Abstract
Post-replicational telomere end processing involves both extension by telomerase and resection to produce 3'-GT-overhangs that extend beyond the complementary 5'-CA-rich strand. Resection must be carefully controlled to maintain telomere length. At short de novo telomeres generated artificially by HO endonuclease in the G2 phase, we show that dna2-defective strains are impaired in both telomere elongation and sequential 5'-CA resection. At native telomeres in dna2 mutants, GT-overhangs do clearly elongate during late S phase but are shorter than in wild type, suggesting a role for Dna2 in 5'-CA resection but also indicating significant redundancy with other nucleases. Surprisingly, elimination of Mre11 nuclease or Exo1, which are complementary to Dna2 in resection of internal double strand breaks, does not lead to further shortening of GT-overhangs in dna2 mutants. A second step in end processing involves filling in of the CA-strand to maintain appropriate telomere length. We show that Dna2 is required for normal telomeric CA-strand fill-in. Yeast dna2 mutants, like mutants in DNA ligase 1 (cdc9), accumulate low molecular weight, nascent lagging strand DNA replication intermediates at telomeres. Based on this and other results, we propose that FEN1 is not sufficient and that either Dna2 or Exo1 is required to supplement FEN1 in maturing lagging strands at telomeres. Telomeres may be among the subset of genomic locations where Dna2 helicase/nuclease is essential for the two-nuclease pathway of primer processing on lagging strands.
Highlights
DNA2 helicase/nuclease participates in telomere maintenance by unknown mechanisms
Yeast dna2 mutants, like mutants in DNA ligase 1, accumulate low molecular weight, nascent lagging strand DNA replication intermediates at telomeres. Based on this and other results, we propose that FEN1 is not sufficient and that either Dna2 or Exo1 is required to supplement FEN1 in maturing lagging strands at telomeres
Nuclease-defective Dna2 Is Impaired in Elongation of a de Novo Telomere—To examine the roles of nucleases in telomere synthesis, we used a strain in which telomere elongation was measured in G2 phase cells at an HO endonuclease-induced DSB having an 81-bp telomere TG/CA seed sequence inserted centromere-proximal to the HO cut site (Fig. 1A) [21]
Summary
DNA2 helicase/nuclease participates in telomere maintenance by unknown mechanisms. Results: dna mutants show slightly shortened telomeric GT-overhangs and accumulate small, nascent lagging strand DNA chains at telomeres. Telomeres are one possibility, because replication forks are known to stall within the duplex GT-rich repeats, indicating they pose difficulties for the processive replisome and because bulk Dna localizes to telomeres from yeast to mammals [42,43,44]. Exo creates long regions of single-stranded DNA at uncapped telomeres in cdc mutants and is involved in resection of a de novo telomere in G2-arrested cells (see Fig. 1). Unlike at DSBs [45, 46, 48, 61], we were surprised to find that inactivation of Mre or Exo did not further reduce overhangs at native telomeres This could be due to a delay in CA-strand fill-in, because we demonstrate that nascent lagging strands accumulate as frag-.
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