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Purification and Characterization of Proteins Important for Homologous Recombination in Eukaryotes

Thesis Info

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Author

Rana Salman Anjum

Program

PhD

Institute

University of Agriculture

City

Faisalabad

Province

Punjab

Country

Pakistan

Thesis Completing Year

2013

Thesis Completion Status

Completed

Subject

Natural Sciences

Language

English

Link

http://prr.hec.gov.pk/jspui/handle/123456789/498

Added

2021-02-17 19:49:13

Modified

2024-03-24 20:25:49

ARI ID

1676727048403

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Successful repair of DNA double strand breaks is crucial; failure of which can lead to tumourigenesis including breast/ovarian cancer. BRCA2 (breast cancer susceptibility protein type 2) and RAD51 from different specie interact in a process called homologous recombination (HR) during the repair of such kind of insults to DNA. BRCA2 being a large protein molecule has different sites of interaction for other proteins like DMC1 (disruption of meiotic control 1). In the present study, we have purified a distinct region in human BRCA2 close to DMC1 binding site which has been shown to undergo CDK (cyclin dependent kinase) based phosphorylation in our kinase assays. Characterization involving dynamic light scattering, limited proteolysis and circular dichroism spectroscopy techniques revealed this region to be mostly disordered. We have also attempted the successful purification of DMC1 (different from the previous method of its purification) employing C-terminus of DMC1 as binding partner for increased solubility. Further studies included the characterization of Trypanosoma brucei BRC repeats (TbBRC) and Rad51 interaction. We observed no interaction of Trypanosoma brucei Rad51 (TbRad51) with its BRC repeats upto 7 BRC repeats based on our in vitro pull down assays. The comparison of human RAD51 and TbRad51 structures highlighted critical differences of residues at hydrophobic cavities for the binding of phenylalanine from BRC repeat. We predicted that TbBRC repeats, collectively, might have WD-40 β-propeller structure. The purification of Trypanosoma brucei 7 BRC repeats rendered this protein highly soluble and stable based on circular dichroism spectroscopy which also revealed the protein having poly-proline structure, a characteristic of unordered proteins. Moreover, we have predicted multiple phosphorylation sites in TbRad51 and compared them with other eukaryotic Rad51 orthologues to get insights into a possible regulatory mechanism for Rad51.
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