Abstract:
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.
