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

Abstract

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...

Mutagenic and Recombinagenic Responses to Defective DNA Polymerase δ Are Facilitated by the Rev1 Protein in pol3-t Mutants of Saccharomyces cerevisiae

“…Unlike USCR, the other mechanisms do not require that DNA replication has proceeded through the recombination substrate, suggesting that direct repeat recombination (DRR) may not be restricted to S and G 2 phases. The results of our experiments were similar to those from a number of studies that have demonstrated that the pol3-t mutation can increase the rate of DRR (Tran et al 1997;Lobachev et al 1998, 13 (27) 2 (4) 0 (0) 8 (17) 0 (0) 0 (0) 19 (40) 1 (2) 3 (6) pol3-t rev1D…”

“…In particular, mutations that disrupt the polymerase function of Pol d confer increased rates of mutation and recombination (Gordenin et al 1992;Tran et al 1995Tran et al , 1996Tran et al , 1997Kokoska et al 1998Kokoska et al , 2000Lobachev et al 1998Lobachev et al , 2000Galli et al 2003), consistent with incom- 18.8 (9.3-24.1) 4.2 9.6 (6.7-11.4) 5.7 pol3-t rev7D…”

“…Strains carrying mutations in the POL1, POL2 (CDC17), and POL3 (CDC2) genes, which encode the catalytic subunits of polymerases a (Budd and Campbell 1987), e (Boulet et al 1989), and d (Morrison et al 1990), respectively, display increased rates of spontaneous mutation and recombination (Aguilera and Klein 1988;Gordenin et al 1992;Ruskin and Fink 1993;Zou and Rothstein 1997;Kirchner et al 2000;Pavlov et al 2001;Galli et al 2003;Fortune et al 2005), supporting the link between defective DNA replication and genome instability. In particular, mutations in the POL3 gene that confer a temperature-sensitive growth defect, most likely by affecting the capacity of the cell to replicate its DNA, also confer elevated rates of spontaneous mutation and recombination with a variety of assays (Gordenin et al 1992(Gordenin et al , 1993Tran et al 1995Tran et al , 1996Tran et al , 1997Tran et al , 1999Kokoska et al 1998;Schweitzer and Livingston 1999;Kokoska et al 2000;Jin et al 2001;Galli et al 2003). One of these mutations, pol3-t, is thought to affect the processivity of Pol d (Gordenin et al 1992;Tran et al 1995;Kokoska et al 2000), which is likely to increase the formation of daughter strand gaps that may be intermediates in the formation of spontaneous mutation and recombination events (Horiuchi et al 1994;Ivessa et al 2000;Michel et al 2001;Minesinger and Jinks-Robertson 2005;Saleh-Gohari et al 2005;Lopes et al 2006).…”

Biased Distribution of Inverted and Direct Alus in the Human Genome: Implications for Insertion, Exclusion, and Genome Stability

“…The present study considerably extends our recent report demonstrating a reduction in closely spaced inverted Alus as compared to direct Alu repeats (Lobachev et al 2000). Based on results from model systems, other factors also are likely to be important in the stability of Alu sites including genetic background (Gordenin et al 1993;Ruskin and Fink 1993;Tran et al 1997;Lobachev et al 1998Lobachev et al , 2000). For example, recombination in mammalian cell constructs containing direct identical Alu repeats is high in p53 defective as compared to p53 wildtype cells (Gebow et al 2000).…”

“…Pairs of large inverted DNA repeats can be unstable, lead to deletions, and stimulate recombination between DNAs surrounding the inverted repeats in yeast (Gordenin et al 1993;Ruskin and Fink 1993;Nag and Kurst 1997;Tran et al 1997;Lobachev et al 1998Lobachev et al , 2000 ) and mammalian cells (Akgun et al 1997;Lewis 1999;Lewis et al 1999). Sequence identity was suggested as a likely factor in the stability of Alu repeats based on results in yeast.…”