Correlation of endonuclease activity at meiotic prophase and cofactor-induced genetic recombination in the basidiomycete Coprinus cinereus

Abstract

An endonuclease was purified from the basidiocarps of the fungus Coprinus cinereus during late premeiotic S-phase, karyogamy, and pachytene stages of meiosis. This endonuclease causes single-strand nicks on supercoiled PM2 DNA. Its activity is dependent on cofactors such as Mg2+ and Ca2+, and is enhanced by hexaminecobalt chloride and dimethylsulfoxide. Treatments with increasing cofactor concentration elicit increasing enzyme activity in vitro and increasing genetic recombination in vivo. The percent recombination that can be induced by cofactor treatment during late premeiotic S-phase, karyogamy and pachytene is correlated with the amount of endonuclease that can be extracted from the respective stages. These results suggest that Mg2+ and Ca2+ induce genetic recombination by increasing the endonuclease activity that creates nicked and gapped DNA substrates for ensuing recombination events. We believe that the meiotic endonuclease of Coprinus is involved in the early phase of genetic recombination. Cold temperature treatment is known to retard repair activity at pachytene and causes a threefold increase in recombination. Double treatments involving Mg2+ and Ca2+ at karyogamy followed by cold temperature at pachytene result in an additive (more than fourfold) increase in recombination frequency. The peak concentration of the nicking endonuclease is found at late premeiotic S and early karyogamy, whereas the peak activity of the repairing DNA polymerase b is found at late pachytene. The staging of these two enzymes is in good accord with the genetic data.Key words: Coprinus, endonuclease, meiosis, genetic recombination, Z-DNA.

Single-strand-specific nucleases

“…Holloman and Holiday [142] observed that certain mutant strains of U. maydis, unable to carry out allelic recombination, showed reduced nuclease levels and hence suggested its role in recombination. High levels of endonuclease activity were observed in the basidiocarp of C. cinereus during the late S-phase and early karyogamy [309]. The authors also demonstrated that cofactors, such as Mg 2þ and Ca 2þ or high temperatures, enhanced the nicking activity during these meiotic phases, leading to increased frequency of recombination.…”

Single-strand-specific nucleases

“…Holloman and Holiday [142] observed that certain mutant strains of U. maydis , unable to carry out allelic recombination, showed reduced nuclease levels and hence suggested its role in recombination. High levels of endonuclease activity were observed in the basidiocarp of C. cinereus during the late S‐phase and early karyogamy [309]. The authors also demonstrated that cofactors, such as Mg 2+ and Ca 2+ or high temperatures, enhanced the nicking activity during these meiotic phases, leading to increased frequency of recombination.…”

Life History and Developmental Processes in the BasidiomyceteCoprinus cinereus

“…In contrast, cold treatment at pachytene prolongs the pachytene stage (285). High and low temperatures appear to affect different steps in recombination-heat treatment at late S phase, at karyogamy, or at pachytene will lead to an increased recombination frequency, indicating that repair activity is not turned on until pachytene (272,280,283,287,403). Cold effects on recombination are very stage specific and are effective only in pachytene with a maximal increase of recombination (272,280,284,285).…”

“…The synchrony of karyogamy and meiosis within the C. cinereus fruiting bodies makes it easy to monitor stage-specific enzyme activities and makes this an excellent system in which to isolate the respective proteins. Thus, a meiotic endonuclease with DNA-nicking activity was purified from basidiocarps during the late premeiotic S phase, karyogamy, and pachytene stages of meiosis (287,290,329). Cloning of the gene encoding this enzyme using antiserum against a gt11 cDNA expression library failed, since the highly expressed galectins copurified with the endonuclease (85,96).…”