STRUCTURAL STUDIES OF DNA REPLICATION INITIATION

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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 continuous movement of the DNA replication fork requires priming at the lagging strand to make each Okazaki fragment as well as a coordinated action of primases and the replicative DNA polymerase. The replication origin of the single-stranded DNA (ssDNA) bacteriophage G4 (G4ori) is a simple system of DNA priming; it needs only the E. coli primase (dnaG) and the single-stranded DNA binding protein (SSB). G4ori consists possibly three stem-loops located immediately on the 5¿¿ side of the initiation trinucleotide 5¿¿-CTG-3¿¿ and, upon saturated binding by SSB tetramers, will form a unique structure recognizable by dnaG primase. The three domains of the modular primase then bind the G4ori initiation site cooperatively to synthesize the primer RNA. A single-stranded 278 nucleotide fragment of G4ori (G4ori278), together with two dnaG primases and four SSB tetramers, can form a minimal active priming complex suitable for structural investigations. We have isolated and obtained crystals of two types of complexes: SSB-G4ori278 and primase-SSB-G4ori278. The SSB-G4ori278 complex does not contain dnaG primase, while the primase-SSB-G4ori278 complex has primase present. Obtaining atomic resolution structures of these complexes will answer many functionally relevant questions on the replication origin of G4 phage DNA and the structure-function of SSB and primase, such as what is the overall structure of the stem-loops of the replication origin and how do the SSB molecules interaction with the ssDNA to form the unique structure of the replication origin. The SSB-G4ori278 structure will also shed light on how SSB interacts with DNA. Furthermore, the structure of the complex with the primase present will show how the primase recognize the replication origin and the interaction of primase and SSB. Obtaining the structure of the primase-SSB-G4ori complex will open many doors for future structural and biochemical studies of DNA priming and replication. For example, it will enable us to study the process of primer synth! esis by obtaining the complex structures of with the primase synthesis intermediates.