This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The main goal of our research has been to determine the structures of DNA replication proteins, until now using single crystal X-ray diffraction. We have recently extended our activities to include solution scattering structural studies of protein:DNA and protein:protein complexes. The proteins are known to interact through structurally specific recognition of fork and flap DNA substrates. SAXS studies of the complexes matched with high resolution single protein models should provide us essential structural information needed to model the complexes and determine experimentally the positioning of the proteins and DNA within the structures. Our single crystal studies have provided high resolution models for several of the elements within these complexes. In the current funded research program, three protein:DNA complexes have been targeted for analysis. The first complex for pilot SAXS study is the T4 RNase H in complex with full length and truncated T4 32 protein with and without DNA. Using SAXS, we would like to 1.) verify that the complexes are uniform in size and shape, 2.) determine, if possible, the relative orientation of the interacting molecules, and 3.) determine the appropriate size of DNA substrate needed to stabilize a homogeneous complex. Information from these SAXS studies in addition to providing solution structural models will also facilitate our research involving single crystal diffraction by deliniating the conditions and preparation of homogenous and stable complexes capable of surviving for the duration of crystallization. Currently we utilize dynamic light scattering to inform us of the approximate size and homogeneity of the sample. Materials for the pilot study are available and have been characterized as monodisperse with light scattering at concentrations similar to those contemplated for SAXS studies.
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