CAREER: Structural Analysis of Branched DNA Recognition

University Of Toledo, Toledo OH

Investigators

Abstract

Bacteriophage T4 is an excellent model system for studies of DNA replication and repair. Recognition of branched DNA is the initial step that determines the fate of DNA replication, repair, and recombination intermediates. The central emphasis of this research is to determine how structure specific recognition of branched DNA is accomplished at the atomic level. Biophysical and single crystal X-ray diffraction studies will be employed to determine intermolecular interactions in DNA replication complexes. T4 RNase H, a member of the rad2 5' nuclease family, has structural similarity to the eukaryotic flap endonucleases needed for processing Okazaki fragments during lagging strand DNA synthesis and for DNA repair events involving strand displacement synthesis. Structure determination of T4 RNase H in complex with branched DNA substrates, and in complex with T4 gene 32 single stranded DNA binding protein, which coordinates T4 RNase H and DNA polymerase activities, will provide valuable insight into this fundamental process. Diffraction data from T4 RNase H (D132N mutant) in complex with fork DNA have been collected and structure determination is underway. T4 gene 59 helicase assembly protein recognizes branched DNA and directs the assembly of T4 41 helicase specifically at DNA replication forks. Structural studies of binary complexes between 59 protein and branched DNA and ternary complexes of 59 and 32 proteins with branched DNA will allow precise analysis of the molecular determinants of these specific interactions. Training in structural biology of macromolecular complexes will involve the use of crystallization robotics, automated imaging, and high brilliance and synchrotron X-ray sources.