Structural Studies of RNases H2 as an Example of Crystal Structure Determination of Protein-Nucleic Acid Complexes.

Abstract

RNases H2 are nucleases that cleave nucleic acids that comprise both RNA and DNA. They specifically recognize and cleave junctions between RNA and DNA using an intricate mechanism that involves substrate-assisted catalysis. Archaeal and eukaryotic RNases H2 also cleave the RNA strands of RNA/DNA hybrids. RNases H2 use their activity to maintain the integrity of genetic information. Particularly important is their ability to initiate the removal of single ribonucleotides from genomic DNA. Single ribonucleotides are very common in replicating cells and pose a serious threat to the stability of genomic DNA. The only known pathway for the error-free removal of single ribonucleotides begins with their recognition and cleavage by RNases H2. The importance of these enzymes is further underscored by the fact that mutations in the human enzyme lead to a severe autoimmune disease, Aicardi-Goutières syndrome. This review summarizes methods for the overproduction and purification of bacterial and human RNases H2. We also describe methods for testing the enzymatic activity of these nucleases and their crystallization both in unliganded form and in complex with nucleic acid substrates. We use these studies to describe general principles of the crystallization and structure determination of protein-nucleic acid complexes, particularly for nucleases. As illustrated by the structural studies of RNases H2, such complex structures can reveal intricate and fascinating aspects of the molecular mechanisms of nucleic acid enzymes.