Yeast mutants with increased bacterial transposon Tn 5 excision

Replication, DNA organization, and mismatch repair (MMR) can influence recombination. We examined the effects of altered replication due to a mutation in the polymerase ␦ gene, long inverted repeats (LIRs) in motifs similar to those in higher eukaryotes, and MMR on intrachromosomal recombination between highly diverged (28%) truncated genes in Saccharomyces cerevisiae. A combination of altered replication and an LIR increased recombination up to 700-fold, while each alone led to a 3-to 20-fold increase. Homeologous recombination was not altered by pms1, msh2, and msh3 mismatch repair mutations. Similar to our previous observations for replication slippage-mediated deletions, there were >5-bp identical runs at the recombination breakpoints. We propose that the dramatic increase in recombination results from enhancement of the effects of altered replication by the LIR, leading to recombinationally active initiating structures. Such interactions predict replication-related, MMR-independent genome changes.Chromosomal recombination has both beneficial and deleterious consequences. During meiosis, recombination is generally considered to be essential to the orderly distribution of chromosomes. In mitotically growing cells of lower organisms, recombination provides for efficient repair of DNA damage, particularly double-strand breaks, through interactions between homologs or sister chromatids. In mammals, somatic recombination is an important component in the development of the immune system.Recombination could also lead to genome instabilities if it were to occur between diverged DNAs. For example, human chromosomes contain many large DNA repeat sequences such as Alus, LINEs, and pseudogenes (11, 39) in which reciprocal exchange would lead to genome rearrangements and/or deletions. This is presumably prevented by divergence between the repeats.In all organisms examined, a high level of DNA divergence can reduce recombination substantially. In addition to the level of DNA divergence, there are many factors (see Discussion) that can influence the likelihood of homeologous recombination; these include the mode of initiation, DNA organization, the recombination system examined, and mismatch repair (MMR). For example, conjugational recombination between Escherichia coli and Salmonella typhimurium, whose DNAs are 16% diverged, is 10 5 -fold lower than for intraspecies crosses (34, 44). However, double-strand break-induced intraplasmid recombination between diverged DNAs is reduced less than 10-fold (2). In yeast, a single-or double-strand break can enhance recombination between highly diverged DNAs during transformation (33,35,36,43).The MMR proteins MutS and MutL in bacteria or their homologs responsible for preventing replication-related mutations in higher organisms are proposed to interact with mismatches in some way to inhibit homeologous recombination, possibly by preventing strand assimilation or strand transfer (37). The role of the MMR system in preventing recombination between highly diverged DNAs appears to depen...

Section: Discussionmentioning

confidence: 99%

“…An insertion of an LIR into a gene can stimulate interchromosomal heteroallelic recombination as much as 10-fold (28). Previously, LIRs were shown to induce deletions between small surrounding direct repeats in bacteria and yeast (17,23,(29)(30)(31)47). In transgenic mice, LIRs are mitotically unstable and lead to insertions or deletions (16).…”

mentioning

confidence: 99%

Altered Replication and Inverted Repeats Induce Mismatch Repair-Independent Recombination between Highly Diverged DNAs in Yeast

Tran

Degtyareva

Gordenin

et al. 1997

“…The reduced expression of Pol1p has been shown to increase the efficiency of mating between two MAT␣ haploids (20). This "illegitimate" mating is usually a consequence of the deletion or mutation of the MAT␣ locus in one of the two mated haploids (10,20,23). To determine whether a reduced expression of Pol3p elevated illegitimate mating, we first used a streak mating assay (Fig.…”

Section: Vol 28 2008 Low Levels Of Pol3p Induce Fragile Site Instabmentioning

confidence: 99%

Reduced Levels of DNA Polymerase δ Induce Chromosome Fragile Site Instability in Yeast

Lemoine

Degtyareva

Kokoska

et al. 2008

Specific regions of genomes (fragile sites) are hot spots for the chromosome rearrangements that are associated with many types of cancer cells. Understanding the molecular mechanisms regulating the stability of chromosome fragile sites, therefore, has important implications in cancer biology. We previously identified two chromosome fragile sites in Saccharomyces cerevisiae that were induced in response to the reduced expression of Pol1p, the catalytic subunit of DNA polymerase ␣. In the study presented here, we show that reduced levels of Pol3p, the catalytic subunit of DNA polymerase ␦, induce instability at these same sites and lead to the generation of a variety of chromosomal aberrations. These findings demonstrate that a change in the stoichiometry of replicative DNA polymerases results in recombinogenic DNA lesions, presumably doublestrand DNA breaks.

“…This mutant (initially named tex1) was isolated in a strain that exhibited an increased excision rate of a bacterial transposon with long terminal repeats inserted within a yeast gene (7). The pol3-t allele, which encodes a mutation (Gly 641 to Ala 641 ) (51) located near the putative nucleotide binding and active-site domains of the enzyme (58), is thought to diminish the rate of lagging-strand synthesis resulting in long stretches of singlestranded DNA on the lagging-strand template (8).…”

mentioning

confidence: 99%

Destabilization of Yeast Micro- and Minisatellite DNA Sequences by Mutations Affecting a Nuclease Involved in Okazaki Fragment Processing (rad27) and DNA Polymerase δ (pol3-t)

Kokoska

Stefanovic

Tran

et al. 1998

175

168

We examined the effects of mutations in the Saccharomyces cerevisiae RAD27 (encoding a nuclease involved in the processing of Okazaki fragments) and POL3 (encoding DNA polymerase ␦) genes on the stability of a minisatellite sequence (20-bp repeats) and microsatellites (1-to 8-bp repeat units). Both the rad27 and pol3-t mutations destabilized both classes of repeats, although the types of tract alterations observed in the two mutant strains were different. The tract alterations observed in rad27 strains were primarily additions, and those observed in pol3-t strains were primarily deletions. Measurements of the rates of repetitive tract alterations in strains with both rad27 and pol3-t indicated that the stimulation of microsatellite instability by rad27 was reduced by the effects of the pol3-t mutation. We also found that rad27 and pol3-01 (an allele carrying a mutation in the "proofreading" exonuclease domain of DNA polymerase ␦) mutations were synthetically lethal.All eukaryotic genomes thus far examined contain many simple repetitive DNA sequences, tracts of DNA with one or a small number of bases repeated multiple times (48). These repetitive regions can be classified as microsatellites (small repeat units in tandem arrays 10 to 60 bp in length) and minisatellites (larger repeat units in tandem arrays several hundred base pairs to several kilobase pairs in length). In this paper, arrays with repeat units 14 bp or less will be considered microsatellites and arrays with longer repeat units will be considered minisatellites.Previous studies show that simple repetitive sequences are unstable relative to "normal" DNA sequences, frequently undergoing additions or deletions of repeat units, in Escherichia coli (24), Saccharomyces cerevisiae (12), and mammals (59). This mutability has two important consequences. First, it results in polymorphic loci that are useful in genetic mapping and forensic studies (15,59). Second, although these repetitive tracts are usually located outside of coding sequences, alterations in the lengths of microsatellites or minisatellites located within coding sequences can produce frameshift mutations or novel protein variants (20,22,26).From studies of the effects of various mutations on microsatellite stability in yeast and E. coli (40) and the analysis of mutational changes caused by DNA polymerase in vitro (21), it is likely that most alterations reflect DNA polymerase slippage events (47). These events involve the transient dissociation of the primer and template strands during the replication of a microsatellite (Fig. 1). If the strands reassociate to yield an unpaired repeat on the primer strand, the net result is an addition of repeats (following a second round of DNA replication). Unpaired repeats on the template strand would result in a deletion by the same mechanism.A number of mutations have been shown to elevate microsatellite instability. In E. coli (24, 46), yeast (44, 45), and mammalian cells (27), mutations in genes affecting DNA mismatch repair dramatically elevate the ins...