Idling by DNA polymerase delta maintains a ligatable nick during lagging-strand DNA replication

Abstract

During each yeast cell cycle, approximately 100,000 nicks are generated during lagging-strand DNA replication. Efficient nick processing during Okazaki fragment maturation requires the coordinated action of DNA polymerase delta (Pol delta) and the FLAP endonuclease FEN1. Misregulation of this process leads to the accumulation of double-stranded breaks and cell lethality. Our studies highlight a remarkably efficient mechanism for Okazaki fragment maturation in which Pol delta by default displaces 2-3 nt of any downstream RNA or DNA it encounters. In the presence of FEN1, efficient nick translation ensues, whereby a mixture of mono- and small oligonucleotides are released. If FEN1 is absent or not optimally functional, the ability of Pol delta to back up via its 3'-5'-exonuclease activity, a process called idling, maintains the polymerase at a position that is ideal either for ligation (in case of a DNA-DNA nick) or for subsequent engagement by FEN1 (in case of a DNA-RNA nick). Consistent with the hypothesis that DNA polymerase epsilon is the leading-strand enzyme, we observed no idling by this enzyme and no cooperation with FEN1 for creating a ligatable nick.

Figure 1.

Strand-displacement synthesis by DNA polymerases. (A) Schematic of the substrate. The 113-mer template is Strep-Bio-V6 (Table 1); PRI is the 5′-³²P-labeled 30-mer primer; BLO is the 5′-phosphorylated 27-mer blocking oligo. The template strand between the PRI and BLO positions is (pT)₂₅ (see Materials and Methods). (B) DNA synthesis by Pol δ-5DV and Pol δ-wt with or without PCNA present. The standard assay (Materials and Methods) was modified to contain only 40 mM NaCl to allow PCNA-independent synthesis to proceed more efficiently. Full-length strand-displacement products should be 83 nt, but 1–2-nt shorter products were mainly observed because the biotin–streptavidin block inhibited progression of the polymerase. The open arrows indicate extension products paused at the nick position (55 nt, position 0) and the closed arrows indicate products at the +3 position (58 nt). (C) Strand opening by various DNA polymerases in the presence of PCNA. Only a close-up of the nick region is shown for each enzyme. The exo-deficient forms of Pol δ and Pol ε also carried out complete strand-displacement synthesis, generally up to ∼30% after 7 min (data not shown). The standard assays contained 100 mM NaCl, RPA, RFC, PCNA, and a threefold molar excess of each DNA polymerase over template-primer. Assays with Sequenase were carried out on naked DNA without accessory factors. Assays were at 30°C for the indicated times and analyzed by 7 M urea/12% PAGE, followed by PhosphorImaging of the dried gel. Strand opening (as percent) is defined as 100 × [products in positions 1 + 2 + 3 + 4]/[products in positions 0 + 1 + 2 + 3 + 4] at the 5-min time point.

Figure 2.

Nucleotide turnover at a nick. (A) Standard dGTP → dGMP turnover assays contained the Strep-Bio-V6 template either with the PRI oligo or with the BLO oligo, or with both, as indicated (see Fig. 1A). Reactions were carried out for the indicated times with Pol δ or with Pol ε. (B) The standard turnover assay, with both the PRI and BLO oligos hybridized, contained wild-type Pol δ and increasing [α-³²P]dGTP, from 2.5 μM to 80 μM. Rates of turnover were calculated from time-course assays and plotted against the dGTP concentration. Half-maximal turnover rates were obtained at 3 μM dGTP. (C) The standard turnover assay, with both the PRI and BLO oligos hybridized, contained wild-type or L523X Pol δ, as indicated, or wild-type Pol δ with FEN1 or with DNA ligase I. (Inset) Sequences of the oligos at the junction positions are shown.

Figure 3.

Efficient turnover by Pol δ requires strand opening. Standard dGTP → dGMP turnover assays contained the Strep-Bio-V6 template primed with the PRI oligo and one of a series of BLO oligos as shown. The C on the template indicated by the arrow is defined as position 0, and therefore blocking oligo pC6 (5′-p-GGTTCC—) has its 5′-junction at the 0 position, and the other oligos accordingly. Blocking oligo pC6-2G (Table 1) is the +2 oligo. Turnover rates with Pol δ (gray) or with Pol ε (cross-hatched) were plotted as a function of the junction position.

Figure 4.

Gap filling and ligation by Pol δ and Pol ε. Assays were as described in Materials and Methods. (A) The scheme shows the proposed sequential action of the factors. However, in the assay PCNA was first loaded by RFC onto RPA-coated primed DNA. Subsequently, Pol δ or Pol ε was added together with DNA ligase and FEN1, where indicated. (B) Comparison of Pol δ and Pol ε. Replication was for the indicated times at 30°C. PCNA was increased to a 10-fold excess over DNA to increase processivity of Pol ε (Burgers 1991). The arrow indicates a replicational pause that is particularly strong with Pol ε. (C) Comparison of Pol δ mutants. All assays were for 7 min at 30°C. The replication products indicated by the arrows are due to aberrant strand displacement. The generation of these products is suppressed in the presence of FEN1.

Figure 5.

Coupled action of Pol δ and FEN1 at a nick. Nick translation assays were as described in Material and Methods. (A) Analysis of nick translation products at a 5′-DNA or a 5′-RNA nick. Nick translation was carried out for 1 min at 30°C. Prolonged incubation, up to 5 min, did not significantly change the distribution of the small oligonucleotide and mononucleotide products (data not shown). Control reactions carried out under otherwise identical conditions were as indicated. In the marker lanes, the DNA oligo was partially digested with snake phosphodiesterase (PDE, lane 5), or with exonuclease V (ExoV, lane 6), which produces a 5′-dinucleotide (Burgers et al. 1988), and the RNA–DNA oligo with PDE (lane 7). (B) Comparison of Pol δ mutants in nick translation. The indicated forms of Pol δ were used on the DNA oligo substrate. Only the mononucleotide to hexanucleotide region is shown. None of the DNA polymerases showed products in this region in the absence of FEN1.

Figure 6.

A binary choice model for Pol δ to maintain a ligatable nick. A DNA gap and displacement of 2 nt by Pol δ is shown. During Okazaki fragment maturation, Pol δ and FEN1 go through multiple cycles of displacement synthesis and FLAP cutting (upper cycle, nick translation) until all RNA has been degraded. pN₁pN₂ is the oligonucleotide released during nick translation, whereas pN₁ and pN₂ indicate mononucleotides released either during nick translation (orange) or during idling (purple).