Overcoming natural replication barriers: differential helicase requirements

Abstract

DNA sequences that form secondary structures or bind protein complexes are known barriers to replication and potential inducers of genome instability. In order to determine which helicases facilitate DNA replication across these barriers, we analyzed fork progression through them in wild-type and mutant yeast cells, using 2-dimensional gel-electrophoretic analysis of the replication intermediates. We show that the Srs2 protein facilitates replication of hairpin-forming CGG/CCG repeats and prevents chromosome fragility at the repeat, whereas it does not affect replication of G-quadruplex forming sequences or a protein-bound repeat. Srs2 helicase activity is required for hairpin unwinding and fork progression. Also, the PCNA binding domain of Srs2 is required for its in vivo role of replication through hairpins. In contrast, the absence of Sgs1 or Pif1 helicases did not inhibit replication through structural barriers, though Pif1 did facilitate replication of a telomeric protein barrier. Interestingly, replication through a protein barrier but not a DNA structure barrier was modulated by nucleotide pool levels, illuminating a different mechanism by which cells can regulate fork progression through protein-mediated stall sites. Our analyses reveal fundamental differences in the replication of DNA structural versus protein barriers, with Srs2 helicase activity exclusively required for fork progression through hairpin structures.

Figure 1.

Schematic of the YEp24 construct and the location of the insert sequences. Indicated sequences were cloned into the AseI site of YEp24 in both orientations. YEp24 plasmids containing either (CGG/CCG)₄₀ or (G₄T₄/C₄A₄) were digested with the restriction enzymes SalI and XbaI, generating a fragment of 4.4 kb. YEp24 plasmids containing either Htel or Ytel sequences were digested with MfeI and NheI, generating a fragment of 4.5 kb.

Figure 2.

Analysis of replication through sequence and protein barriers in WT and srs2Δ cells. Replication of (a) CGG and CCG repeats, (b) Oxytel sequence, (c) Htel sequence, (d) Ytel sequence. Sequence nomenclature refers to the lagging strand template. Solid arrows indicate location of the stall due to the insert. Quantification of the percentage of stalled intermediates compared to the Y arc signal is shown in the graphs at right. (e) Densitogram showing relative peak intensities of the stall by CGG, Oxytel, Htel and Ytel sequences with G rich sequences on the lagging strand template. The following number of experiments were done for each strain: WT CGG, 7; WT CCG, 4; srs2Δ CGG, 5; srs2Δ CCG, 4; WT Ytel, 3; all others, 2. Error bars indicate standard error of the mean (SEM). Stars indicate a significant difference between the wild-type and mutants (Student's t-test, **P ≤ 0.01; *P ≤ 0.05).

Figure 3.

Direct repeat recombination assay to measure the breakage rate of CGG and CCG repeats in WT and srs2Δ cells. (a) Schematic of the genetic assay. The construct contained 81 repeats of CGG in either orientation and the URA3 gene, integrated at the LYS2 locus on yeast chromosome II such that they are flanked by 708 bp of duplicated LYS2 sequence. Recombination between the LYS2 duplication will result in loss of the URA3 gene and resistance to the drug 5-FOA. (b) Mean rates (shown on top of the bars) ± SEM were compared by a pooled variance t-test. Asterisks indicate significant difference between the indicated categories. **P ≤ 0.01, *P ≤ 0.05.

Figure 4.

Determination of the Srs2 activity needed for fork progression past a CGG barrier and unwinding efficiencies. (a) Analysis of replication intermediates was performed and quantified as in Figure 2a. Percentage stall is the average of 4, 3, and 2 experiments for Δ875-902, K41R and 1-998, respectively, ± SEM. WT and srs2Δ values as in Figure 2a. (b) Srs2-mediated unwinding of substrates with no hairpin or a (CGG)₁₁ or (CTG)₁₁ triplet is shown. ATP was omitted in lane 6 and the helicase defective srs2-K41R mutant was examined in lane 7. Heat-denatured substrate (HD) was analyzed in lane 8. The mean values ±SD from three independent experiments of analyzing wild-type Srs2 activity are plotted on the graph. (c) Unwinding of the same substrates by the Sgs1 helicase. The helicase defective sgs1-K706A mutant was examined in lane 6, and heat-denatured substrate (HD) was analyzed in lane 7. The mean values ±SD from three independent experiments of analyzing wild-type Sgs1 activity are plotted.

Figure 5.

Analysis of replication through hairpin and G4 forming sequences in sgs1Δ, pif1Δ and rrm3Δ cells. Labels and symbols as in Figure 2. Percent stall is the average of at least two experiments ± SEM.

Figure 6.

Analysis of replication through a telomere protein barrier in sgs1Δ, pif1Δ and rrm3Δ cells. Labels and symbols as in Figure 2. Percent stall is the average of at least two experiments ± SEM. White arrow indicates presumptive recombination structures.

Figure 7.

Effect of HU on replication of CGG and Ytel sequences. (a) Replication of CGG sequences in WT cells in the presence (+) and absence (−) of 0.2 M HU. Percent stall is the average of four experiments ± SEM. (b) Replication of Ytel sequences in the presence and absence of 0.2 M HU in WT and mutant cells. Percent stall is the average of at least two experiments ± SEM. Comparisons were made to −HU control of the same strain, *P ≤ 0.05.