Cancer, mutation, and aging are common consequences of the failed enzymatic repair of DNA lesions. DNA damage is produced not only by environmental agents such as radiation but also by endogenous toxicants present in the normal cell. Almost every type of DNA repair enzyme (e.g., endonucleases for apurinic sites, mismatch repair enzymes, photolyases, ligases, glycosylases, methyl transferases, recombinases) has first been discovered and/or extensively characterized in bacteria, then found to be universal, and finally studied in mammalian cells. This proposal is for the study of a unique bacterial Dnase that can recognize lesions in irradiated DNA. Endonuclease V of Escherichia coli in a Dnase of wide-ranging specificity. Among its preferred substrates are DNAs that have been heavily irradiated by UV, treated with OsO4 (which produces some lesions similar to those of ionizing radiation), partially depurinated, alkali-treated, uracil- substituted, or treated with the carcinogen 7-bromobenz[alpha]anthracene. Although its ability to recognize a variety of DNA lesions suggests that it should be an extraordinarily useful and therefore ubiquitous DNA repair enzyme, its study has been severely limited by its paucity and by the unavailability of enzyme-deficient mutants. The objectives of this proposal are to clone its gene, to produce and characterize mutants, and to purify and characterize the enzyme from overproducers. The gene will be cloned via reverse genetics. To accomplish this, purified endonuclease V protein will first be prepared and then partially sequenced. Oligonucleotide probes will be generated to match the possible coding sequences, and the probes will be used to detect the cloned gene in an ordered genomic library. The cloned gene will be mutated and transferred to the chromosome. In order to detect the importance of the enzyme in various pathways of DNA repair, the mutants will be tested for their sensitivities to the lethal and mutagenic effects of DNA-damaging agents. The gene will be also subcloned in expression vectors for overproduction, purification, and subsequent study of the enzyme. A goal of the enzyme characterization will be to discover a common feature in its substrates or cleavage sites, thereby revealing its ultimate specificity an possibly enabling its subsequent use as a reagent for detecting certain types of lesions or structural features in DNA.
| Schouten, K A; Weiss, B (1999) Endonuclease V protects Escherichia coli against specific mutations caused by nitrous acid. Mutat Res 435:245-54 |
| Guo, G; Weiss, B (1998) Endonuclease V (nfi) mutant of Escherichia coli K-12. J Bacteriol 180:46-51 |
