RAD51AP1 mediates RAD51 activity through nucleosome interaction

Elena Pires,Neelam Sharma,Platon Selemenakis,Bo Wu,Yuxin Huang,Dauren S Alimbetov,Weixing Zhao,Claudia Wiese

doi:10.1016/j.jbc.2021.100844

Abstract

RAD51-associated protein 1 (RAD51AP1) is a key protein in the homologous recombination (HR) DNA repair pathway. Loss of RAD51AP1 leads to defective HR, genome instability, and telomere erosion. RAD51AP1 physically interacts with the RAD51 recombinase and promotes RAD51-mediated capture of donor DNA, synaptic complex assembly, and displacement-loop formation when tested with nucleosome-free DNA substrates. In cells, however, DNA is packaged into chromatin, posing an additional barrier to the complexities of the HR reaction. In this study, we show that RAD51AP1 binds to nucleosome core particles (NCPs), the minimum basic unit of chromatin in which approximately two superhelical turns of 147 bp double-stranded DNA are wrapped around one histone octamer with no free DNA ends remaining. We identified a C-terminal region in RAD51AP1, including its previously mapped DNA-binding domain, as critical for mediating the association between RAD51AP1 and both the NCP and the histone octamer. Using in vitro surrogate assays of HR activity, we show that RAD51AP1 is capable of promoting duplex DNA capture and initiating joint-molecule formation with the NCP and chromatinized template DNA, respectively. Together, our results suggest that RAD51AP1 directly assists in the RAD51-mediated search for donor DNA in chromatin. We present a model, in which RAD51AP1 anchors the DNA template through affinity for its nucleosomes to the RAD51-ssDNA nucleoprotein filament.

Highlights

RAD51AP1 is an intrinsically unfolded protein [2] and is likely to undergo induced folding upon binding to specific partners, which will make it well-ordered [3]
The nucleosome core particle (NCP) is the minimum basic unit of chromatin in which approximately two superhelical turns of 147 bp double-strandedDNA are wrapped around one histone octamer with no free DNA ends remaining [12, 13]
RAD51AP1 interacts with nucleosomes deposition using the 601 Widom 147 bp dsDNA fragment and human histone octamers, as previously described [12, 14], and used electrophoretic mobility shift assays (EMSAs) to assess the RAD51AP1-NCP association

Summary

Introduction

RAD51AP1 is an intrinsically unfolded protein [2] and is likely to undergo induced folding upon binding to specific partners, which will make it well-ordered [3]. Intrigued by our earlier findings that showed affinity of purified human RAD51AP1 to chromatinized DNA in the immobilized template assay [11], we decided to test complex formation between RAD51AP1 and the NCP. RAD51AP1 interacts with nucleosomes deposition using the 601 Widom 147 bp dsDNA fragment and human histone octamers, as previously described [12, 14], and used electrophoretic mobility shift assays (EMSAs) to assess the RAD51AP1-NCP association (for schematic of the assay see Fig. 1A).

Results

Conclusion