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 × 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.
Highlights
In eukaryotes, DNA replication is initiated at multiple origins
ARS elements are more AþT-rich than the genomic average, and contain regions of low local thermodynamic stability that are thought to be necessary for function [2,3]
We hypothesize that the superhelical stresses that occur in vivo play a role in regulating the strand opening needed to initiate replication
Summary
DNA replication is initiated at multiple origins. Potential sites in the genome of the yeast Saccharomyces cerevisiae that may serve this function are referred to as autonomously replicating sequences, or ARS elements [1]. ARS elements are more AþT-rich than the genomic average, and contain regions of low local thermodynamic stability that are thought to be necessary for function [2,3]. The duplex unwinding required for replication initiation occurs as an isothermal process within topologically constrained domains of DNA. Under these conditions susceptibility to strand opening is not dependent only on local thermodynamic stability. We hypothesize that the superhelical stresses that occur in vivo play a role in regulating the strand opening needed to initiate replication. This suggests that ARS elements should have an increased local susceptibility to superhelically induced duplex destabilization (SIDD). Experiments on four specific ARS-containing regions have shown that each does experience local denaturation when negatively supercoiled [4]
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