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R H Haynes

Publications and source records attributed to R H Haynes.

At least 73 records · Page 4Linked to original sources

Repair of UV-induced DNA damage and survival in yeast. I. Dimer excision.

The amount of pyrimidine dimer UV photoproduct lost from the DNA of irradiated yeast cells during dark incubation has been measured in various conditions. It was found that no dimers were lost when cells were incubated in saline. When the cells were incubated, with aeration, in a full growth medium, dimers were lost, most excision being complete within 4 h. Not all dimers were lost and the number lost was a function of UV dose. Maximum loss, amounting to 50 000 dimers per genome was observed after 4000 or 6000 erg/mm2 of UV. At higher doses, the number excised declined. Making the assumptions that dimers are the principal lethal product of UV, that a single dimer remaining in its genome is enough to prevent a cell from multiplying and that excision is the principal dark-repair process in yeast, these data were incorporated into the repair term of an expression relating survival to repair8 and it was found that the survival of yeast at doses up to 2000 erg/mm2 of UV could be quite accurately predicted. This is the first time it has been possible to account for survival in terms of measured repair. It is suggested that the divergence of the predicted and observed curves at higher doses is due to other processes known to exist in yeast.

DNA Repair↗

Changes in the ultraviolet sensitivity of Escherichia coli during growth in batch cultures.

The ultraviolet (UV) sensitivity of Escherichia coli B/r harvested at various times during growth in batch cultures was measured. The results showed a period of increased UV sensitivity in late log phase, just before the cultures entered stationary phase. This increase in sensitivity was associated with a decreased shoulder in the UV survival curves. The postirradiation division delay of survivors was shortest for cells harvested during the period of maximal sensitivity. This period of increased UV sensitivity during late log phase was not found in the radiation-sensitive, repair-deficient mutant B(s-1) (a strain which is unable to excise pyrimidine dimers from UV-damaged deoxyribonucleic acid). These results suggest that the variation in UV sensitivity of E. coli B/r as a function of time of harvesting of the cells from batch cultures is related to the varying capacities of these populations to repair UV-damaged deoxyribonucleic acid. Further experiments designed to elucidate the mechanism underlying this variation in UV sensitivity indicated that it arises from the partial depletion of nutrients in the medium during late log phase. We suggest that growth in such depleted media leads to a depression in the intercellular concentration or activity of one or more of the repair enzymes concerned with the repair of damaged deoxyribonucleic acid.

Cell Division↗

Repair-induced changes in yeast radiosensitivity.

Potentially lethal X-ray or ultraviolet damage in the diploid yeast, Saccharomyces cerevisiae, can be reversed if the irradiated cells are incubated in distilled water or buffer for a number of hours prior to plating. This phenomenon is called liquid-holding recovery. We found that the liquid-holding procedure served not only to restore the viability of the irradiated cells, but also to alter their sensitivity to further doses of radiation. Specifically, the ultraviolet sensitivity of cells which had undergone liquid-holding recovery was markedly decreased, whereas their X-ray sensitivity appeared to be slightly increased. These sensitivity changes were qualitatively the same irrespective of whether the initial radiation exposure was to X rays or ultraviolet light. (In contrast, the radiation sensitivity of cells which had undergone maximal photoreactivation was essentially the same as that of untreated controls.) It is suggested that these changes in radiosensitivity are the result of structural alterations induced in the cell's deoxyribonucleic acid by the execution of at least the initial steps of a deoxyribonucleic acid repair process during the liquid-holding period.

DNA↗

The repair of DNA.

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DNA, Bacterial↗

MluI site-dependent transcriptional regulation of the Candida albicans dUTPase gene.

The Candida albicans dUTP pyrophosphatase (dUTPase) gene DUT1 has been isolated by genetic complementation in S. cerevisiae. It was found to encode a 17-kDa protein similar in amino-acid sequence to dUTPases isolated from other systems. The gene was adapted for expression in E. coli and yielded a soluble and highly-active enzyme which is easily purified. The 5' flanking sequence of DUT1 contains an MluI site typical of MCB cell-cycle-dependent UAS elements of budding and fission yeast. We found the gene to be cell-cycle-regulated when expressed in S. cerevisiae, and deletion of the MluI site resulted in a large reduction of DUT1 transcription in C. albicans. These results suggest that MCB elements are functionally conserved in this pathogenic fungus. Based on the vital role that dUTPase plays in DNA replication, the C. albicans enzyme may be a potentially useful target for the development of novel anti-fungal compounds.

Amino Acid Sequence↗

Analysis of non-linearities in mutation frequency curves.

Mutation frequency curves for ultraviolet light and other mutagens often exhibit non-linear, as well as linear components. A common pattern observed for UV-induced reversion of auxotrophy in yeast is a biphasic, linear-quadratic (or higher order) response. The non-linear component in such a biphasic frequency curve can arise in two distinct, but non-mutually exclusive, ways: (i) as a result of the existence of two-hit processes in the molecular mechanism(s) of mutagenesis; and (ii) as a result of the possible stochastic dependence of mutation and killing, such that the probability of clone formation by the mutant cells differs from that of the non-mutant cells in the population. We describe here a simple mathematical method for distinguishing between these two sources of non-linearity. It is based on the calculation of a quantity that we call 'apparent survival.' This is given, for any mutagen dose chi, by the ratio of the mutant yield to the corresponding linear component of mutation frequency. If the apparent survival rises to values greater than unity before declining at high doses, then there must exist positive two-hit (or higher order) components in the mutational mechanism. If the final slope of the apparent survival curve differs from that of the measured survival curve, then there also exists some degree of stochastic dependence between mutation and killing.

Dose-Response Relationship, Radiation↗