A 5'- to 3'-exonuclease of about 45 kDa has been purified from various mammalian sources and shown to be required for the completion of lagging strand synthesis in reconstituted DNA replication systems. RTH1 encodes the yeast Saccharomyces cerevisiae counterpart of the mammalian enzyme. To determine the in vivo biological role of RTH1-encoded 5'- to 3'-exonuclease, we have examined the effects of an rth1 delta mutation on various cellular processes. rth1 delta mutants grow poorly at 30 degrees C, and a cessation in growth occurs upon transfer of the mutant to 37 degrees C. At the restrictive temperature, the rth1 delta mutant exhibits a terminal cell cycle morphology similar to that of mutants defective in DNA replication, and levels of spontaneous mitotic recombination are elevated in the rth1 delta mutant even at the permissive temperature. The rth1 delta mutation does not affect UV or gamma-ray sensitivity but enhances sensitivity to the alkylating agent methyl methanesulfonate. The role of RTH1 in DNA replication and in repair of alkylation damage is discussed.

The RAD3 gene of Saccharomyces cerevisiae is required for excision repair of ultraviolet-damaged DNA and is essential for cell viability. The RAD3-encoded protein shares a high degree of homology with the human ERCC2(XPD) gene product. Mutations in XPD, besides causing the cancer-prone syndrome xeroderma pigmentosum, can also result in Cockayne's syndrome and trichothiodystrophy. To investigate the role of RAD3 in viability, we examined here the effect of a recessive, temperature-sensitive (ts) conditional lethal mutation of the gene on transcription by RNA polymerase II. Upon transfer to the restrictive temperature, the rad3-ts mutant rapidly ceases growth and poly(A)+ RNA synthesis is inhibited drastically. Messenger RNA levels of all the genes examined, HIS3, TRP3, STE2, MET19, RAD23, CDC7, CDC9 and ACT1, decline rapidly upon loss of RAD3 activity. The synthesis of heat-shock-inducible HSP26 mRNA and galactose-inducible GAL7 and GAL10 mRNAs is also drastically inhibited in the rad3-ts mutant at the restrictive temperature. The RNA polymerase II transcriptional activity in extract from the rad3-ts14 strain is thermolabile, and this in vitro transcriptional defect can be fully corrected by the addition of homogeneous RAD3 protein. These findings indicate that RAD3 protein has a direct and essential role in RNA polymerase II transcription.

An outbreak strain of Escherichia coli O157:H7 was inoculated onto closely related but structurally distinct types of lettuce (Lactuca sativa): Boston (butterhead lettuce), iceberg (crisphead lettuce), and green leaf and red leaf (colored variants of looseleaf lettuce). The E. coli O157:H7 was inoculated either onto the surface of cut leaf pieces or into a homogenized leaf suspension. Samples were gamma irradiated, and the radiation sensitivity of the inoculated bacteria was expressed as a D-value (the amount of ionizing radiation necessary to reduce the bacterial population by 90% [kGy]). The recovery of bacteria from nonirradiated leaf pieces was also measured. When inoculated onto the leaf surface, E. coli O157:H7 had significantly stronger radiation sensitivity on red leaf lettuce (D = 0.119 +/- 0.004 [standard error]) and green leaf lettuce (D = 0.123 +/- 0.003) than on iceberg lettuce (D = 0.136 +/- 0.004) or Boston lettuce (D = 0.140 +/- 0.003). When E. coli O157:H7 was inoculated into a homogenized leaf suspension, its sensitivity was significantly stronger on iceberg lettuce (D = 0.092 +/- 0.002) than on green leaf lettuce (D = 0.326 +/- 0.012), Boston lettuce (D = 0.331 +/- 0.009), or red leaf lettuce (D = 0.339 +/- 0.010), with a threefold difference. Significantly fewer bacteria were recovered from the surface of iceberg lettuce than from the surfaces of the other types of lettuce examined. Following radiation doses of up to 0.5 kGy, the texture (maximum shear strength) of lettuce leaves was measured along the midrib and along the leaf edge for each type of lettuce. There was no meaningful change in texture for any type of lettuce for either leaf section examined at any dose up to 0.5 kGy. These data show (i) that relatively subtle differences between lettuce types can significantly influence the radiation sensitivity of associated pathogenic bacteria and (ii) that doses of up to 0.5 kGy do not soften lettuce leaves.

Listeria monocytogenes, a psychrotrophic foodborne pathogen, is a frequent postprocessing contaminant of ready-to-eat (RTE) meat products, including frankfurters and bologna. Ionizing radiation can eliminate L. monocytogenes from RTE meats. When they are incorporated into fine-emulsion sausages, sodium diacetate (SDA) and potassium lactate (PL) mixtures inhibit the growth of L. monocytogenes. The radiation resistance of L. monocytogenes, and its ability to proliferate during long-term refrigerated storage (9 degrees C), when inoculated into beef bologna that contained 0% SDA-0% PL, 0.07% SDA-1% PL, and 0.15% SDA-2% PL, were determined. The radiation doses required to eliminate 90% of the viable L. monocytogenes cells were 0.56 kGy for bologna containing 0% SDA-0% PL, 0.53 kGy for bologna containing 0.07% SDA-1% PL, and 0.46 kGy for bologna containing 0.15% SDA-2% PL. L. monocytogenes was able to proliferate on bologna containing 0% SDA-0% PL during refrigerated storage, but the onset of proliferation was delayed by the addition of the SDA-PL mixtures. An ionizing radiation dose of 3.0 kGy prevented the proliferation of L. monocytogenes and background microflora in bologna containing 0.07% SDA-1% PL and in bologna containing 0.15% SDA-2% PL over 8 weeks of storage at 9 degrees C. Little effect on lipid oxidation and color of the control bologna, or bologna containing SDA-PL mixtures, was observed upon irradiation at either 1.5 or 3.0 kGy.

Nucleotide excision repair (NER) in eukaryotes requires the assembly of a large number of protein factors at the lesion site which then coordinate the dual incision of the damaged DNA strand. However, the manner by which the different protein factors are assembled at the lesion site has remained unclear. Previously, we have shown that in the yeast Saccharomyces cerevisiae, NER proteins exist as components of different protein subassemblies: the Rad1-Rad10 nuclease, for example, forms a tight complex with the damage recognition protein Rad14, and the complex of Rad1-Rad10-Rad14 can be purified intact from yeast cells. As the Rad1-Rad10 nuclease shows no specificity for binding UV lesions in DNA, association with Rad14 could provide an effective means for the targeting of Rad1-Rad10 nuclease to damage sites in vivo. To test the validity of this idea, here we identify two rad1 mutations that render yeast cells as UV sensitive as the rad1⌬ mutation but which have no effect on the recombination function of Rad1. From our genetic and biochemical studies with these rad1 mutations, we conclude that the ability of Rad1-Rad10 nuclease to associate in a complex with Rad14 is paramount for the targeting of this nuclease to lesion sites in vivo. We discuss the implications of these observations for the means by which the different NER proteins are assembled at the lesion site.Nucleotide excision repair (NER) is a highly conserved repair system among eukaryotes. In the yeast Saccharomyces cerevisiae, a combination of Rad14, Rad4-Rad23, RPA, TFIIH, Rad1-Rad10, and Rad2 mediates the dual incision of the damaged DNA strand, releasing an ϳ30-nucleotide (nt) lesioncontaining DNA fragment (6). Rad14, Rad4-Rad23, and RPA function at the damage recognition step. The Rad3 and Rad25 DNA helicases, which are the components of TFIIH, unwind the duplex DNA around the lesion, and the Rad1-Rad10 and Rad2 nucleases incise the damaged DNA strand on the 5Ј and the 3Ј side of the lesion, respectively (24). In humans, a combination of their respective counterparts, XPA, XPC-HR23B, RPA, TFIIH, XPF-ERCC1, and XPG, mediates the dual incision of the damaged DNA strand (19,20). Similar to that in yeast, in humans, XPA, XPC-HR23B, and RPA act in damage recognition, the XPD and XPB helicases in TFIIH unwind the damaged duplex, and the XPF-ERCC1 and XPG nucleases incise the damaged strand on the 5Ј and the 3Ј side of the lesion, respectively (26, 27).Previously, we have shown that the yeast NER proteins exist in vivo as components of separate protein subassemblies, named nucleotide excision repair factors (NEFs) 1 to 4. NEF1 is comprised of the damage recognition protein Rad14 and the Rad1-Rad10 nuclease, which incises the damaged strand on the 5Ј side of the lesion (7). NEF2, the Rad4-Rad23 complex (6), is involved in damage recognition, and NEF3, comprised of TFIIH and the Rad2 endonuclease (12), promotes DNA unwinding and 3Ј incision (24). In yeast, the repair of the nontranscribed DNA strand and of transcriptionally inactive regions additionally req...

The RADI and RADIO genes of Saccharomyces cerevisiae are required for excision repair of ultraviolet light-damaged DNA, and they also function in a mitotic recombination pathway that is distinct from the double-strandbreak recombination pathway controlled by RAD52. Here, we show that the RAD1 and RADi1 proteins are complexed with each other in vivo. Immunoprecipitation of yeast cell extracts with either anti-RAD1 antibody or anti-RAD10 antibody coprecipitated quantitative amounts of both RAD1 and RADiO proteins. The level of coprecipitable RAD1 and RADiO increased when both proteins were overproduced together, but not if only one of the proteins was overproduced. The RAD1/RAD10 complex is highly stable, being refractory to 1 M NaCl and to low concentrations of SDS. By hydroxylamine mutagenesis, we have identified a radi mutant allele whose encoded protein fails to complex with RADiO. The interactiondefective radi mutant resembles the radi or radiO null mutant in defective DNA repair and recombination, implying that complex formation is essential for the expression of biological activities controlled by RADI and RADIO.In humans, the genetic disorder xeroderma pigmentosum (XP) causes extreme sensitivity to sunlight and XP individuals suffer from a high incidence of skin cancers. XP cells exhibit defects in the incision step of excision repair of DNA damaged by ultraviolet (UV) light. Cell fusion studies with skin fibroblasts from XP patients have identified seven XP complementation groups, A-G (1, 2). Eight complementation groups have been identified among UV-sensitive Chinese hamster ovary (CHO) cell lines, and mutants from five of these groups are incision-defective (3). Cross-complementation of UV sensitivity of mutant CHO cell lines by human DNA has led to the isolation of the ERCCI, ERCC2, and ERCC3 genes (4-6), and ERCC3 and XP-B represent the same gene (6). Recently, the XPAC cDNA was cloned by using as hybridization probe a mouse gene that corrects the excision repair defect of XP-A cell lines (7).The yeast Saccharomyces cerevisiae resembles humans in the genetic complexity of excision repair, and at least 10 genes-RADJ, RAD2, RAD3, RAD4, RADIO, RADI4, RAD7, RAD16, RAD23, and MMSJ9-are involved in this process. The first 6 of these genes are indispensable for the incision step of repair and they very likely encode proteins that collectively mediate the recognition and endonucleolytic scission of damaged DNA (8, 9). Characterization ofgenomic deletion mutations has revealed a role of some of these genes in cellular processes unrelated to excision repair. Specifically, RAD3 is also essential for cell viability (10, 11), and RADI and RADIO function in a mitotic recombination pathway that is distinct from the RAD52 double-strand-break recombination pathway (12).The proteins encoded by the human ERCCI, ERCC2, and XPAC genes share remarkable homology with the products of the yeast RADIO, RAD3, and RADI4 genes, respectively (4,5,9), and a yeast gene homologous to the human ERCC3 (XPBC) gene has also been ...

Frankfurter is a generic term for a cured and cooked sausage, which may consist of almost any meat type and include a wide variety of nonmeat fillers and additives. When several “brands” or types of commercially available frankfurters were surface‐inoculated with Listeria monocytogenes and vacuum‐packed, gamma radiation D‐values ranged from 0.49 kGy to 0.71 kGy, with an average D‐value of 0.61 kGy. Differences in gamma radiation D‐value were observed for nine of twenty one pair‐wise comparisons (α= 0.01) as determined by analysis of covariance. Therefore, frankfurter formulation may affect radiation D‐values for surface inoculated L. monocytogenes. If low dose gamma irradiation, cold pasteurization, were to be used for control of L. monocytogenes on frankfurters, gamma radiation dosage should be based on individual product formulation.

Ultraviolet Light (254 nm) is a U.S. Food and Drug Administration‐approved nonthermal intervention technology that can be used for decontamination of food surfaces. In this study, the use of ultraviolet light (UV‐C) at doses of 0.5–4.0 J/cm2 to inactivate a cocktail of Salmonella spp., Listeria monocytogenes and Staphylococcus aureus that were surface‐inoculated on frankfurters, bratwurst, shell eggs, chicken drumsticks, boneless skinless chicken breasts, boneless pork chops, tomatoes and jalapeno peppers was investigated. The pathogens displayed similar sensitivities to UV‐C on individual food products. Pathogen reductions ranged from approximately 0.5 log/g on raw meat and poultry to almost 4 log/g on tomatoes, while the pathogens were not recovered from stainless steel at a UV‐C dose of 0.4 J/cm2. Use of UV‐C light should be given serious consideration as a technology for routine surface decontamination of food contact surfaces and appropriate food products. PRACTICAL APPLICATIONS Ultraviolet light (UV‐C) is an U.S. Food and Drug Administration‐approved intervention technology that can be used to inactivate pathogenic bacteria in liquid foods and water, food contact surfaces, and food surfaces. This work indicates than UV‐C would be an effective technology for inactivation of foodborne pathogens on the surfaces of frankfurters and sausages immediately prior to packaging, shell eggs immediately prior to cracking in the production of liquid egg products, and smooth skinned produce such as tomatoes and jalapeno peppers prior to further processing. This work provides pathogen inactivation kinetics for food processors and government regulatory agencies.