Expression of the Escherichia coli dam methylase in Saccharomyces cerevisiae: effect of in vivo adenine methylation on genetic recombination and mutation.

Abstract

The Escherichia coli DNA adenine methylase (dam) gene has been introduced into Saccharomyces cerevisiae on a yeast-E. coli shuttle vector. Sau3AI, MboI, and DpnI restriction enzyme digests and Southern hybridization analysis indicated that the dam gene is expressed in yeast cells and methylates GATC sequences. Analysis of digests of total genomic DNA indicated that some GATC sites are not sensitive to methylation. The failure to methylate may reflect an inaccessibility to the methylase due to chromosome structure. The effects of this in vivo methylation on the processes of recombination and mutation in mitotic cells were determined. A small but definite general increase was found in the frequency of mitotic recombination. A similar increase was observed for reversion of some auxotrophic markers; other markers demonstrated a small decrease in mutation frequency. The effects on mutation appear to be locus (or allele) specific. Recombination in meiotic cells was measured and was not detectibly altered by the presence of 6-methyladenine in GATC sequences.Methylation of DNA bases has been demonstrated to play an important role in the processes of DNA replication, repair, and recombination in procaryotes and gene expression in eucaryotes (1, 4). In Escherichia coli a methyl group is added, after replication, to the N6 position of adenine in 5'-GATC-3' sequences by DNA adenine methylase produced by the dam gene (15,28). It has been proposed that the transient undermethylation of newly replicated strands allows a mismatch repair system to preferentially remove the new (incorrect) information when replication errors occur (13,37,42). Recent experiments using heteroduplexes of phage X DNA, methylated in vitro and transformed into E. coli, have confirmed that the mismatched base on the undermethylated strand is preferentially repaired (36).In E. coli, the consequences of losing the ability to methylate adenine are profound. The lack of methylation in dam-strains leads to increased frequencies of recombination and spontaneous mutation (2, 27, 29) and increased sensitivity to methyl methanesulfonate (29) and UV (27). Furthermore, the dam mutation is lethal in combination with recBIC or lexA mutations (2, 29). All of these phenotypes can be understood in terms of the mismatch correction system being unable to distinguish which strand to attack when a mismatch is created during replication. Specifically, increased mutation could occur when the correct base was removed, and increased recombination could result from single-strand gaps and breaks or from double-strand breaks generated when excision tracks on both DNA strands overlap (37). Overproduction of the dam enzyme also leads to increased mutation frequencies (19). Herman and Modrich argued that increased levels of the dam methylase would result in a DNA molecule (19) Table 1. To monitor the effect of dam on recombination, the yeast diploid strain MH16 was constructed containing six heteroallelic loci and two recessive drug resistance loci. The former allow us to examine mi...

Saccharomyces telomeres assume a non-nucleosomal chromatin structure.

“…The extent of adenine methylation that occurs in vivo can be measured by comparing the relative cleavage of isolated genomic DNA with restriction enzymes that cleave at the sequence modified by the dam methylase: Cleavage at GATC by SauSA is insensitive to methylation, whereas cleavage at GATC by Dpnl occurs only if the site is methylated on both strands. The persistence of large (>10 kb) DNA fragments in Dpnl digestions of genomic DNA from cells expressing the dam methylase suggests that some regions of the yeast genome are inaccessible to the methylase activity (Hoekstra and Malone 1985).…”

“…Other than minor effects on mitotic recombination rates, yeast cells expressing the methylase grow normally (Hoekstra and Malone 1985). The extent of adenine methylation that occurs in vivo can be measured by comparing the relative cleavage of isolated genomic DNA with restriction enzymes that cleave at the sequence modified by the dam methylase: Cleavage at GATC by SauSA is insensitive to methylation, whereas cleavage at GATC by Dpnl occurs only if the site is methylated on both strands.…”

“…coli dam methylase gene was integrated at the LYS2 gene on yeast chromosome II-R as described (Gottschling 1992), where it is expressed constitutively, presumably from a fortuitous promoter within the bacterial DNA. The dam methylase gene was isolated as a 1.5-kb Hindlll-PvuII fragment from pMFHl (Hoekstra and Malone 1985).…”

Differential Requirement of DNA Replication Factors for Subtelomeric ARS Consensus Sequence Protosilencers in Saccharomyces cerevisiae

“…DamI methylaseexpressing yeast strains were generated by transforming cells with XhoI-linearized pDP6-Dam1 (Hoekstra and Malone 1985). Transformants expressing DamI methylase and their genomic DNAs were tested for sensitivity to DpnI.…”

Excision repair functions in Saccharomyces cerevisiae recognize and repair methylation of adenine by the Escherichia coli dam gene.

“…The E. coli dam product methylates at a position that is different from that of most exogenous agents, such as methyl methanesulfonate. Previously, we have shown that the yeast cell expresses a cloned dam gene and methylates its chromosomal DNA (15). Methylation causes a general twofold increase in mitotic recombination and a moderate increase in mutation frequencies at some loci.…”