Susceptibility to Superhelically Driven DNA Duplex Destabilization: A Highly Conserved Property of Yeast Replication Origins

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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. Several DNA functions require the two strands of the DNA duplex to transiently separate. Examples include the initiation of gene expression and of DNA replication. Here the authors examine the strand separation properties of the DNA duplex at autonomously replicating sequences (ARS elements), which are the potential replication origins in yeast. In vivo, susceptibility to strand separation does not depend only on local DNA properties such as adenine plus thymine content or thermodynamic stability. Rather, stresses imposed on the DNA in vivo couple together the strand-opening behaviors of all base pairs that experience them. The authors use computational methods for analyzing stress-driven strand separation to examine the susceptibility to opening of 39 experimentally well-characterized ARS elements. They show that these ARS elements have strikingly increased susceptibilities to stress-induced separation relative to the surrounding sequences. On average, these ARS elements require 4.78 kcal/mol less free energy to separate than do surrounding sequences, making them more than 2,000 times easier to open. This enhanced susceptibility to stress-driven strand separation has obvious implications for the mechanisms that begin the process of replication. This property is also shared by bacterial and viral replication start points, suggesting that it may be a general attribute of replication origins.