Matt D. McAlister, Garrett W. Buzzard, and Michael G. Sehorn
Create a BCCIP truncation series through PCR Mutagenesis
The Sehorn lab has identified several candidate regions of BCCIP that mediate its interaction with PCNA. In addition to these mutated variants, we are creating a series of truncations that will further allow us to identify the interaction domains within BCCIP.
Grow a working stock of purified BCCIP variant proteins
The tests and assays necessary to uncover the underlying mechanisms of BCCIP require a working stock of purified protein. This stock includes both the wild type (WT) for all isoforms, the PIP box mutants, and the truncation series.
Test BCCIP truncation variants for physical interaction with PCNA
Prior research in the Sehorn lab has indicated physical interaction between BCCIPβ (but not BCCIPα or BCCIP Core) and GST-PCNA. After optimizing the conditions for a pull-down assay with all four of these proteins, we can narrow in on the region(s) of BCCIP responsible for the interaction by performing pull-down assays with the PIP box mutants and the truncation series.
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Identify BCCIP and PCNA binding domains
Currently, BCCIPα and BCCIPβ indicate moderate physical interaction with PCNA, while BCCIP Core does not.
Propose a mechanism for BCCIP’s observed effects
Kelso, et al. (2016). The β-isoform of BCCIP promotes ADP release from the RAD51 presynaptic filament and enhances homologous DNA pairing.
High Speed Centrifuge
Column Chromatography: Nickel, Glutathione
Pull-Down Assay: Nickel, Glutathione S-Transferase
Work performed by Hilda Chan.
Molecular Weight of BCCIPβ
Size of Expressed BCCIPβ Protein
Length of BCCIPβ
This study aims to identify the binding domains and propose a mechanism for BCCIPβ’s effect on RAD51. To achieve this, multiple isoforms of BCCIP were tested for physical interaction with specific proteins in the HR repair pathway and then a series of BCCIPβ variants were generated through mutation and truncation. For each variant, the differential effects on RAD51 and HR should provide insight into the mechanistic unpinning of BCCIPβ’s observed effects.