STRUCTURES OF PROTEIN-DNA COMPLEXES INVOLVED IN EUKARYOTIC 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. Before DNA replication can take place, specialized AAA+ ATPases known as initiators must separate the template strands. The monomeric structure of one eukaryotic-type archaeal Cdc6/Orc1 initiator has been reported, but very little is known about how origin binding and oligomer formation leads to initiation. We have recently used selenomethionine phasing to solve the structure to 3.3¿ of a complex between a Cdc6/Orc1 hetereodimer and a genomic initiation sequence. We hope to use the synchrotron facilities at SSRL to 1) improve the experimental phases via heavy atom derivatives and 2) improve the resolution of this structure, allowing us to gain insight into the control of replication initiation at an atomic level. Initial experiments have suggested that proper cryoprotection is critical for resolution, and we plan to perform a systematic screen of cryoprotectants and osmotic strengths to improve resolution. Once replication has initiated and the replisome has begun duplicating the genome, topological stress builds ahead of the replication fork. The homodimeric type II topoisomerases (TopoII) relieve this stress through a complex series of movements that culminate in one DNA strand being passed through the other. We have recently obtained a 3.2¿ native dataset from a crystal of the S. cerevisiae TopoII trapped in the middle of a strand cleavage reaction with orthovanadate. We plan to use the SSRL facilities to both extend the resolution of this data and obtain phases from SAD/MAD/SIRAS experiments. Though certain platinum salts yield stable derivatives, initial experiments with these crystals have not yet yielded usable phase information.