Involvement of Sir2/4 in silencing of DNA breakage and recombination on mouse YACs during yeast meiosis

Abstract

Yeast artificial chromosomes (YACs) that contain human DNA backbone undergo DNA double-strand breaks (DSBs) and recombination during yeast meiosis at rates similar to the yeast native chromosomes. Surprisingly, YACs containing DNA covering a recombination hot spot in the mouse major histocompatibility complex class III region do not show meiotic DSBs and undergo meiotic recombination at reduced levels. Moreover, segregation of these YACs during meiosis is seriously compromised. In meiotic yeast cells carrying the mutations sir2 or sir4, but not sir3, these YACs show DSBs, suggesting that a unique chromatin structure of the YACs, involving Sir2 and Sir4, protects the YACs from the meiotic recombination machinery. We speculate that the paucity of DSBs and recombination events on these YACs during yeast meiosis may reflect the refractory nature of the corresponding region in the mouse genome.

Figure 1.

Absence of meiotic DSBs on YACs containing mouse MHC class III sequences, in rad50S strains. Analysis of two yeast strains containing two different YACs, YAC99 (200 kb) and YAC100 (450 kb). Chromosome-size DNA was extracted from samples after 0 and 6 h in meiosis, separated by PFGE, and blotted. The blots were Southern-hybridized to mouse-DNA probe B2 (A) and to yeast chromosome-III-specific probe YCR48w (B).

Figure 2.

Accumulation of meiotic DSBs on YACs containing DNA sequences from mouse chromosome 2, compared with absence of meiotic DSBs on YAC containing mouse MHC class III sequences. Analysis of yeast strains containing YACs with inserts of different mouse DNA sequences, YAC328 (500 kb), YAC35 (370 kb), YAC109 (150 kb), and YAC100 (450 kb). Chromosome-size DNA was extracted from cell samples undergoing meiosis, after 0 and 6 h, separated by PFGE, and blotted onto membranes. The membranes were hybridized to mouse-DNA specific probe B2.

Figure 3.

CoHR profile matches on YAC99 and YAC100. Five High-quality CoHR-profile-hits (1–5) found on an 80-kb sequence from the MHC class III region (sequence AF397035). Both the forward and reverse DNA strands were checked for the presence of sequences similar to the preferred DSB-associated CoHR (Blumental-Perry et al., 2000), using the program ProfileGap from the GCG package. CoHR2, 4 and 5 are present in both, YAC99 and YAC100. CoHR3 is present only in YAC99, which may also contain CoHR1 (CoHR1 and 3 are absent on YAC100).

Figure 4.

Effect of sir mutations on meiotic DSBs on MHC class III YAC. Analysis of three double mutant haploid strains that contain YAC100: sir2Δ rad50S, sir3Δ rad50S, sir4Δ rad50S. Haploids carrying sir mutations initiate meiosis, like MATa/MATα diploids. Chromosome-size DNA was extracted from cell samples after 0 and 6 h in sporulation medium, separated by PFGE, blotted, and hybridized to a YAC-specific probe (pBR322 DNA).