Evidence that base stacking potential in annealed 3' overhangs determines polymerase utilization in yeast nonhomologous end joining

Abstract

Nonhomologous end joining (NHEJ) directly rejoins DNA double-strand breaks (DSBs) when recombination is not possible. In Saccharomyces cerevisiae, the DNA polymerase Pol4 is required for gap filling when a short 3' overhang must prime DNA synthesis. Here, we examined further end variations to test specific hypotheses regarding Pol4 usage in NHEJ in vivo. Surprisingly, Pol4 dependence at 3' overhangs was reduced when a nonhomologous 5' flap nucleotide was present across from the gap, even though the mismatched nucleotide was corrected, not incorporated. In contrast, a gap with a 5' deoxyribosephosphate (dRP) was as Pol4-dependent as a gap with a 5' phosphate, demonstrating the importance of the downstream base in relaxing the Pol4 requirement. Combined with prior observations of Pol4-independent NHEJ of nicks with 5' hydroxyls, we suggest that base stacking interactions across the broken strands can stabilize a joint, allowing another polymerase to substitute for Pol4. This model predicts that a unique function of Pol4 is to actively stabilize template strands that lack stacking continuity. We also explored whether NHEJ end processing can occur via short- and long-patch pathways analogous to base excision repair. Results demonstrated that 5' dRPs could be removed in the absence of Pol4 lyase activity. The 5' flap endonuclease Rad27 was not required for repair in this or any situation tested, indicating that still other NHEJ 5' nucleases must exist.

Figure 1

(A) Short and long patch pathways of mammalian BER. (B) Analogous modes of 5’ processing in putative short and long patch pathways of NHEJ.

Figure 2

The requirement of Pol4 for gap filling is relaxed in the presence of 5’ flaps. (A) and (B) OMPs were used to create DSBs with the indicated structures. Repair is expressed as the ratio of Ade+ colonies to Leu+ colonies. Ade+ colonies arise from accurate repair of the OMP, whereas Leu+ colonies result from a simple religation NHEJ event (see Materials and Methods). A reduction in this ratio indicates a defect in end processing. Each point represents the mean ± standard deviation from three independent transformations. Vertical lines on sequences denote the overhang position, and annealed nucleotides are shown in gray.

Figure 3

5’ dRP lesions demand gap filling by Pol4, but do not require the Pol4 lyase activity or Rad27. (A) Schematic for construction of OMPs with 5’ dRP termini. Deoxyuracil residues are indicated in gray. Treatment with T4 PNK followed by UDG forms a 5’ dRP. (B) Polymerase domain point mutant pol4-D367E eliminates repair of 5’ dRP-containing DSBs only when a missing base must be synthesized. Lyase domain mutant pol4-KK247∷248RR does not impair rejoining when 5’ dRPs are present. (C) Additional mutation of rad27 does not impair joining. Data are expressed as in Figure 2.

Figure 4

Rad27 is not required for rejoining of DSBs with gaps, flaps, and complex end structures. (A), (B) and (C) Joints with the indicated structures were created in OMPs, and data are expressed as in Figure 2. (D) pBX2 was cut with BglII and XhoI, forming the joint pictured within the ADE2 gene. PCR on 16 Ade+ colonies was used to differentiate flap joining from blunt joining, and data were corrected to show only the efficiency of flap joints in each strain.

Figure 5

3’ dideoxynucleotide termini inhibit NHEJ. (A) Addition of the 3’ dideoxynucleotide was verified using pTW582, which forms compatible overhangs upon BstXI digestion. The linearized plasmid was incubated with T4 DNA ligase before (lane 4) and after TdT treatment (lane 5). (B) BstXI-digested plasmids were transformed into wild-type and pol4Δ yeast before and after TdT treatment. Data are expressed as in Figure 2.

Figure 6

A model for Pol X polymerase action in NHEJ. (A) When a base exists on the 5’ terminal position of a template strand nick or mismatched 5’ flap, stacking interactions (dashed lines) are hypothesized to stabilize that strand sufficiently for its use by a polymerase other than Pol4. P = primer strand, T = template strand, green = incoming dNTP. (B) When gaps with no potential for base stacking exist on both strands, proteins motifs in Pol4 (cyan colored loop) are hypothesized to provide critical bridging contacts in the template strand gap. (C) Depiction of the extensive base stacking observed in a crystal structure of Pol λ (PDB 1XSN, [35]). The 0 (template nucleotide), -1 and +1 template strand positions are labeled. Pol λ residues are labeled and colored by element (yellow = carbon, blue = nitrogen, red = oxygen). Grey = DNA, green = incoming dNTP. (D) A different view of PDB 1XSN to highlight the position of Loop I (colored in cyan) relative to the expected position of a template strand gap in NHEJ (the -4 nucleotide colored in magenta, which might be absent in an NHEJ joint). The template strand (T) is otherwise yellow, the primer strand (P) white, the incoming dNTP green, and the protein in blue cartoon diagram.