Deciphering the Role of BCCIP in Homologous Recombination, Genomic Stability, and Cancer

Matt D. McAlister, Garrett W. Buzzard, and Michael G. Sehorn

Introduction

DNA’s essential role as a repository for genetic information can be compromised through a variety of disturbances that damage its structure. These damaged segments, called lesions, are not anomalous events — most cells will face tens of thousands of lesions every day.
Single-strand breaks are often readily repaired using the undamaged complementary strand as a template, but double-strand breaks (DSBs) can be catastrophic for a cell.
To address DSBs, cells deploy a form of repair called homologous recombination (HR). This high-fidelity process locates and utilizes a sister chromatid as a repair template, allowing the cell to maintain genomic stability.
The RAD51 recombinase forms a helical nucleoprotein filament on single-stranded DNA (ssDNA) exposed by a DSB. This filament plays a central role in HR: it allows a damaged strand of DNA to repair itself by invading an intact homologous counterpart and searching for a suitable repair template.
A mediator protein, BCCIPβ (BRCA2 and CDKN1A Interacting Protein), promotes RAD51’s active state by allowing it to bind and retain ATP more efficiently. PCNA (Proliferating Cell Nuclear Antigen) acts as a clamp during DNA replication, greatly improving the efficiency and duration of a given replicative event. It also acts as a loading platform for various proteins involved in HR.

Objectives

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.

Glutathione S-transferase (GST) Pull-Down Assay

Created with BioRender.com

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

The B-isoform of BCCIP promotes ADP release from the RAD51 presynaptic filament and enhances homologous DNA pairing

Kelso, et al. (2016). The β-isoform of BCCIP promotes ADP release from the RAD51 presynaptic filament and enhances homologous DNA pairing.

Failure to repair DNA damage can result in cell cycle arrest, cell death, and tumor formation. Accumulation of DNA damage may also underlie age-related functional decline, as more robust DNA repair pathways have been correlated with longer lifespans in mammals³.

Methods

Create a BCCIP Truncation Series
PCR Mutagenesis
Confirm Expression of the Variant BCCIP Proteins
Coomassie Stain
Western Blot
Grow, Isolate, and Purify BCCIP Truncation Series
High Speed Centrifuge
Column Chromatography: Nickel, Glutathione
Demonstrate Physical Interaction
Pull-Down Assay: Nickel, Glutathione S-Transferase

Progress

Test BCCIP for Physical Interaction with PCNA

Create a Series of Truncated Variants of BCCIP

Confirm Expression of the Variant BCCIP Proteins

Grow, Isolate, and Purify BCCIP Truncation Series

Test BCCIP Variants for Physical Interaction with PCNA

COVID-19 disrupted the schedule for validating and growing the BCCIP (α, β, and Core) truncation series. Instead, this summer’s work focused on pull-down assays demonstrating physical interaction between the BCCIP isoforms and PCNA.

Results

BCCIPβ and PCNA (Coomassie)

Physical Interaction Between BCCIPβ and PCNA (Qualitative)

Work performed by Hilda Chan.

BCCIPβ and PCNA (Western)

BCCIP (α and β) and PCNA

BCCIP (α, β, and Core) and PCNA #1

BCCIP (α, β, and Core) and PCNA #2

36 kDa

Molecular Weight of BCCIPβ

322 Amino Acids

Size of Expressed BCCIPβ Protein

942 Base Pairs

Length of BCCIPβ

Conclusions

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.

References

Droz-Rosario, Roberto, Huimei Lu, Jingmei Liu, Ning-Ang Liu, Shridar Ganesan, Bing Xia, Bruce G. Haffty, and Zhiyuan Shen. 2017. “Roles of BCCIP Deficiency in Mammary Tumorigenesis.” Breast Cancer Research: BCR 19 (1): 115.
Kelso, Andrew A., Steven D. Goodson, Leah E. Watts, Leanna L. Ledford, Sarah M. Waldvogel, J. Nathaniel Diehl, Shivani B. Shah, Amanda F. Say, Julie D. White, and Michael G. Sehorn. 2017. “The β-Isoform of BCCIP Promotes ADP Release from the RAD51 Presynaptic Filament and Enhances Homologous DNA Pairing.” Nucleic Acids Research 45 (2): 711–25.
Tian, Xiao, Denis Firsanov, Zhihui Zhang, Yang Cheng, Lingfeng Luo, Gregory Tombline, Ruiyue Tan, et al. 2019. “SIRT6 Is Responsible for More Efficient DNA Double-Strand Break Repair in Long-Lived Species.” Cell 177 (3): 622–38.e22.