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 sae2Delta 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.

Figure 1.

HO endonuclease induction of DSB repair events in Mush18/21. (A) The substrate consists of inverted repeats comprised of two truncated but overlapping alleles of TRP1, a full-length CAN gene, and a truncated can1-5′Δ allele, separated by a full-length HIS3 gene. The extent of the inverted repeat sequence is marked by the gray double-headed arrow. Between the trp1-3′Δ allele and the full-length CAN1 gene, there is a unique recognition sequence for the HO endonuclease. The normal HO recognition sequence at the MAT locus has been mutated (MATα-inc). The substrate is inserted near the MATα locus on chromosome III. (B) Structure of the palindromic duplications. Note that the can1-5′Δ, HIS3, and a variable amount of the 3′ end of the CAN1 gene have been duplicated. The increase in the extent of the inverted repeat is indicated by the gray double-headed arrow.

Figure 2.

Southern blot analysis of a palindromic gene duplication event. (A) Schematic of the palindromic molecule showing the location of the BamH1 (B) and PvuII (P) sites. (B) Southern blot analysis of BamH1-digested DNA. (Bd) BamH1-digested DNA that was denatured by boiling prior to electrophoresis; (G) BglI; (E) EcoR1; (S) BstEII; (A) AvaII. (A,D) The blot was probed with unique sequences from the 3′ end of the CAN1 gene (dotted lines). (C) Location of restriction sites in relevant region of the native CAN1 gene. (D) Schematic showing interpretation of the Southern blot shown in panel B.

Figure 3.

DNA sequence analysis of palindromic junctions. (A) Schematic of a palindromic event with the location of the region sequenced shown by the boxed region. (B) Sequence analysis of the same event analyzed in Figure 2. (Top) Sequence of the relevant region of the native CAN gene. (Center) Bisulfite-modified sequence of the junction. (Bottom) Deduced junction sequence. Sequence in italics diverges from the native sequence. Underlined sequences indicate short inverted repeats preceding the point of divergence. (C) Interpretation of sequence analysis with inverted repeats shown in red and loop region shown in blue. BsrF1 indicates the site used for vectorette PCR analysis. (D) Summary of sequence from 24 independent junctions. Sequence shown is a portion of the CAN1 gene, where the BamH1 site of the starting substrate is at position 1 (not shown). The end point of each different palindromic junction is shown with the stem regions highlighted in red and the loop region highlighted in blue. For each junction the homology ends at the second inverted repeat sequence followed immediately by the complement to the sequence preceding the first inverted repeat (as drawn in C). The shortest palindrome is the first sequence highlighted and the longest palindrome is the last sequence highlighted. The number of independent events mapping to each of the six junctions is shown above the sequence. Junction sequences from rad50Δ cells are noted in parentheses, the remaining 20 events are derived from sae2Δ cells. EcoR1 defines the site at which homology to the can1-5′Δ allele begins. BsrF1 indicates the location of the sites used for verifying the relevant events by vectorette PCR.

Figure 4.

Model for palindrome formation. (A) DSB repair substrate. (B) Recombination is induced by turning on the expression of the HO endonuclease. (C) After invasion, DNA synthesis proceeds by BIR toward the end of the molecule. (New DNA synthesis is shown in black.) (D) A free 3′ end folds back intramolecularly via short (4-6 bp), closely spaced (2-9 bp), inverted repeats. The paired repeats function to prime DNA synthesis. Eventually, the newly primed strand becomes ligated to the other newly synthesized DNA. (E) Branch migration of the intermediate leads to a palindromic molecule with a gap that is subsequently filled in. The Watson and Crick strands of the DNA molecules are indicated by 5′ and 3′.

Figure 5.

Model for palindromic amplification using dispersed repeats. (A) A spontaneous chromosomal DSB can lead to invasion at an inverted dispersed repeat (blue triangles), which initiates BIR. (B) Extension by BIR toward the broken end followed by foldback priming can lead to palindrome formation. (C-E) Subsequent breakage cycles, possibly induced by the presence of the palindrome itself, can lead to further amplification of the array.