W. Dean Rupp

Discontinuities in the DNA synthesized in an Excision-defective strain of Escherichia coli following ultraviolet irradiation

Although Escherichia coli K12 uvrA6 is defective in the excision of pyrimidine dimers from its DNA, 37% of cells survive a dose of ultraviolet light which is equivalent to about 50 pyrimidine dimers per 107 nucleotides. The amount of tritiated thymidine incorporated into the DNA of irradiated cells indicates that pyrimidine dimers in the DNA inhibit DNA synthesis but are not permanent blocks. Zone sedimentation of single-strand DNA was performed in alkaline sucrose gradients. To minimize degradation by shearing, the DNA was released from spheroplasts layered on top of the gradients. Newly synthesized, denatured DNA from unirradiated cells sediments with a molecular weight of greater than 100 × 106, whereas the newly synthesized, denatured DNA from cells irradiated with 60 ergs/mm2 has a molecular weight of about 14 × 106. During subsequent incubation of the irradiated cells, the sedimentation rate of the DNA synthesized immediately after irradiation increases and approaches that of normal DNA. However, at any time during this incubation period, the incorporation of tritiated thymidine into fast-sedimenting DNA is minimal, suggesting that the daughter-strand DNA synthesized after ultraviolet-irradiation contains gaps, or alkalilabile bonds. These dicontinuities disappear during further incubation, as a higher rate of sedimentation is found. The number of these daughter-strand defects is similar to the number of pyrimidine dimers in an equivalent length of parental DNA.

Exchanges between DNA strands in ultraviolet-irradiated Escherichia coli☆

Abstract Ultraviolet induced pyrimidine dimers remain in the DNA of excision-defective mutants of Escherichia coli for at least several hours after irradiation. If kept in the dark, the survival of colony-forming ability of these mutant bacteria depends upon recombinational repair, a mechanism which is controlled by the gene recA . In u.v.-irradiated bacteria, newly synthesized DNA strands are of low molecular weight, but these strands are subsequently joined into high molecular weight chains, as is shown by sedimenting the bacterial DNA in alkaline sucrose gradients. These longer chains may contribute to cell survival by acting as templates for the synthesis of high molecular weight strands during further replication. At times greater than 90 minutes after irradiation, the molecular weight of newly synthesized DNA covers a wide range and extends fully to the maximum found with unirradiated cells, thus indicating the presence of high molecular weight templates virtually free from damaged bases. Evidently the u.v. photoproducts tend to remain in the original strands where they were formed, rather than becoming distributed equally among all the strands with each subsequent replication. Recombinational repair may depend upon genetic exchanges between sister duplexes. To see whether such exchanges could be detected after u.v.-irradiation, cells were grown for several generations in medium containing 13 C and 15 N. They were then exposed to various doses of ultraviolet light and grown for less than a generation in light medium containing [ 3 H]thymidine, so that part of their DNA was hybrid in density, with the 3 H label in the light strand. Under these conditions, light 3 H-labeled chains will become linked to heavy strands if exchanges occur between sister duplexes. The DNA was extracted from unirradiated cells, heat denatured and centrifuged in neutral cesium chloride gradients, and was found to separate into heavy and light bands. Denatured DNA from u.v.-irradiated cells did not separate completely, but contained strands of intermediate density which separated into heavy and light components only after shearing to 5 × 10 5 molecular weight, thus indicating that segments of molecular weight greater than 5 × 10 5 had been exchanged. To judge from the fraction of the 3 H-labeled molecules that were of intermediate density, one exchange occurred for every one to two pyrimidine dimers in the DNA replicated in the 3 H-labeled medium. Sister exchanges involving a single strand of each duplex, may insert the correct bases sequence into the gap opposite each pyrimidine dimer, and thus promote the reconstruction of a genome with the complete base sequence needed for the survival of colony-forming ability.

Identification of the uvrA gene product

Abstract The proteins synthesized by the plasmid pDR2000, a recombinant plasmid with the Escherichia coli uvrA and ssb (lexC) genes cloned on pBR322, were examined using the maxicell procedure developed by Sancar et al. (1979). The proteins synthesized by this plasmid include those expected from pBR322 plus two others, a small protein (Mr = 18,500) and a large one (Mr = 114,000). The molecular weight and DNA binding properties of the 18,500 Mr protein indicate that it is the product of the ssb gene. Derivatives of pDR2000 were prepared in which the uvrA gene was inactivated by insertion of the γδ transposon (Tn1000). In each of these derivatives the 114,000 Mr protein was not made, indicating that it is the product of the uvrA gene. By extrapolating from the relative amounts of ssb and uvrA proteins synthesized in maxicells, we estimate that a normal cell contains only about 20 uvrA polypeptide chains. The uvrA protein binds to both single and double-stranded DNA. A procedure is described for purifying uvrA protein to radiochemical homogeneity, and subsequent gel filtration of this isolated native uvrA protein shows that it is a monomer. When γδ is inserted into the uvrA gene, truncated uvrA polypeptides resulting from premature termination at nonsense codons in the inserted sequence are observed. From the sizes of these truncated polypeptides and the sites of the insertions determined by restriction mapping, the direction of transcription of the uvrA gene was determined to be counter-clockwise on the E. coli genetic map.

The uvrB gene of Escherichia coli has both lexA-repressed and lexA-independent promoters

We have found that the uvrB gene of Escherichia coli is transcribed from at least two promoters, which we call P1 and P2. Transcription from P1 begins with an ATP at +1, and transcription from P2 begins primarily with a GTP at -31. A binding site for the lexA protein (LEXA), located between the -35 sequence and Pribnow box of P2, regulates transcription from this promoter. In vitro, LEXA inhibits transcription from P2 but has no detectable effect on transcription from P1. A third promoter, P3, was also detected at -341; transcription from P3 is toward uvrB but terminates in vitro in the region of the LEXA binding site. The binding of LEXA to P2 inhibits transcription from the P3 promoter even though several hundred nucleotides separate the two promoters. The data suggest that a transcribing RNA polymerase stalls when it reaches the repressor-operator complex but remains bound to the DNA, causing a jamming of RNA polymerases between P3 and the repressor-operator complex at P2. The physiological significance of P3 is unknown.

Identification of the uvrB gene product

Abstract We have constructed plasmids carrying various restriction fragments of the biouvrB region of the Escherichia coli chromosome. By analyzing the proteins synthesized in maxicells from the uvrB + plasmid pDR1494, and its derivatives containing γδ sequences inserted in the uvrB gene, we have determined that the uvrB gene is about two kilobase-pairs in length, that it is transcribed clockwise on the standard E. coli genetic map, and that it codes for a single polypeptide of M r = 84,000. The number of uvrB polypeptides in a normal cell is estimated to be about 140. We have also found that the uvrB gene is cut by Eco RI near its promoter.

Cloning of uvrA, lexC and ssb genes of Escherichia coli.

Abstract We have constructed a recombinant plasmid carrying a DNA fragment of the E. coli chromosome that specifically complements the uvrA , lexC and ssb mutations of this bacterium. Preliminary experiments indicate that this complementation is due to the presence of the structural genes on this plasmid.

Asymmetric segregation of the complementary sex-factor DNA strands during conjugation in Escherichia coli☆

Abstract When breaks are introduced into double-stranded covalently closed circular sex-factor DNA molecules, the complementary strands can be separated in a CsCl-poly (U, G) equilibrium density gradient. Using this technique, double-stranded circular sex-factor DNA isolated from donor and recipient cells after mating was analyzed to determine the fate of the complementary strands. Only one strand of the sex-factor DNA is transferred from donor to recipient cells, this being the denser strand in CsCl-poly (U, G). A complement to this strand is synthesized in the recipient and a covalently closed sex-factor DNA molecule is formed. The sex-factor strand not transferred to the recipient remains in the donor where it acquires a complement during mating and forms a covalently closed double-stranded circle. These results show that DNA synthesis associated with mating occurs in both the donor and recipient cells. The asymmetric segregation of the complementary sex-factor DNA strands could be generated by a mechanism such as the rolling circle model of DNA synthesis proposed by Gilbert & Dressler (1968).

Identification of individual sex-factor DNA strands and their replication during conjugation in thermosensitive DNA mutants of Escherichia coli

Abstract Covalent circular sex-factor DNA has been isolated from donor and recipient cells during the conjugation of normal and temperature-sensitive DNA mutants of Escherichia coli . Single strands of sex-factor DNA were centrifuged in cesium chloride-poly(U,G) gradients to give two components that have been identified by annealing experiments as the separated complementary strands. When matings are performed with either DNA temperature-sensitive donor or recipient cells, the inhibition of vegetative DNA synthesis at the restrictive temperature does not interfere with transfer and circularization of the sex-factor DNA. If DNA is isolated from temperature-sensitive donor cells mated at the restrictive temperature, a specific stimulation of sex-factor DNA synthesis can be demonstrated. By separating the complementary strands of the sex-factor in a cesium chloride-poly (U,G) gradient, this DNA synthesis has been found to be asymmetric. The sex-factor DNA strand which is synthesized in the donor has the same polarity as the strand which is transferred to the recipient.

Properties and regulation of the UVRABC endonuclease

This report summarizes the cloning of the uvrA, uvrB and uvrC genes of E. coli, the identification and isolation of the gene products, the regulation of the genes, and reconstitution of active UVRABC endonuclease from the individually isolated components.

Analysis of mRNA synthesis following induction of the Escherichia coli SOS system.

Escherichia coli responds to impairment of DNA synthesis by inducing a system of DNA repair known as the SOS response. Specific genes are derepressed through proteolytic cleavage of their repressor, the lexA gene product. Cleavage in vivo requires functional RecA protein in a role not yet understood. We used mRNA hybridization techniques to follow the rapid changes that occur with induction in cells with mutations in the recA operator or in the repressor cleavage site. These mutations allowed us to uncouple the induction of RecA protein synthesis from its role in inducing the other SOS functions. Following induction with ultraviolet light, we observed increased rates of mRNA synthesis from five SOS genes within five minutes, maximum expression ten to 20 minutes later and then a later decline to near the initial rates. The presence of a recA operator mutation did not significantly influence these kinetics, whereas induction was fully blocked by an additional mutation in the repressor cleavage site. These experiments are consistent with activation of RecA protein preceding repressor cleavage and derepression of SOS genes. The results also suggest that the timing and extent of induction of individual SOS genes may be different.