Mismatch correction catalyzed by cell-free extracts of Saccharomyces cerevisiae.

Abstract

Heteroduplex DNA substrates containing a 4-or 7-base-pair insertion/deletion mismatch or each of the eight possible single-base-pair mismatches were constructed. Extracts of mitotic Saccharomyces cerevisiae cells catalyzed the correction of mismatched nucleotides in a reaction that required Mg2' and had a partial requirement for ATP and the four dNTPs. The insertion/deletion mismatches and the AC and G-T mismatches were repaired efficiently, while the six other single-base-pair mismatches were repaired poorly or at undetectable rates. Mismatch correction was accompanied by the specific incorporation of less than 20 nucleotides at or near the site of the repaired mismatch.Mispaired nucleotides in DNA are thought to arise by at least three different mechanisms. Misincorporation during DNA replication can lead to mispairs in DNA and mismatch correction can increase replication fidelity (1-4). Deamination of DNA bases can form mispairs, and such lesions can be repaired by N-glycosylases (5, 6). Mispaired bases can be formed during genetic recombination; and correction of mispairs, or the failure of correction, can explain gene conversion, post-meiotic segregation, localized negative interference, and map expansion (7)(8)(9)(10)(11)(12).Transformation experiments have provided direct evidence for mismatch correction (1)(2)(3)(4)(13)(14)(15)(16)(17). In Escherichia coli several mismatch correction systems have been identified. The E. coli dam-instructed system has been postulated to repair mispairs produced during DNA replication and genetic recombination (1-4, 18). A second system will repair fully dam methylated DNA and may act during genetic recombination (19,20). Both reactions involve excision/resynthesis tracts that are several thousand nucleotides long (1,19,20 (23,24). However, cytosine methylation may play a role in mismatch correction in mammalian cells (25).The development of E. coli in vitro mismatch correction systems have provided assays for use in purifying the proteins required for mismatch correction (26,27 Fig. 1, and the DNA sequences of the polylinker region of the heteroduplex substrates are presented in Fig. 2.A 4-base-pair (bp) insertion that inactivated the Xba I cleavage site of M13mpll was constructed as described (30).Abbreviations: bp, base pair(s); kb, kilobase(s); RFI, covalently closed replicative form DNA.

Mapping the Polarity of Changes That Occur in Interrupted CAG Repeat Tracts in Yeast

“…Possibly, small loops containing one or two repeat units on the template strand that are not repaired during one round of replication collapse during the next round of replication into larger hairpins that are too large for the mismatch machinery to recognize (1,17,28,32). Because the small changes that occur in this mutant background do not exhibit a bias in polarity, neither should the large changes resulting from their collapse.…”

Reconstitution of long and short patch mismatch repair reactions using Saccharomyces cerevisiae proteins

“…MMR assays were based on modification of previously published procedures (16,17,35,54). Proteins were diluted if necessary with 7.5 mM Hepes, pH 7.…”

DNA Mismatch Correction

“…Such sites, which are resistant to cleavage by the endonuclease, are rendered sensitive to the enzyme by mismatch correc tion on the appropriate DNA strand. This method has been used to demon strate the methyl-directed and methyl-independent pathways in cell-free ex tracts of E. coli (27 , 57, 100), and more recently to detect mismatch repair in extracts of yeast (101). Since the E. coli methyl-directed pathway has been the most extensively studied in this respect, the remainder of this section is devoted to this system.…”