A mechanism of palindromic gene amplification in Saccharomyces cerevisiae

Abstract

Selective gene amplification is associated with normal development, neoplasia, and drug resistance. One class of amplification events results in large arrays of inverted repeats that are often complex in structure, thus providing little information about their genesis. We made a recombination substrate in Saccharomyces cerevisiae that frequently generates palindromic duplications to repair a site-specific double-strand break in strains deleted for the SAE2 gene. The resulting palindromes are stable in sae2⌬ cells, but unstable in wild-type cells. We previously proposed that the palindromes are formed by invasion and break-induced replication, followed by an unknown end joining mechanism. Here we demonstrate that palindrome formation can occur in the absence of RAD50, YKU70, and LIG4, indicating that palindrome formation defines a new class of nonhomologous end joining events. Sequence data from 24 independent palindromic duplication junctions suggest that the duplication mechanism utilizes extremely short (4-6 bp), closely spaced (2-9 bp), inverted repeats to prime DNA synthesis via an intramolecular foldback of a 3 end. In view of our data, we present a foldback priming model for how a single copy sequence is duplicated to generate a palindrome.[Keywords: DNA palindrome; gene amplification; MR complex; SAE2; Saccharomyces cerevisiae; BFB] Supplemental material is available at http://www.genesdev.org.

Intrastrand Annealing Leads to the Formation of a Large DNA Palindrome and Determines the Boundaries of Genomic Amplification in Human Cancer

“…This model is analogous to the intramolecular mechanism of palindrome formation mediated by the short DNA IR in Tetrahymena and to the small IR-assisted intramolecular mechanism for palindrome formation in yeast (Fig. 3b, left) (6,20,26). Microhomology represents a very short DNA IR (Fig.…”

“…3b, right), and the length of homology determines the efficiency of intrastrand annealing. This process is likely to be independent of NHEJ, since an intramolecular mechanism using a short stretch of homology can occur in the absence of RAD50, YKU70, and LIG4 in S. cerevisiae (20,26).…”

Minding the gap: The underground functions of BRCA1 and BRCA2 at stalled replication forks

“…3). BIR was first described as a RAD51-independent error-prone DSBR pathway and is known to mediate several types of chromosomal instability in yeast [55][56][57][58]. BIR is as processive as conventional chromosomal replication and it therefore probably entails formation of a replication fork at the recombination joint [59] (Fig.…”

Budding Yeast Sae2 is an In Vivo Target of the Mec1 and Tel1 Checkpoint Kinases During Meiosis

“…In fact, similarly to the rad50s and mre11s alleles, SAE2 deletion impairs the repair of hairpin-capped DSBs 37 and increases the percentage of DSB repair events that lead to inverted repeat duplication in mitotic cells, without affecting the overall recombination rate. 38,39 Moreover, both MRX and Sae2 are required to ensure efficient repair by single-strand annealing not only of meiotic but also of mitotic DSBs, 40,41 and they act in the same epistasis group to allow mitotic DSB resection. 42 Finally, the lack of Sae2 increases MRX persistence at DNA breaks, 42,43 and concomitantly prevents the switch off of both Tel1-and Mec1-dependent DNA damage checkpoints during mitosis, 20,42 suggesting that Sae2 may negatively regulate both Tel1-and Mec1-dependent signalling by modulating MRX association at damaged DNA.…”