Okazaki Fragment Maturation in Yeast

Abstract

To address the different functions of Pol δ and FEN1 (Rad27) in Okazaki fragment maturation, exonuclease-deficient polymerase Pol δ-01 and Pol δ-5DV (corresponding to alleles <i>pol3–01</i>-(D321A, E323A) and <i>pol3–5DV</i>-(D520V), respectively) were purified and characterized in this process. In the presence of the replication clamp PCNA, both wild-type and exo⁻ Pol δ carried out strand displacement synthesis with similar rates; however, initiation of strand displacement synthesis was much more efficient with Pol δ-exo⁻. When Pol δ-exo⁻ encountered a downstream primer, it paused with 3–5 nucleotides of the primer displaced, whereas the wild type carried out precise gap filling. Consequently, in the absence of FEN1, Pol δ exonuclease activity was essential for closure of simple gaps by DNA ligase. Compared with wild type, Okazaki fragment maturation with Pol δ-exo⁻proceeded with an increased duration of nick translation prior to ligation. Maturation was efficient in the absence of Dna2 and required Dna2 only when FEN1 activity was compromised. In agreement with these results, the proposed generation of double strand breaks in<i>pol3-exo</i> ⁻ <i>rad2</i>7 mutants was suppressed by the overexpression of <i>DNA2</i>. Further genetic studies showed that <i>pol3-exo</i> ⁻ <i>rad27</i> double mutants were sensitive to alkylation damage consistent with an <i>in viv</i>o defect in gap filling by exonuclease-deficient Pol δ.