SFI UREKA Site in Molecular Recognition
Translating Design into Application

Biophysical Characterisation

Project 6:        DNA binding activities of human replication protein A

Supervisor:     Dr. Heinz-Peter Nasheuer, Department of Biochemistry

Project Summary

Replication protein A (RPA) is the main single-stranded DNA binding protein in eukaryotic cells and is an essential enzyme complex for eukaryotic DNA replication. RPA forms a three subunit protein complex consisting of p70, p32 and p14 (Broderick, S., K. Rehmet, C. Concannon and H. P. Nasheuer (2009). "Eukaryotic single-stranded DNA binding proteins: central factors in genome stability." Subcell Biochem 50: 143-63). The two smallest subunits of RPA, p32 and p14 form a stable heterodimeric complex (p32/p14) that contains weak ssDNA binding activity, whereas the large subunit p70 contains the high affinity DNA binding sites. To fulfil its functions, RPA performs multiple physical interactions with DNA and proteins.

To-date, the research group has expressed human RPA and its subcomplexes as soluble proteins in bacteria and in insect cells. Recently, we have determined that a C-terminal mutant of p32 modulates the RPA bind to single-stranded FX174-DNA (FX174-ssDNA). In the proposed project, we will study the DNA binding of RPA with biochemical and molecular biological methods. The RPA heterotrimer will be expressed with a 6xHis tag in E. coli, and purified using metal chelate affinity chromatography. In addition, mutants of RPA will be prepared to determine the DNA binding. After purification of these proteins, the binding of wild-type RPA and the mutant protein complex will be compared by analysing their interaction with FX174-ssDNA using gel mobility shift assays. To this end the protein-DNA complexes will be form in vitro and then separated by native polyacrylamide gel electrophoresis. Next these mobility shifts will be quantified and compared. These findings will allow us to understand the functions of specific regions and subunits of RPA in DNA replication in more detail.

During their project the students will produce mutants of RPA, express them in E. coli and purify them according to methods established in the research group. Then the student will analyze and compare the binding of the purified proteins with DNA. In addition to their contribution to the research of group, the student will learn various modern molecular biological and biochemical techniques and will learn to develop ideas and strategies on all levels of laboratory work.

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