Book of Abstracts: Albany 2011
June 14-18 2011
©Adenine Press (2010)
Elucidating the Intrinsic Sequence Specificity of Dnase I Using High-Throughput Sequencing
Deoxyribonuclease I (DNAse I) is an endonuclease that degrades DNA through hydrolysis of the phosphodiester bond (1). Though it is widely used to probe interactions between proteins and DNA both in vitro and in vivo(2), DNAse I is perhaps the ideal molecule to study in order to gain an understanding of the true richness of the sequence specificity of a DNA-binding factor. Since DNAse I cleaves the phosphodiester backbone, it leaves a precise record of its binding location along the chromosome. High-throughput sequencing technology allows for millions of such records to be amassed. Current state-of-the-art representations of the sequence specificities of DNA-binding factors employ algorithms to construct position weight matrices (PWMs). Implicit in the use of a PWM is the assumption that the total energy of the interaction between the DNA-binding factor and its binding site is merely the sum of the energies of the contacts to the individual nucleotides. Given the wealth of data that can be obtained with high-throughput sequencing, one can dispense with the independence assumption and determine the sequence specificity for each possible binding site. It is certain that these sequence specificities will provide an unprecedented opportunity to learn about the validity and drawbacks of the independence assumption.
Our analysis of the sequence specificity of DNAse I reveals that at least three base pairs up- and downstream of the cleavage site contribute to the cleavage rate. The depth of information provided by high-throughput sequencing allowed us accurately and comprehensively to model interactions between base positions within this range. We find that the rate at which DNA is cleaved is strand-specific and varies by more than two orders of magnitude between different sequence combinations. Our analysis also reveals a marked dependency between the first and second nucleotide positions downstream of the cleavage site, which is likely related to the local geometry of the DNA minor groove. Finally, comparison of the predicted specificities of DNaseI inferred independently from yeast and human genomic DNA cleavage patterns exposed species-specific differences in cleavage rates that were isolated to positions flanking CpG dinucleotides, indicating that the enzymatic efficiency of DNaseI may be significantly modulated by DNA methylation status events(3).
1Department of Electrical Engineering, Columbia University, New York, NY 10027