Break-induced replication and telomerase-independent telomere maintenance require Pol32

Abstract

Break-induced replication (BIR) is an efficient homologous recombination process to initiate DNA replication when only one end of a chromosome double-strand break shares homology with a template. BIR is thought to re-establish replication at stalled and broken replication forks and to act at eroding telomeres in cells that lack telomerase in pathways known as 'alternative lengthening of telomeres' (reviewed in refs 2, 6). Here we show that, in haploid budding yeast, Rad51-dependent BIR induced by HO endonuclease requires the lagging strand DNA Polalpha-primase complex as well as Poldelta to initiate new DNA synthesis. Polepsilon is not required for the initial primer extension step of BIR but is required to complete 30 kb of new DNA synthesis. Initiation of BIR also requires the nonessential DNA Poldelta subunit Pol32 primarily through its interaction with another Poldelta subunit, Pol31. HO-induced gene conversion, in which both ends of a double-strand break engage in homologous recombination, does not require Pol32. Pol32 is also required for the recovery of both Rad51-dependent and Rad51-independent survivors in yeast strains lacking telomerase. These results strongly suggest that both types of telomere maintenance pathways occur by recombination-dependent DNA replication. Thus Pol32, dispensable for replication and for gene conversion, is uniquely required for BIR; this finding provides an opening into understanding how DNA replication re-start mechanisms operate in eukaryotes. We also note that Pol32 homologues have been identified both in fission yeast and in metazoans where telomerase-independent survivors with alternative telomere maintenance have also been identified.

Perinuclear tethers license telomeric DSBs for a broad kinesin- and NPC-dependent DNA repair process

“…In addition, deletion of the BIR factor DNA Pola subunit Pol32, but not control Pole subunit Dpb3, significantly decreased subtelomeric DSB survival to levels similar to those seen in Rad52-deficient cells (Fig. 1b) 39 . Moreover, disruption of Rad52, Lrs4, Cik1 or Nup84 specifically decreased BIR-dependent DSB survival (Fig.…”

“…BIR is physiologically important for the maintenance of telomeres in the absence of telomerase via a recombination-based mechanism that is akin to the 'alternative lengthening of telomeres' seen in telomerase-negative human cancers 39,[52][53][54] . Furthermore, eroded telomeres in telomerase-negative yeast cells relocate to nuclear pore complexes, suggesting that the dynamics of telomere localization at the nuclear periphery may be critical for the telomerase-independent maintenance of chromosome ends 23 .…”

“…6j). This initial accelerated senescence is reminiscent of telomerase-negative rad52D or pol32D cells, although these latter cells typically fail to exhibit recovery 39,52,53 . This is consistent with the fact that the loss of Rad52 or Pol32 decreases BIR-dependent repair more drastically than the loss of kinesin-14.…”

Smc5–Smc6 complex suppresses gross chromosomal rearrangements mediated by break-induced replications

“…To determine whether the smc6-9 mutation affects the BIR efficiency, the BIR efficiency was measured by using the recently developed BIR assay [40]. The smc6-9 mutation did not show any significant difference in the BIR efficiency (Fig.…”

“…BIR assay was performed following exactly same procedures as previously described [40]. Briefly, exponentially growing yeast cells in yeast-peptone (YP) with 2% succinic acid and 1% glycerol were plated on YP with 2% glucose or YP with 2% galactose and incubated for two days until visible colonies were grown.…”