Tzu-chien V. Wang

Enzymatic Photoreactivation of Escherichia coli after Ionizing Irradiation: Chemical Evidence for the Production of Pyrimidine Dimers

By comparing the results for a uvrA recAand a uvrA uvrB recA phr strain, we conclude that the photoreactivation (PR) observed after ionizing irradiation involves the same enzyme that is responsible for the PR of ultraviolet (uv) radiation-induced cyclobutadipyrimidines (Py Py). A comparison of the photoreactivable sectors in a uvrA recA strain after uv irradiation and 137Cs γ irradiation indicates that 10 krad of γ radiation produces about 0.07 Jm−2-equivalents of 254-nm radiation-induced photoreactivable damage. After 400 krad, an acid hydrolysate of the isolated DNA revealed the presence of thymine-containing Py Py, as evidenced by their chromatographic properties and their photochemical reversibility. The yield of thymine dimers after 400 krad of γ irradiation, as detected chromatographically, was in close agreement with the yield that was predicted from our PR studies. Thus, the PR observed after ionizing irradiation in strains that are blocked in excision repair and in postreplication repair is d...

Effect of recB21, uvrD3, lexA101 and recF143 mutations on ultraviolet radiation sensitivity and genetic recombination in ΔuvrB strains of Escherichia coli K-12

SummaryThe interaction of the recB21, uvrD3, lexA101, and recF143 mutations on UV radiation sensitization and genetic recombination was studied in isogenic strains containing all possible combinations of these mutations in a ΔuvrB genetic background. The relative UV radiation sensitivities of the multiply mutant strains in the ΔuvrB background were: recF recB lexA> recF recB uvrD lexA, recF recB uvrD>recA>recF uvrD lexA> recF recB, recF uvrD>recF lexA>recB uvrD lexA>recB uvrD> recB lexA, lexA uvrD>recB>lexA, uvrD>recF; three of these strains were more UV radiation sensitive than the uvrB recA strain. There was no correlation between the degree of radiation sensitivity and the degree of deficiency in genetic recombination. An analysis of the survival curves revealed that the recF mutation interacts synergistically with the recB, uvrD, and lexA mutations in UV radiation sensitization, while the recB, uvrD, and lexA mutations appear to interact additively with each other. We interpret these data to suggest that there are two major independent pathways for postreplication repair; one is dependent on the recF gene, and the other is dependent on the recB, uvrD, and lexA genes.

Mechanism of sbcB-suppression of the recBC-deficiency in postreplication repair in UV-irradiated Escherichia coli K-12

SummaryThe mechanism by which an sbcB mutation suppresses the deficiency in postreplication repair shown by recB recC mutants of Escherichia coli was studied. The presence of an sbcB mutation in uvrA recB recC cells increased their resistance to UV radiation. This enhanced resistance was not due to a suppression of the minor deficiency in the repair of DNA daughter-strand gaps or to an inhibition of the production of DNA double-strand breaks in UV-irradiated uvrA recB recC cells; rather, the presence of an sbcB mutation, enabled uvrA recB recC cells to carry out the repair of DNA double-strand breaks. In the uvrA recB recC sbcB background, a mutation, at recF produced a huge sensitization to UV radiation, and it rendered cells deficient in the repair of both DNA daughter-strand gaps and DNA double-strand breaks. Thus, an additional sbcB mutation in uvrA recB recC cells restored their ability to perform the repair of DNA double-strand breaks, but the further addition of a recF mutation blocked this repair capacity.

Discontinuous DNA replication in a lig-7 strain of Escherichia coli is not the result of mismatch repair, nucleotide-excision repair, or the base-excision repair of DNA uracil

After pulse-labeling with 3H-thymidine for 30 s at 42 degrees C, the newly-synthesized DNA from uvrB5 lig-7, uvrB5 lig-7 ung-1 (or ung152), uvrB5 lig-7 mutL218 (or mutS215), and uvrB5 lig-7 ung-1 mutL218 (or mutS215) cells sedimented very slowly in alkaline sucrose gradients. The bulk of these DNA molecules were smaller than 2,000 nucleotides long (i.e., about the size of Okazaki fragments), and none of the 3H-radioactivity was found to sediment as high-molecular-weight DNA. These results indicate that the apparent discontinuous DNA replication observed in lig-7 strains is not the result of mismatch repair, nucleotide-excision repair, or the base-excision repair of DNA uracil.

The roles of RecBCD, Ssb and RecA proteins in the formation of heteroduplexes from linear-duplex DNA in vitro

SummaryThe formation of heteroduplexes from linear duplex DNA, where one molecule possesses a DNA doublestrand break, was assayed by agarose gel electrophoresis. Using unlabeled whole-length linear duplex DNA and 3H-labeled half-length linear duplex DNA (obtained from plasmid pACYC184), the appearance of 3H-labeled DNA that migrated as whole-length linear DNA was taken as evidence for formation of heteroduplex DNA. When the DNA mixtures were incubated with RecA, RecBCD, or Ssb proteins, or any double or triple combination of these proteins under a variety of reaction conditions, no heteroduplex DNA was detected. However, heteroduplex DNA was detected when the DNA mixtures were first incubated briefly with the RecBCD and Ssb proteins under reaction conditions that allow unwinding to proceed, and then the MgCl2 concentration was raised such that renaturation could proceed. The inclusion of the RecBCD and Ssb proteins was sufficient to catalyze the slow formation of heteroduplex DNA, but the presence of RecA protein greatly increased the kinetics. The roles of the RecBCD, Ssb and RecA proteins in heteroduplex formation in vitro are discussed.

Rich growth medium enhances ultraviolet radiation sensitivity and inhibits cell division in ssb mutants of Escherichia coli K-12

Abstract— –DNA single‐strand binding protein mutants (ssb) of Escherichia coli K‐12 exhibit much greater UV radiation sensitivity when plated on rich medium (RM) than when plated on minimal medium (MM). However, when comparing UV‐irradiated ssb‐113 cells (previously known as exrB and lexC113) incubated in RM vs MM, no difference was observed in DNA degradation or in the repair of incision breaks that arose during excision repair. Although UV‐irradiated ssb‐113 cells incubated in RM did resume DNA synthesis slightly sooner than those incubated in MM, and there was a small increase in the production of DNA double‐strand breaks in these cells, the most dramatic difference noted, however, was the much enhanced filamentation of irradiated cells incubated in RM vs MM. Therefore, we suggest that most of the RM‐enhanced U V radiation sensitivity in ssb‐113 cells is due to an inhibitory effect of RM on cell division rather than on DNA repair.

PHOTOREACTIVATION OF Escherichia coli IRRADIATED WITH IONIZING RADIATION

ABSTRACT By comparing the results for a uvrA recA and a uvrA uvrB recA phr strain, we conclude that the photoreactivation after ionizing irradiation involves the same enzyme that is responsible for the photoreactivation of UV-induced cyclobutadipyrimidines. A comparison of the photoreactivable sectors in a uvrA recA strain after UV and 137Cs-y-irradiation indicates that 10 krads of γ-radiation produces about 0.07 Jm−2-equivalents of 254 nm-induced photoreactivable damage. After 400 krads, an acid hydrolysate of the isolated DNA revealed the presence of thymine-containing cyclobutadipyrimidines, as evidenced by their chromatographic properties and their photochemical reversibility. Thus, the photoreactivation observed after ionizing radiation in strains that are blocked in excision repair and in postreplication repair is due to the production of trace amounts of cyclobutadipyrimidines.

New DNA Repair Systems and New Insights on Old Systems in Escherichia coli

One can deduce the extreme importance of maintaining the integrity of cellular DNA, simply by noting the numerous and diverse types of systems that a cell has at its disposal for the repair of damaged DNA (for reviews, see references 1-3). There is a repair system that requires visible light (i. e., photoreactivation), and several systems that can work in the absence of light. There are repair systems that can function in the absence of DNA replication (e. g., excision repair), and systems that can only function after damaged DNA has been replicated (i. e., postreplication repair). There are systems for the repair of DNA base damage, and systems for the repair of single-strand and double-strand breaks in DNA. Certain alterations in DNA can be repaired by more than one type of repair system, suggesting that cells have “backup ” systems for DNA repair. Some of these repair systems are constitutive and some are inducible. Finally, some of these repair systems are error-free and some are error-prone (i. e., the repair is not accurate and, therefore, produces mutations). Within the space limitations for this review, we will describe some new DNA repair systems and discuss new insights on some old repair systems in Escherichia coli.