Mutations affecting the biosynthesis of S-adenosylmethionine cause reduction of DNA methylation in Neurospora crassa.
A temperature-sensitive methionine auxotroph of Neurospora crassa was found in a collection of conditional mutants and shown to be deficient in DNA methylation when grown under semipermissive conditions. The defective gene was identified as met-3, which encodes cystathionine-gamma-synthase. We explored the possibility that the methylation defect results from deficiency of S-adenosylmethionine (SAM), the presumptive methyl group donor. Methionine starvation of mutants from each of nine complementation groups in the methionine (met) pathway (met-1, met-2, met-3, met-5, met-6, met-8, met-9, met-10 and for) resulted in decreased DNA methylation while amino acid starvation, per se, did not. In most of the strains, including wild-type, intracellular SAM peaked during rapid growth (12-18 h after inoculation), whereas DNA methylation continued to increase. In met mutants starved for methionine, SAM levels were most reduced (3-11-fold) during rapid growth while the greatest reduction in DNA methylation levels occurred later. Addition of 3 mM methionine to cultures of met or cysteine-requiring (cys) mutants resulted in 5-28-fold increases in SAM, compared with wild-type, at a time when DNA methylation was reduced approximately 40%, suggesting that the decreased methylation during rapid growth in Neurospora is not due to limiting SAM. DNA methylation continued to increase in a cys-3 mutant that had stopped growing due to methionine starvation, suggesting that methylation is not obligatorily coupled to DNA replication in Neurospora.
Institute of Molecular Biology, University of Oregon, Eugene 97403, USA.
C J
CJ
Roberts
E U
EU
Selker
D000596:Amino Acids; D003470:Culture Media / Q000737:chemistry; D004271:DNA, Fungal / Q000378:metabolism*; D005838:Genotype; D008715:Methionine / Q000096:biosynthesis / Q000172:deficiency; D008745:Methylation; D009154:Mutation; D009492:Neurospora crassa / Q000235:genetics* / Q000254:growth & development / Q000378:metabolism; D012436:S-Adenosylmethionine / Q000096:biosynthesis* / Q000172:deficiency; D013696:Temperature
D000596:Amino Acids; D003470:Culture Media; D004271:DNA, Fungal; D012436:S-Adenosylmethionine; D008715:Methionine