Susceptibility to superhelically driven DNA duplex destabilization: a highly conserved property of yeast replication origins

Abstract

Strand separation is obligatory for several DNA functions, including replication. However, local DNA properties such as A+T content or thermodynamic stability alone do not determine the susceptibility to this transition in vivo. Rather, superhelical stresses provide long-range coupling among the transition behaviors of all base pairs within a topologically constrained domain. We have developed methods to analyze superhelically induced duplex destabilization (SIDD) in genomic DNA that take into account both this long-range stress-induced coupling and sequence-dependent local thermodynamic stability. Here we apply this approach to examine the SIDD properties of 39 experimentally well-characterized autonomously replicating DNA sequences (ARS elements), which function as replication origins in the yeast Saccharomyces cerevisiae. We find that these ARS elements have a strikingly increased susceptibility to SIDD relative to their surrounding sequences. On average, these ARS elements require 4.78 kcal/mol less free energy to separate than do their immediately surrounding sequences, making them more than 2,000 times easier to open. Statistical analysis shows that the probability of this strong an association between SIDD sites and ARS elements arising by chance is approximately 4 x 10(-10). This local enhancement of the propensity to separate to single strands under superhelical stress has obvious implications for origin function. SIDD properties also could be used, in conjunction with other known origin attributes, to identify putative replication origins in yeast, and possibly in other metazoan genomes.

Figure 1. Comparison of Stress-Induced Duplex Destabilization Calculations with Assessments of Thermodynamic Stability

(A) The SIDD profile showing the G(x) values computed for a 3-kbp region (525701–528700) on Chromosome X of yeast at superhelix density σ = −0.055. (B) The SIDD profile of a 38-bp deletion mutant of the same region [28], at the same superhelicity. The deletion is at positions 526489–526526, indicated by the red arrow. This deletion causes drastic changes of SIDD properties throughout the region, even 2 kbp away. This is an effect of the global coupling induced by the superhelical stresses. (C) Thermodynamic stability profiles of the same regions as computed by WEB-THERMODYN [12,13], both before (black) and after (red) the deletion. The only effect of this deletion, whose location is indicated by the red arrow, is to displace the downstream profile by 38 bp. However, as shown in (B), the SIDD profile is profoundly altered throughout the region.

Figure 2. The Cumulative Distribution of Destabilization Levels G(x) for the Entire Yeast Genome

For each value of G on the horizontal axis, this curve plots the number of base pairs (expressed as a percent of the genome) needing that amount of free energy (or less) to strand separate. G = 10 kcal/mol is sufficient to open any base pair in the genome.

Figure 3. ARS Elements Are More Destabilized Than Other Parts of the Genome

(A) The SIDD profile of a region (bp 166936–168740) of Chromosome 6, containing ARS606 (position marked in red). (B) For contrast, the SIDD profile of a randomly chosen but representative genomic location of equal length (Chromosome 6, bp 21000–22834). Graphs of all 39 ARS elements are presented in Protocol S1.

Figure 4. Duplex Destabilization at ARS Elements Compared with Duplex Destabilization at the Surrounding Sequences

This histogram shows the distributions of ARS elements (red), and of comparison regions (black) whose G_min values fall in the indicated ranges. (Here, as elsewhere, the lower the G_min value, the more destabilized the region.) The comparison regions were chosen to have the same lengths as the ARS element they flank, and to be positioned 250 bp away from it on either side. There being twice as many comparison regions as ARS elements, these distributions are normalized to show the fraction of sites of each type falling within each interval. Equivalent results were obtained when the comparison regions were chosen to directly abut the ARS elements, so the localization of destabilization at ARS elements is not simply a consequence of their positions within intergenic regions. (ARS elements 302, 303, and 320 on Chromosome III were positioned very close [20 bp separate ARS302 from ARS303, and ARS320 directly abuts ARS303], so for the purpose of these statistical tests these three were regarded as a single site.)

Figure 5. Localization of ARS Elements

(A) Replication timing profile of Chromosome 3. The two peaks indicated by red stars are predicted with high confidence to contain replication origins. (Data replotted from [23].) (B and C) SIDD profiles of the two peak regions (from [A]) are plotted to high resolution, along with locations of the known ARS element (red) and the DNA segments within which ORC and MCM proteins were shown to bind [22] (yellow). (B) shows the profile around ARS 310, and (C) shows that of ARS 314.