Book of Abstracts: Albany 2011
June 14-18 2011
©Adenine Press (2010)
Getting a Firm Grip on DNA: Understanding DNA Recognition at the Finger-Finger Interface of Zinc Finger Proteins
Zinc finger nucleases hold tremendous potential for site-specifically editing genomes in a variety of organisms (1). However, their utility is predicated on the ability to efficiently create sequence-specific C2H2 Zinc Finger Proteins (ZFPs) for a wide variety of target sequences. Each zinc finger module typically binds to a 3 bp core DNA element (DNA triplet). Zinc finger modules have been characterized that can specify most of the sixty-four possible DNA triplets. These modules can be assembled into multi-finger zinc finger proteins (modular assembly) that bind extended target sites (9-12 bp), where typically three to four zinc fingers are employed for efficient DNA recognition. However, modularly-assembled ZFPs often show poor specificity presumably due to incompatible specificity determinants at the finger-finger interface (2). To understand the influence of these interactions on DNA recognition, we employed bacterial one-hybrid selections (3,4) to identify groups of amino acid residues at the interface of two-finger modules that specify all sixteen 2 bp interfaces between the two DNA triplets. In total, we could identify two-finger modules that specify >90% of these targets. Further analysis of the selected modules suggests the presence of complex interactions at the finger-finger interface that contribute to context-dependent effects on specificity. These selected finger pairs are functional in vivo, as zinc finger nucleases employing these modules generate targeted lesions in zebrafish. Ultimately, understanding finger-finger interactions will allow the rational design of multi-finger ZFPs for their use as artificial proteins and aid the assignment of specificities for naturally-occurring zinc finger proteins.
Ankit Gupta1, 2
1Program in Gene Function and Expression