# Phosphoregulation of RAD51AP1 function in homology-directed repair

## TL;DR

This study shows that the DNA repair protein RAD51AP1 is regulated by phosphorylation, which affects its function in DNA repair and cancer.

## Contribution

The study reveals that phosphorylation of RAD51AP1 at specific sites regulates its activity in homology-directed DNA repair.

## Key findings

- Phosphorylation at S277/282 affects RAD51AP1's ability to stimulate D-loop formation and bind DNA.
- RAD51AP1-S277/282D fully rescues RAD51AP1 deficiency in DNA replication assays.
- RAD51AP1-S277 is a target of CDK2, suggesting a regulatory role in HDR.

## Abstract

Homology-directed DNA repair (HDR) is critical for genome stability and tumor suppression. HDR is initiated by the RAD51 single-stranded (ss)DNA nucleoprotein filament, which conducts the homology search and invades a homologous DNA template, creating a displacement-loop (D-loop). The RAD51 filament is assisted in these processes by several proteins. One such protein is RAD51-associated protein 1 (RAD51AP1), which binds DNA and RNA and directly interacts with RAD51. Of note, RAD51AP1 overexpression is associated with poor prognosis in several different cancer types. Here, we show that RAD51AP1 activity is regulated by phosphorylation. RAD51AP1 bearing S277/282A mutations is more proficient in the stimulation of D-loop formation than wild-type RAD51AP1 or phosphomimetic RAD51AP1-S277/282D. In EMSAs, RAD51AP1 with S277/282A mutations more avidly binds ssDNA, double-stranded (ds)DNA, and the nucleosome core particle than wild-type RAD51AP1 or RAD51AP1-S277/282D. In cells, RAD51AP1-S277/282A confers no rescue of RAD51AP1 deficiency in toxicity tests and DNA replication assays. In contrast, RAD51AP1-S277/282D fully rescues RAD51AP1 deficiency. We provide evidence that RAD51AP1-S277 is a CDK2 target and propose a model in which RAD51AP1-S277/282 phosphorylation ensures RAD51AP1 flexibility for dynamic engagement in consecutive steps of the HDR reaction. Our results provide new mechanistic insights into RAD51AP1 regulation by a CDK.