A. Structural mechanisms for progression of DNA processing assemblies.
Replication Protein A (RPA), the abundant eukaryotic single-strand DNA (ssDNA) binding protein, was chosen as the starting point for our studies because it is a central component of the assemblies that perform DNA replication, recombination and repair (DNA-RRR). The observation of functional interactions between RPA and a surprisingly large number of different DNA-RRR proteins indicates that RPA does much more than simply bind ssDNA. It is now well accepted that RPA functions in a highly interwoven series of steps involving binding of ssDNA, internal structural rearrangements, and interactions with other proteins. Previous work from our laboratory (Mer et al., 2000) revealed RPA uses a common binding mode for DNA damage recognition proteins from three different DNA repair pathways. These results suggested that RPA serves as a scaffold for the ordered assembly and disassembly of DNA-RRR complexes. To develop a more complete view of RPA function, it is essential to delineate its interactions with other proteins and address the coupling between its ssDNA and protein binding activities. To this end, projects have been devised to investigate RPA's interactions with proteins that function in the early stages of DNA replication and recombination, and to examine the effects of these RPA-protein networks on the binding of ssDNA. By piecing together RPA's actions in DNA-RRR, we will build a basic understanding of RPA's function and obtain insights into the transitions that enable the progression of DNA processing by the replication, recombination and repair machinery (Stauffer & Chazin, 2004b). Importantly, our studies will also provide insights into the mechanisms by which mutations in the constituent proteins cause functional defects that can lead to cancer and other diseases. This research is supported by NIH RO1 GM65484.
1. Replication- interactions with SV40 T-antigen helicase and DNA polymerase α/primase.
The study of this coupled set of proteins is carried out in collaboration with Ellen Fanning (Dept. of Biological Science) and is intended to provide insight into RPA's mode of action in the context of the progression of DNA replication using the well characterized model system, simian virus 40 (SV40). The network of the SV40 T-antigen (T-ag) helicase, RPA and human DNA polymerase α/primase (DNA pol-prim) proteins carries out the earliest stages of DNA replication: origin recognition, unwinding, and primer synthesis. The first goal in this project is to map out the RPA binding sites on T-ag and DNA pol-prim. Then, the binding domains are subcloned, expressed and purified for basic biophysical analysis as well as structure determination using a combination of crystallography, NMR and computational docking. The information obtained is then used to design mutations for functional studies in vitro and in vivo in collaboration with the Fanning laboratory.
2. Recombination-interactions with Rad51 and Rad52. (Stauffer & Chazin, 2004a)
Studies of the human RPA/Rad51/Rad52 system are designed to reveal structural mechanisms at the early stages of DNA recombination processes. The eukaryotic recombinase Rad51 is required for the initial strand invasion steps of DNA recombination. Rad51's activity is stimulated by the presence of RPA and Rad52, another RPA-binding protein that we have studied previously. The design of this project purposely parallels studies of the early stages of replication so that we can compare RPA's mode of action in the two systems. Hence, we follow the same basic strategy of mapping interaction sites, subcloning and producing proteins, biophysical analysis, and structure determination. Mutations will also be designed, in this case for functional analysis using an in vitro DNA unwinding assay.
3. Coupling of RPA protein interactions with ssDNA binding activity. (Bhattacharya 2002, Arunkumar 2003)
To enhance our overall understanding of RPA's activities, the interplay of ssDNA and protein binding must be factored into models of RPA's mode of action. RPA and T-ag form a cooperative network in the early stages of replication that closely parallels that of RPA and Rad51 in the context of recombination. A series of experiments has been designed to systematically examine the RPA affinities of ssDNA, T-ag and Rad51, first as binary, then as the ternary RPA/ssDNA/T-ag and RPA/ssDNA/Rad51 systems. In parallel, the effects of ssDNA and protein binding partners on RPA structure and dynamics are being determined using a combination of NMR chemical shift perturbation, cross saturation, residual dipolar couplings and heteronuclear relaxation experiments.
last updated April 26, 2004