ULTRA HIGH RESOLUTION DIFFRACTION STUDIES OF FEN-1 AND OTHER DNA REPLICATION

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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 proposed diffraction studies seek to obtain higher resolution data from proteins in the DNA replication pathway. These proteins are of interest in modeling and understanding the macromolecular complexes that function in DNA replication and repair. Multidisciplinary structural studies of the complexes (protein:DNA and protein:protein) and their constituents are underway using diffraction and scattering techniques with X-rays and neutrons. However the bases of all our studies are maximal resolution X-ray structural models of the protein elements that comprise these molecular machines. Of primary interest for this proposal are the proteins FEN-1, and the elements and ternary complex of RNase H:gp32-N:DNA. We seek ultra-high resolution diffraction data (<0.9A) of flap endonuclease 1 (FEN-1) protein from the Aeropyrum pernix. This extremophile protein that binds flap lagging strand DNA during replication has diffracted to 1.4¿ in previous experiments. However larger crystals combined with flash-cooling conditions that better preserve crystalline order have resulted in measurement of much higher resolution diffraction data in-house than has been seen previously. This protein is also the subject of low temperature neutron diffraction studies, both with H/D exchanged and perdeuterated proteins. Combined with different cooling regimes during diffraction, the protein will provide a useful benchmark for comparison of different data measurement techniques. The 5¿ to 3¿ exonuclease (RNase H) and the single-stranded DNA binding protein (gp32) from bacteriophage T4 form a transient complex during DNA replication. The X-ray structures of T4 RNase H and of the core of T4 gp32 are known. In solution, gp32 missing the N-terminus binds to RNase H and forms a stable complex in the absence of DNA. Crystal diffraction extends to 3.5¿ with a long cell dimension. High flux beamline data are essential to resolving the structures of this complex.