Genomic integrity is critical to maintain the normal function of organisms. Pathological DNA double strand breaks (DSBs) has been regarded as the most toxic type of DNA lesions that affects the stability of the genome and cell fate [1]. To maintain genomic stability following DSBs, cells require efficient and proper DNA repair processes. There are two indispensable pathways responsible for DSB repair in eukaryotic cells: non-homologous end-joining (NHEJ) and homologous recombination (HR) [2]. NHEJ does not require a homologous template and can potentially re-ligate any type of damaged DNA ends throughout the cell cycle, and therefore it is the most essential DSB repair pathway [3].

The initial step of NHEJ is the rapid binding of Ku70 and Ku80 heterodimer to the DSB ends. Ku binds double-stranded DNA (dsDNA) ends with high affinity and relocates to DNA lesions within 1 second, acting as a DSB sensor [4], [5]. Then the Ku70 and Ku80 heterodimer binds to DNA-dependent protein kinase catalytic subunit (DNA-PKcs) to form the so called DNA-PK complex, which recruits downstream NHEJ core factors, including X-ray repair cross-complementing protein 4 (XRCC4), XRCC4-like factor (XLF), DNA ligase 4 (LIG4), Artemis nuclease, DNA polymerase λ (Pol λ) and μ (Pol μ), to the dsDNA end to facilitate DNA end processing and ligation [6]. In addition to these NHEJ factors, several new proteins have recently been discovered to play essential roles in NHEJ, such as Paralog of XRCC4 and XLF (PAXX), Modulator of retroviral infection (MRI), Intermediate filament family orphan (IFFO1), ERCC excision repair 6 like 2 (ERCC6L2), transactivation response DNA-binding protein of 43 kDa (TDP-43) and Wiskott-Aldrich Syndrome Protein and SCAR Homolog (WASH) [7], [8], [9], [10], [11], [12], [13].

As a key member of Wiskott-Aldrich Syndrome Protein (WASP) protein family, WASH facilitates the generation of branched filamentous actin (F-actin) by activation of the actin-related protein 2/3 (Arp2/3) complex through its conserved C-terminal VCA (verprolin, central, acid) domain [14]. WASH, also named WASH complex subunit 1 (WASHC1), was originally reported to exist in a FAM21 containing multiprotein complex that regulates retromer-dependent endosomal sorting and trafficking in a manner dependent F-actin generation by the Arp2/3 complex [15], [16]. FAM21, known as WASH complex subunit 2 (WASHC2), directs the WASH complex to endosomes through a direct interaction between FAM21 C-terminal fragment aa 357-1341 containing multiple L-F-[D/E]3-10-L-F motifs and the retromer protein VPS35 [17]. In addition to the critical function of cytoplasmic WASH on endosomal trafficking, there is a large body of evidence indicating that nuclear WASH plays multi-faceted roles in the regulation of gene transcription, maintenance of global nuclear architecture and DNA damage repair [13], [18], [19], [20], [21]. Of note, we have recently reported that nuclear WASH localizes to DNA damage sites by interacting with Ku and loss of WASH leads to a decrease in the efficiency of the NHEJ and an impairment in DSB repair [13]. Intriguingly, nuclear FAM21 function might be independent of WASH. Our previous study indicates that nuclear FAM21, but not WASH, participates in NF-κB-dependent gene transcription [22]. Thus, whether FAM21 mediates WASH localization to DSB sites and is involved in WASH-dependent DSB repair requires further investigation.

Here, we show that FAM21 localizes to DNA damage sites by interacting with Ku and the interaction between FAM21 C-terminal region and Ku is requisite for the recruitment of FAM21. Furthermore, FAM21 promotes the recruitment of WASH to DNA damage sites. Taken together, these observations reveal that nuclear FAM21 mediates WASH localization and WASH-dependent DNA repair.