Maintaining Genome Stability: The Role of Helicases and Deaminases

(Aim 1). Study the in vitro functions of MCM proteins from archaea and yeast cells using the genetically engineered protein constructs. In this aim, we will also extend our prior success in the X-ray structural studies of an N-terminal fragment of an archaea MCM by attempting to crystallize MCM proteins from yeast. (Aim 2). Examine in vivo effects of helicase function and in particular MCM roles in maintaining genome integrity in response to damage. This aim will use existing and newly generated mutants, which can be achieved through genetic screening and site-directed mutagenesis based on the 3-dimensional structure of MCM, to investigate how MCMs contribution to genome stability during chemical damage. (Aim 3). Express, purify and crystallize the proteins of deaminases. We will focus on AID and APOBEC3G to obtain purified deaminase proteins for the in vitro biochemical, functional, and structural studies. (Aim 4). Examine the functions and substrate specificity of AID and identify other factors required for the coupling of deamination with other processes of DNA synthesis and RNA transcription. The experiments will be carried out in a cell free assay