-coupled repair (TCR) is a subpathway of nucleotide excision repair (NER) that acts specifically on lesions in the transcribed strand of expressed genes. First reported in mammalian cells, TCR was then documented in Escherichia coli. In this organism, an RNA polymerase arrested at a lesion is displaced by the transcription repair coupling factor, Mfd. This protein recruits the NER lesion-recognition factor UvrA, and then dissociates from the DNA. UvrA binds UvrB, and the assembled UvrAB* complex initiates repair. In mutants lacking active Mfd, TCR is absent. A gene transcribed by the bacteriophage T7 RNA polymerase in E. coli also requires Mfd for TCR. The CSB protein (missing or defective in cells of patients with Cockayne syndrome, complementation group B) is essential for TCR in humans. CSB and its homologs in higher eukaryotes are likely functional equivalents of Mfd.

The complex choreography of transcription-coupled repair.

A quarter of a century has elapsed since the discovery of transcription-coupled repair (TCR), and yet our fascination with this process has not diminished. Nucleotide excision repair (NER) is a versatile pathway that removes helix-distorting DNA lesions from the genomes of organisms across the evolutionary scale, from bacteria to humans. TCR, defined as a subpathway of NER, is dedicated to the repair of lesions that, by virtue of their location on the transcribed strands of active genes, encumber elongation by RNA polymerases. In this review, we will report on newly identified proteins, protein modifications, and protein complexes that participate in TCR in Escherichia coli and in human cells. We will discuss general models for the biochemical pathways and how and when cells might choose to utilize TCR or other pathways for repair or bypass of transcription-blocking DNA alterations.

Enhanced transforming activity of pSV2 plasmids in human cells depends upon the type of damage introduced into the plasmid

When pSV2-gpt or pSV2-neo plasmids are introduced into human cells by calcium phosphate coprecipitation, the yield of stable transformants (Gpt+ or Neo+) is increased by irradiating the respective plasmid DNA in vitro with UV (254 nm). To identify specific lesions that can increase the transforming activity of plasmids in human cells we examined pSV2 plasmids containing different types of damage. Of the lesions tested, cyclobutane pyrimidine dimers produced the greatest increase, and can nearly fully account for the effect of 254 nm UV on transformation. The enhancement of transformation produced by UV was not altered by the additional treatment of the plasmid DNA with T4 endonuclease V, an enzyme that nicks DNA specifically at pyrimidine dimers. Treatment of plasmid DNA with osmium tetroxide to produce thymine glycols, or with acid and heat to produce apurinic sites did not affect transformation frequency. The enhancement occurred in all the human cell lines tested, whether they contained or not sequences homologous to those in the plasmids, and was independent of the repair capacity of the recipient cells.

Expression and nucleotide excision repair of a UV-irradiated reporter gene in unirradiated human cells

It has been suggested that reactivation of damaged reporter genes introduced into cultured mammalian cells reflects transcription-coupled nucleotide excision repair. To evaluate this possibility directly, we introduced a UV-irradiated shuttle vector, pCMV beta, into unirradiated human cells and compared expression of the reporter gene (lacZ) with repair of cyclobutane pyrimidine dimers (CPDs). Expression of the irradiated reporter gene was more UV resistant in XPC cells, which are deficient in global genome repair, than in CSB cells, which are deficient in transcription-coupled repair. These results are consistent with the idea that repair of the reporter gene is primarily dependent upon transcription-coupled repair. However, when the plasmid DNA was analyzed for removal of CPDs, no clear evidence was obtained for transcription-coupled repair either in XPC cells or in cells with normal repair capacity.

[25] Purification and properties of a pyrimidine dimer-specific endonuclease from E. coli infected with bacteriophage T4

Publisher Summary The chapter explains purification and properties of a pyrimidine dimer-specific endonuclease from E. coli infected with bacteriophage T4. The pyrimidine dimer-specific endonuclease of bacteriophage T4 is coded by the v (denV) gene. The enzyme has been called endonuclease V of phage T4 or the T4 uv endonuclease. For detecting pyrimidine dimers a preparation of enzyme free of activity against unirradiated DNA or DNA containing other types of damage is required. The chapter describes several methods for assaying T4 endonuclease V as well as the partial purification and general properties of the enzyme. The chapter discusses a reproducible procedure for obtaining stable preparations of the enzyme which can be used as a specific probe for pyrimidine dimers. For the purpose of purification, the enzyme can be assayed by one of three general methods— namely, coupled nuclease assay, filter binding assay, and DNA nicking assay. The chapter further discusses enzyme purification.

The duration of recovery and DNA repair in excision deficient derivatives of Escherichia coli K-12 after ultraviolet irradiation

SummaryOur results indicate that cells of excision deficient (uvr) mutants of Escherichia coli K-12 which survive exposure to ultraviolet radiation may require several hours to complete their recovery. For example, the duration of the recovery period for cells exposed to 63 ergs mm-2 at 254 nm was about 5 hours, the equivalent of slightly more than 4 generations of the unirradiated controls. During the recovery period the rate of cell division was reduced (Figs. 3 and 4), the cells gradually regained resistance to complex medium (Figs. 1 and 3), and they became refractory to photoreactivation (Fig. 1). Over the same period of time their pattern of DNA synthesis changed. More intact molecules, similar to those found in unirradiated controls, and relatively fewer discontinuous molecules were synthesized (Figs. 6 and 7).