Repeat expansion in the budding yeast ribosomal DNA can occur independently of the canonical homologous recombination machinery

Abstract

Major eukaryotic genomic elements, including the ribosomal DNA (rDNA), are composed of repeated sequences with well-defined copy numbers that must be maintained by regulated recombination. Although mechanisms that instigate rDNA recombination have been identified, none are directional and they therefore cannot explain precise repeat number control. Here, we show that yeast lacking histone chaperone Asf1 undergo reproducible rDNA repeat expansions. These expansions do not require the replication fork blocking protein Fob1 and are therefore independent of known rDNA expansion mechanisms. We propose the existence of a regulated rDNA repeat gain pathway that becomes constitutively active in asf1Δ mutants. Cells lacking ASF1 accumulate rDNA repeats with high fidelity in a processive manner across multiple cell divisions. The mechanism of repeat gain is dependent on highly repetitive sequence but, surprisingly, is independent of the homologous recombination proteins Rad52, Rad51 and Rad59. The expansion mechanism is compromised by mutations that decrease the processivity of DNA replication, which leads to progressive loss of rDNA repeats. Our data suggest that a novel mode of break-induced replication occurs in repetitive DNA that is dependent on high homology but does not require the canonical homologous recombination machinery.

Mechanisms of Gene Duplication and Amplification

“…This result was interpreted as evidence that the copy number change occurs by gene conversion-like events in which the two ends at a single break is repaired by invading an unbroken homolog at distant sites in the rrn array to increase the copy number in the broken chromosome. However, these events do not seem to require standard homologous recombination enzymes as expected for such exchanges (Houseley and Tollervey 2011). This may suggest that recombination defects and frequent damage to rrn sequences may activate alternative annealing pathways in yeast, as described above for bacteria (Reams et al 2014).…”

Regulation of ribosomal DNA amplification by the TOR pathway

“…(20). However, only loss of histone chaperone Asf1 has been shown to cause non-HR-dependent amplification, and Asf1 affects many processes in addition to H3 K56 acetylation (32), leaving it unclear whether H3 K56 acetylation actually regulates the non-HRdependent pathway.…”

“…Loss of Dun1 has little effect on Rad52-dependent HR but completely inhibits the non-HR-dependent amplification pathway (20,36,37). rDNA 35 dun1Δ cells underwent rDNA amplification after the introduction of pFOB1, but again, to a lesser extent than wild-type (Fig.…”

“…This may overlap with the Sir2-regulated pathway but must be distinct, as complete de-regulation of rDNA BIR in sir2Δ mutants does not cause constitutive rDNA amplification (13). In contrast, constitutive rDNA amplification has been reported in cells lacking histone H3 K56 acetyltransferase activity, suggesting H3 K56 acetylation may control entry to an rDNA amplification pathway (20,21). Surprisingly, rDNA amplification can occur in the absence of critical HR proteins, including strand exchange factor Rad52 (20), suggesting two mechanistically separable pathways exist: the HR-dependent BIR pathway and a non-HR-dependent amplification pathway.…”

“…This method of regulation may seem unlikely, as loss of Hst3 and Hst4 leads to general genome instability (31, 34); however, the loss of HDAC activity need not be complete. HR proteins are excluded from the nucleolus (48), and the highly repetitive rDNA is an excellent substrate for non-HR-dependent recombination (20). Therefore, a reduction in H3 K56 HDAC activity that has little effect on the rest of the genome could well drive non-HRdependent rDNA amplification.…”