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A cytosine methyltransferase homologue is essential for repeat-induced point mutation in Neurospora crassa.

ABSTRACT: During sexual development, Neurospora crassa inactivates genes in duplicated DNA segments by a hypermutation process, repeat-induced point mutation (RIP). RIP introduces C:G to T:A transition mutations and creates targets for subsequent DNA methylation in vegetative tissue. The mechanism of RIP and its relationship to DNA methylation are not fully understood. Mutations in DIM-2, a DNA methyltransferase (DMT) responsible for all known cytosine methylation in Neurospora, does not prevent RIP. We used RIP to disrupt a second putative DMT gene in the Neurospora genome and tested mutants for defects in DNA methylation and RIP. No effect on DNA methylation was detected in the tissues that could be assayed, but the mutants showed recessive defects in RIP. Duplications of the am and mtr genes were completely stable in crosses homozygous for the mutated potential DMT gene, which we call rid (RIP defective). The same duplications were inactivated normally in heterozygous crosses. Disruption of the rid gene did not noticeably affect fertility, growth, or development. In contrast, crosses homozygous for a mutation in a related gene in Ascobolus immersus, masc1, reportedly fail to develop and heterozygous crosses reduce methylation induced premeiotically [Malagnac, F., Wendel, B., Goyon, C., Faugeron, G., Zickler, D., et al. (1997) Cell 91, 281-290]. We isolated homologues of rid from Neurospora tetrasperma and Neurospora intermedia to identify conserved regions. Homologues possess all motifs characteristic of eukaryotic DMTs and have large distinctive C- and N-terminal domains.

SUBMITTER: Freitag M

PROVIDER: S-EPMC124379 | biostudies-literature | 2002 Jun

REPOSITORIES: biostudies-literature

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A cytosine methyltransferase homologue is essential for repeat-induced point mutation in Neurospora crassa.

Freitag Michael M Williams Rebecca L RL Kothe Gregory O GO Selker Eric U EU

Proceedings of the National Academy of Sciences of the United States of America 20020618 13

During sexual development, Neurospora crassa inactivates genes in duplicated DNA segments by a hypermutation process, repeat-induced point mutation (RIP). RIP introduces C:G to T:A transition mutations and creates targets for subsequent DNA methylation in vegetative tissue. The mechanism of RIP and its relationship to DNA methylation are not fully understood. Mutations in DIM-2, a DNA methyltransferase (DMT) responsible for all known cytosine methylation in Neurospora, does not prevent RIP. We u ...[more]

PMID: 12072568

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Project description:Numerous cytogenetic observations have shown that homologous chromosomes (or individual chromosomal loci) can engage in specific pairing interactions in the apparent absence of DNA breakage and recombination, suggesting that canonical recombination-mediated mechanisms may not be the only option for sensing DNA/DNA homology. One proposed mechanism for such recombination-independent homology recognition involves direct contacts between intact double-stranded DNA molecules. The strongest in vivo evidence for the existence of such a mechanism is provided by the phenomena of homology-directed DNA modifications in fungi, known as repeat-induced point mutation (RIP, discovered in Neurospora crassa) and methylation-induced premeiotically (MIP, discovered in Ascobolus immersus). In principle, Neurospora RIP can detect the presence of gene-sized DNA duplications irrespectively of their origin, underlying nucleotide sequence, coding capacity or relative, as well as absolute positions in the genome. Once detected, both sequence copies are altered by numerous cytosine-to-thymine (C-to-T) mutations that extend specifically over the duplicated region. We have recently shown that Neurospora RIP does not require MEI-3, the only RecA/Rad51 protein in this organism, consistent with a recombination-independent mechanism. Using an ultra-sensitive assay for RIP mutation, we have defined additional features of this process. We have shown that RIP can detect short islands of homology of only three base-pairs as long as many such islands are arrayed with a periodicity of 11 or 12 base-pairs along a pair of DNA molecules. While the presence of perfect homology is advantageous, it is not required: chromosomal segments with overall sequence identity of only 35-36 % can still be recognized by RIP. Importantly, in order for this process to work efficiently, participating DNA molecules must be able to co-align along their lengths. Based on these findings, we have proposed a model, in which sequence homology is detected by direct interactions between slightly-extended double-stranded DNAs. As a next step, it will be important to determine if the uncovered principles also apply to other processes that involve recombination-independent interactions between homologous chromosomal loci in vivo as well as to protein-free DNA/DNA interactions that were recently observed under biologically relevant conditions in vitro.

Dataset Information

A cytosine methyltransferase homologue is essential for repeat-induced point mutation in Neurospora crassa.

Publications

A cytosine methyltransferase homologue is essential for repeat-induced point mutation in Neurospora crassa.

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