Tarmo Nuutinen

University of Eastern Finland, Joensuu Campus
Supervisor: Juhani Syvšoja
Funding: other
Date: 11/08/2003

Structural and functional studies on p12 and p17 - two small subunits of dna polymerase epsilon (pol-epsilon)

p12 and p17 form dimer which associates with dimer of two largest subunits of pol-epsilon, p261 and p59 to form functionally active enzyme. Exact roles of two replicative DNA polymerases, pol-delta and pol-epsilon, in the bulk replication of DNA remains partially unclear. Interestingly, p17 with p15 have been found also from huCHRAC-complex, which is known of its chromatin remodelling activity. p12 and p17 contains histone fold motifs similar to those in CCAAT-binding factors subunits C and A, respectively. Presumably, motifs enable interactions between subunits and with other proteins with formed hybrid surface of dimer, interaction with DNA could be also possible, but not proofed.

Goals of my studies with these protein are to find biological roles of p12 and p17 and to solve structures of p12, p17 and possibly structure of the dimer using X-ray crystallography. I will start with purification of p12 and p17 expressed in E. coli followed by crystallization. I will also utilize antibodies produced as my M.Sc. work using immunofluorescence microscopy and immunoelectron microscopy to determinate their counterparts and localization in cell. To determinate counterparts and the sites of interactions two-hybrid-assay with different constructs will be used and results could be fit to the results of X-ray crystallography.