Project 7: DNA Binding Activities of Human Primase
Supervisor: Dr. Heinz-Peter Nasheuer, Department of Biochemistry
Project Summary: DNA polymerase alpha-primase (pol-prim) is an essential enzyme complex in eukaryotic DNA replication and forms a four subunit protein complex
1
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The two smallest subunits of pol-prim, p58 and p48, form a stable heterodimeric complex (p58/p48) that contains primase activity, which synthesizes short oligoribonucleotides during DNA replication, which is essential for the process, since no DNA polymerase can start DNA synthesis de novo . To fulfil its functions, primase performs multiple physical interactions with DNA and proteins.
To-date, the research group has expressed human pol-prim and its subcomplexes as soluble proteins in bacteria and in insect cells. Recently, we have determined that polprim and the p58/p48 complex bind to single-stranded
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X174-DNA (
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X174-ssDNA) with high affinity. In the proposed project, we will study the DNA binding of primase with biochemical and molecular biological methods. The primase heterodimer and both subunits will be expressed with a 6xHis tag in E. coli , and purified using metal chelate affinity chromatography. In addition, N- and C-terminal deletion mutants of the primase subunits will be prepared to determine the DNA binding regions of primase. Then these mutants will be expressed in E. coli and purified. After purification of the proteins, the binding of primase, its subunits and the mutant proteins will be compared by analysing their interaction with
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X174-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 primase in DNA replication in more detail.
During their project the student will produce mutants of primase, express them in E. coli and purify them according to methods established in the research group. Then the students will analyze and compare the binding of the purified proteins with DNA. In addition to their contribution to the research of group, the students will learn various modern molecular biological and biochemical techniques and will learn to develop ideas and strategies on all levels of laboratory work.
References
1. Nasheuer et al. (2006) in Genome Integrity: Facets and Perspectives, D. H.
Lankenau (ed.); Springer, Berlin) p. 27 – 68;