Crystal Structure of hSMUG1 and Its Interaction surface with DKC1

Abstract

SMUG1 is DNA repair protein, and Dyskerin (DKC1) is involved in maintaining telomeres. A recent publication has shown that SMUG1 interacts with DKC1 and contribute and the complex can be involved in RNA quality control (Jobert et al., 2013). RNA quality control is critical for the integrity and function of long-lived RNA molecules like ribosomal rRNA. This process removes erroneously modified RNA molecules, and is an important part of the stress response in cells. Mutations in DKC1 leads to an accelerated aging disease, Dyskeratosis Congenita, that renders the affected individuals at high risk of developing several types of cancer. In order to understand the molecular basis for the role of SMUG1 in RNA quality control, we want to determine the experimental 3-dimentional structure of the complex between SMUG1 and DKC1. In this study, we were able to achieve high purity of human SMUG1, by testing different expression vectors. The purified protein was used for crystallization and for solving the structure. For the human DKC1, we also tried different expression vectors and truncated forms of hDKC1. We were able to get good expression, however, the protein was not soluble. Thus, other expression system such as insect cells or yeast should be tried in the future for hDKC1. The purified hSMUG1 was used for cocrystallization with a 10mer oligonucleotide containing a single uracil in the middle. Through a series of crystallization screens, we were able to get optimized crystals of the hSMUG1-Asn85Ala:DNA complex. By X-ray crystallography, the 3-dimentional structure of the complex was solved at 2.48 Å resolution. The 3D model of hSMUG1 is important to study the protein-DNA interaction in detail, particularly with respect to uracil recognition and base excision. However, in this structure, the base excision repair was not carried out by hSMUG1, and also the uracil was not flipped into the active site pocket. Therefore, more cocrystallization needs to be done with different types of hSMUG1 and different substrates.