Biochemical Characterization of RADX in Homologous Recombination and Replication Fork Repair

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

Introduction

  • Homologous recombination (HR) maintains genomic stability through the high fidelity, template-driven repair of DNA double-strand breaks (DSBs).
  • RAD51 forms a helical nucleoprotein filament on exposed single-stranded DNA (ssDNA). This filament plays a central role in HR: it allows a damaged strand of DNA to repair itself by invading an intact homologous counterpart. The role of RAD51 is not limited to HR, however. It also has a less-recognized role in promoting the repair of stalled replication forks.
  • RADX is a ssDNA-binding protein that antagonizes RAD51 and disrupts the formation of its helical filament. Ostensibly, RADX should regulate both HR and replication fork repair.
  • However, current in vivo results only demonstrate a regulatory role for RADX at replication forks. The underlying, biochemical basis of this specificity is unknown.

Objectives

Upregulation and downregulation of RADX disrupt RAD51 nucleoprotein filament at replication forks, but have shown no effect on homology-driven repair of double strand breaks (Dungrawala et al. 2017).

When binding single-stranded DNA (ssDNA), RADX outcompetes RAD51 at thousand-fold lower concentrations (Bhat et al. 2018). To test whether this strong ssDNA binding is responsible for RADX’s disruption of the RAD51 nucleoprotein filament, we have created a DNA binding-deficient variant of RADX.

The tests and assays necessary to uncover the underlying mechanisms of RADX require a working stock of purified protein. This stock includes both the wild type (WT) and the DNA-binding deficient mutant (OBM). The variant is designated OBM because the site of the mutation was at an Oligonucleotide / Oligosaccharide-Binding (OB) fold, making it an OB-Mutant (OBM).

Preliminary research has not detected an interaction between RADX and RAD51 (Dungrawala et al. 2017). Given the novelty of this protein and the infancy of the research around it, we think it’s wise to further investigate this question. Clarifying this point will narrow down which research we will pursue in the future. If, for example, we find no evidence of physical interaction then we may begin investigating binding-partners that could mediate the observed effects of RADX.

We currently have two running hypotheses: 1) RADX is present at both replication forks and at double-strand breaks, but we only observe effects at replication forks because a protein or other regulatory molecule plays a protective role; 2) RADX is specifically recruited to stalled replication forks by an characterized binding-partner or a preference for specific DNA substrates. Both of these possibilities have different empirical consequences that we can uncover through experiment.

Methods

  • Create a DNA-Binding Deficient Variant of RADX (OBM)

    PCR Mutagenesis

  • Confirm Expression of the Variant RADX Protein

    Coomassie Stain
    Western Blot

  • Isolate and Purify RADX

    High Speed Centrifuge
    Nickel Column
    Glutathione Column
    Ion Exchange Columns
    Size Exclusion Column

Progress

Create a DNA-Binding Deficient Variant of RADX (OBM)
Confirm Expression of the Variant RADX Protein
Isolate and Purify RADX
Investigate RADX for Physical Interaction with RAD51
Identify Optimal DNA-Binding Substrate for RADX
Test RAD51 Binding Partners for RADX Protection
Test RADX Interaction Partners for Replication Fork Recruitment

Future work depends on a large, working stock of RADX.

Results

97 kDa

Molecular Weight of RADX

855 Amino Acids

Size of Expressed RADX Protein

2,565 Base Pairs

Length of RADX Gene

4 Papers

Published on RADX

Conclusions

RADX is a novel single-strand binding protein that’s enriched at replication forks. As a RAD51 antagonist, RADX is capable of disrupting homology-driven repair (HDR). However, in functional cells, it does not. This suggests at least two possibilities: 1) RADX is enriched only at replication forks, or 2) an unknown regulatory mechanism protects RAD51 from RADX at the site of DSBs but not replication forks. To this end, we have created a DNA-binding deficient mutant of RADX, which we successfully cultivated and purified. We’ll follow this procedure to purify a stock of WT RADX, and then proceed to test 1) physical interaction, 2) optimal DNA binding substrate, and 3) RAD51 binding partners that could play a protective role against the antagonism of RADX.

References

  • Bhat, Kamakoti P., Archana Krishnamoorthy, Huzefa Dungrawala, Edwige B. Garcin, Mauro Modesti, and David Cortez. 2018. “RADX Modulates RAD51 Activity to Control Replication Fork Protection.” Cell Reports 24 (3): 538–45.
  • Dungrawala, Huzefa, Kamakoti P. Bhat, Rémy Le Meur, Walter J. Chazin, Xia Ding, Shyam K. Sharan, Sarah R. Wessel, Aditya A. Sathe, Runxiang Zhao, and David Cortez. 2017. “RADX Promotes Genome Stability and Modulates Chemosensitivity by Regulating RAD51 at Replication Forks.” Molecular Cell 67 (3): 374–86.e5.