Rejoining of DNA double-strand breaks as a function of overhang length

Abstract

The ends of spontaneously occurring double-strand breaks (DSBs) may contain various lengths of single-stranded DNA, blocking lesions, and gaps and flaps generated by end annealing. To investigate the processing of such structures, we developed an assay in which annealed oligonucleotides are ligated onto the ends of a linearized plasmid which is then transformed into Saccharomyces cerevisiae. Reconstitution of a marker occurs only when the oligonucleotides are incorporated and repair is in frame, permitting rapid analysis of complex DSB ends. Here, we created DSBs with compatible overhangs of various lengths and asked which pathways are required for their precise repair. Three mechanisms of rejoining were observed, regardless of overhang polarity: nonhomologous end joining (NHEJ), a Rad52-dependent single-strand annealing-like pathway, and a third mechanism independent of the first two mechanisms. DSBs with overhangs of less than 4 bases were mainly repaired by NHEJ. Repair became less dependent on NHEJ when the overhangs were longer or had a higher GC content. Repair of overhangs greater than 8 nucleotides was as much as 150-fold more efficient, impaired 10-fold by rad52 mutation, and highly accurate. Reducing the microhomology extent between long overhangs reduced their repair dramatically, to less than NHEJ of comparable short overhangs. These data support a model in which annealing energy is a primary determinant of the rejoining efficiency and mechanism.

FIG. 1.

Oligonucleotide-modified plasmid assay. (A) Plasmid modification scheme. pTW423 is digested with BglII and XhoI and purified, removing the polyterminator. Annealed oligonucleotides are then ligated onto the BglII and XhoI ends, restoring the ADE2 coding sequence. Precise in-frame repair of the break yields Ade⁺ colonies. (B) Primer extension assay to determine the oligonucleotide ligation efficiency. Annealed oligonucleotides (Oligos) were added to the ligation reaction mixture at concentrations of 50, 100, 500, 1,000, and 5,000-fold molar excess over the concentration of the linearized plasmid (indicated by the thickness of the triangle over the lanes). Primer extension was performed after ligation as described in Materials and Methods.

FIG. 2.

Rejoining of DSBs with 3′ overhangs. (A) DSBs with 3′ overhangs are repaired more efficiently as the single-stranded overhang length increases. To control for transformation efficiency, repair is expressed as the ratio of Ade⁺ colonies arising from accurate repair of the linearized plasmid over Leu⁺ colonies resulting from uptake of a cotransformed supercoiled plasmid. This ratio can exceed 1 in part because 10-fold-more linear plasmid than circular plasmid is transformed. Each point represents the mean ± standard deviation (error bar) from four independent transformations. (B) DSB repair is independent of Yku70 and Dnl4 when overhangs are 6 nucleotides and longer. (C) Repair of DSBs with overhangs 8 nucleotides and longer is partially compromised in a rad52Δ strain. (D) In the absence of Rad52, DSBs with overhangs of >8 nucleotides are not repaired by Yku70-dependent NHEJ. Due to the decreased transformation efficiency of the yku70Δ rad52Δ strain, we were able to reliably assay only DSBs with overhangs of 6 nucleotides or longer at the plasmid concentration used. (E) Adding 5′ phosphates to DSBs enhances the repair of DSBs with 4- but not 13-base overhangs. Different carrier DNA and plasmid preparations were used in the experiment shown panel E than those used in the experiments shown in panels A to D, resulting in a different range in the linear to supercoiled plasmid transformation ratio. Variability in this ratio is common when changing reagents in yeast transformation experiments, as carrier DNA has nonequivalent effects on the uptake of linear and supercoiled DNA. This does not affect the interpretation of the data within an experiment, and similar trends are observed in panel E versus panels A to D.

FIG. 3.

Rejoining of DSBs with 5′ overhangs. The experiments are analogous to those shown in Fig. 2. Data are expressed as the ratio of Ade⁺ to Leu⁺ colonies as in Fig. 2.

FIG. 4.

Partially filling 5′ overhangs inhibits their repair by both the Rad52-dependent and NHEJ pathways. (A) The oligonucleotide ligation technique was used to construct DSBs with partially filled 5′ 13-base overhangs. Substrate pTW423-513F is expected to equilibrate between two different annealing states. Data are expressed as the ratio of Ade⁺ to Leu⁺ colonies as in Fig. 2. Each point represents the mean ± standard deviation (error bar) from four independent transformations. (B) 4-Base 5′ overhangs were filled one base at a time with Klenow polymerase in vitro (see Materials and Methods). Data are expressed as colony counts from linearized pES16 normalized to colony counts from parallel transformations with supercoiled pES16. For comparison, these ratios were further normalized so that the total repair rate of the unfilled overhangs equaled 100%. Each point represents the mean ± standard deviation (error bar) from three independent transformations. Accurate repair yields white Ade⁺ colonies, whereas imprecise repair results in red Ade⁻ colonies.

FIG. 5.

Increasing the GC content of overhangs relaxes the requirement for NHEJ. Oligonucleotide-modified plasmids were designed to contain DSBs with 3′ 4-base overhangs with one, two, or three GC base pairs. Data are expressed as the ratio of Ade⁺ to Leu⁺ colonies as in Fig. 2. Each point represents the mean ± standard deviation (error bar) from four independent transformations.

FIG. 6.

Rejoining of DSBs with 3′ phosphate termini. Oligonucleotide ligation was used to generate plasmids containing DSBs with 3′ overhangs of 4, 6, 8, and 13 bases terminating in 3′ hydroxyls (A) or 3′ phosphates (B). The tpp1Δ apn1Δ apn2Δ genotype is represented as taaΔ. Data are expressed as the ratio of Ade⁺ to Leu⁺ colonies as in Fig. 2.

FIG. 7.

Inefficient repair of DSBs with long overhangs but short regions of homology. DSBs with long overhangs but limited base-pairing potential, which form gap structures upon annealing, were created by oligonucleotide ligation. Data are expressed as the ratio of Ade⁺ to Leu⁺ colonies as in Fig. 2. No Ade⁺ colonies were recovered for substrate pTW423-313/9.

FIG. 8.

A model for rejoining of DSBs as a function of overhang length. Three pathways can rejoin DSBs. NHEJ, catalyzed by the Ku and DNA ligase IV complexes, is mainly used when joint stabilization is energetically unfavorable, such as when overhangs are less than 4 bases. DSBs with overhangs of 6 or more bases can anneal and be rejoined without the need for Ku and DNA ligase IV. Longer overhangs (>10 bases) can also function as substrates for Rad52, which promotes rejoining through an SSA-like pathway. See text for further discussion.