Unraveling the molecular basis of protein search on ssDNA track and binding specificity of the ssDNA sequences.

Abstract

The genetic materials of cellular organisms are organized as double-stranded DNA (dsDNA), which transiently gets unwrapped and forms single-stranded DNA (ssDNA) intermediates. These intermediates serve as a template for the various cellular processes. In its isolated form, ssDNA is vulnerable to nucleolytic degradation and prone to adopt unwanted secondary structures. To avoid the mess, cells employ specialized ssDNA binding proteins (SSBs), which bind with high affinity and both specifically and non-specifically to ssDNA to stabilize these intermediates. To understand their biophysics, we have developed a computational model that has been extensively parametrized by studying ssDNA binding on replication protein A, a major eukaryotic SSB, which is later tested on numerous other protein-ssDNA complexes. Our study delineates the molecular mechanism of target search by SSBs on the ssDNA track, which is otherwise difficult to capture through experimental approaches due to their binding promiscuity. The SSB-ssDNA model is then used as a testbed to develop a hybrid simulation technique for calculating protein-ssDNA binding thermodynamics for many ssDNA sequences in a single run. The method explores both sequence specificity of ssDNA and conformational space simultaneously by simulating a joint probability distribution, which makes searching through ssDNA sequence space computationally efficient. Our results show that the method agrees well with the experiments in predicting the relative binding specificity of ssDNA sequences for a given SSB without performing a costly, time-consuming DNA microarray experiment.