DNA METHYLATION ASSOCIATED WITH REPEAT-INDUCED POINT MUTATION IN NEUROSPORA-CRASSA

Repeat-induced point mutation (RIP) is a process that efficiently dete cts DNA duplications prior to meiosis in Neurospora crassa and peppers them with G:C to A:T mutations. Cytosine methylation is typically ass ociated with sequences affected by RIP, and methylated cytosines are n ot limited to CpG dinucleotides. We generated and characterized a coll ection of methylated and unmethylated am(RIP) alleles to investigate t he connection(s) between DNA methylation and mutations by RIP. Alleles of am harboring 84 to 158 mutations in the 2.6-kb region that was dup licated were heavily methylated and triggered de novo methylation when reintroduced into vegetative N. crassa cells. Alleles containing 45 a nd 56 mutations were methylated in the strains originally isolated but did not become methylated when reintroduced into vegetative cells. Th is provides the first evidence for de novo methylation in the sexual c ycle and for a maintenance methylation system in Neurospora cells. No methylation was detected in am alleles containing 8 and 21 mutations. All mutations in the eight primary alleles studied were either G to A or C to T, with respect to the coding strand of the am gene, suggestin g that RTP results in only one type of mutation. We consider possibili ties for how DNA methylation is triggered by some sequences altered by RIP.