, Volume 211, Issue 1, pp 41-48

An endo-exonuclease activity of yeast that requires a functional RAD52 gene

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Summary

Extracts of Rad+ and radiation-sensitive (rad) mutants of the yeast Saccharomyces cerevisiae were examined for total Mg2+-dependent alkaline deoxyribonuclease activity and the presence of a nuclease that crossreacts immunologically with an antiserum raised against an endoexonuclease from Neurospora crassa, an enzyme exhibiting both deoxyribo- and ribonuclease activities. No significant differences were observed in total deoxyribonuclease activity between Rad+ and rad mutants. The antibody precipitable activity, however, was found to be 30%–40% of the total alkaline deoxyribonuclease activity in logarithmically growing Rad+ cells. Extracts of stationary phase cells were lacking in antibody precipitable activity. Using immunoblot methods, a 72 kDa crossreacting protein was identified from logarithmically growing cells that was absent from stationary phase cells. In all radiation-sensitive mutants examined, except rad52, at least 20% of total activity was precipitable. Extracts from logarithmically growing rad52 mutants, including a rad52::LEU2 insertion mutant, exhibited less than 10% of the Rad+ precipitable activity; however, some crossreacting material was detected. Although, the level of endo-exonuclease activity is influenced by the RAD52 gene, it is not the product of this gene. The total deoxyribonuclease and the antibody precipitable endo-exonuclease activities were also followed during meiosis. Unlike the Rad+ strain which had previously been shown to have increased levels of total and immunoprecipitable endo-exonuclease as cells underwent meiosis, the rad52 mutant exhibited no increases in either category of nuclease activity. Given the importance of the RAD52 gene in repair, recombination and mutagenesis, the endo-exonuclease may be a significant component of these processes.

Communicated by B. Kilbey