Dna2 Is Involved in CA Strand Resection and Nascent Lagging Strand Completion at Native Yeast Telomeres

Abstract

Background: DNA2 helicase/nuclease participates in telomere maintenance by unknown mechanisms. Results: dna2 mutants show slightly shortened telomeric GT-overhangs and accumulate small, nascent lagging strand DNA chains at telomeres. Conclusion: Unlike at DSBs, additional nucleases largely compensate for the simultaneous loss of Dna2 and Mre11 in producing GT. Significance: FEN1, Dna2, or Exo1 is necessary to mature lagging strands at telomeres.

A critical role for Dna2 at unwound telomeres

“…Interestingly, the telomeres of chk1 Δ dna2 Δ, mec1 Δ dna2 Δ, rad17 Δ dna2 Δ, and ddc1 Δ dna2 Δ cells were long, and in fact longer and more diffuse, than pif1 Δ strains, known to have very long telomeres (Schulz and Zakian, 1994) (Figure 3c, Figure S4, Figure S6). In contrast, and as reported before, rad9 Δ dna2 Δ telomeres were slightly shorter than the wild type length (Budd and Campbell, 2013). Rad9 is unique amongst checkpoint proteins because it binds chromatin and inhibits nuclease activity at telomeres and DSBs (Bonetti et al, 2015, Ngo and Lydall, 2015).…”

“…Furthermore, in fission yeast Dna2 was shown to be involved in the generation of G-rich ssDNA at telomeres (Tomita et al, 2004). Importantly, it was reported that dna2 Δ rad9 Δ cells have abnormally low levels of telomeric 3’ G-rich ssDNA (Budd and Campbell, 2013). Consistent with what was reported for rad9 Δ dna2 Δ, chk1 Δ dna2 Δ, mec1 Δ dna2 Δ, rad17 Δ dna2 Δ, ddc1 Δ dna2 Δ and pif1 Δ dna2 Δ cells all showed low levels of 3’ G-rich ssDNA at telomeres in comparison with DNA2 strains (Figure 3a-b, Figure S3, Figure S4).…”

“…By this logic the suppression of dna2 Δ by rad9 Δ implies that dna2 Δ might cause telomere-specific rather than general chromosome replication defects. Furthermore, Dna2 localises to human and yeast telomeres (Choe et al, 2002, Chai et al, 2013, Lin et al, 2013), and pif1 Δ, which suppresses dna2 Δ, affects a helicase that is active at telomeres and affects telomere length (Dewar and Lydall, 2010, Budd and Campbell, 2013, Lin et al, 2013, Phillips et al, 2015). Thus, several lines of evidence suggest that Dna2 might play critical function(s) at telomeres.…”

Processing of eukaryotic Okazaki fragments by redundant nucleases can be uncoupled from ongoing DNA replicationin vivo

“…Therefore, although the nuclease activity of Dna2 is essential for viability(Budd et al, 296 2000), the absence of Dna2 during S-phase is not obviously detrimental to exponentially 297 dividing S. cerevisiae cells. It is possible that Dna2 is required for the cleavage of only a small 298 number of Okazaki fragment 5' flaps -either long flaps generated by low levels of ongoing 299 nuclease cleavage, or those in specific, late-replicating repeat regions such as telomeres(Budd and Campbell, 2013;Markiewicz-Potoczny et al, 2018) or the rDNA repeat(Villa et al, 2016).…”

Sharpening the ends for repair: mechanisms and regulation of DNA resection

“…Extensive resection by Apollo is inhibited by the binding of Pot1b to the ssDNA [201]. In yeast, Dna2 has also been suggested to be involved in limited processing of the 5′ strand to maintain the telomere length and telomerase binding [203,204]. While 5′ strand resection is important for telomere maintenance, over-resection could lead to telomere shortening, senescence, and other deleterious consequences [198].…”