RAD54L regulates replication fork progression and nascent strand degradation in BRCA1/2-deficient cells.

RAD54L is a DNA motor protein with critical roles in homologous recombination DNA repair (HR). In vitro, RAD54L was also shown to catalyze the reversal and restoration of model replication forks. Little, however, is known about the role of RAD54L in regulating the dynamics of DNA replication in cells. Here, we show that RAD54L functions as a fork remodeler and restrains the progression of replication forks in human cells. Analogous to HLTF and FBH1, and consistent with a role in fork reversal, RAD54L catalyzes the slowing of fork progression in response to replication stress. In BRCA1/2-deficient cells, RAD54L activity leads to nascent strand DNA degradation, and loss of RAD54L reduces DNA double-strand break formation. Using a separation-of-function mutation, we show that RAD54L-mediated fork restraint depends on its ability to catalyze branch migration. Our results reveal a new role for RAD54L in regulating the dynamics of replication forks in cells and highlight the impact of RAD54L function on the treatment of patients with BRCA1/2-deficient tumors.

A multiplexed, paired-pooled droplet digital PCR assay for detection of SARS-CoV-2 in saliva.

In response to the SARS-CoV-2 pandemic, we developed a multiplexed, paired-pool droplet digital PCR (MP4) screening assay. Key features of our assay are the use of minimally processed saliva, 8-sample paired pools, and reverse-transcription droplet digital PCR (RT-ddPCR) targeting the SARS-CoV-2 nucleocapsid gene. The limit of detection was determined to be 2 and 12 copies per µl for individual and pooled samples, respectively. Using the MP4 assay, we routinely processed over 1,000 samples a day with a 24-h turnaround time and over the course of 17 months, screened over 250,000 saliva samples. Modeling studies showed that the efficiency of 8-sample pools was reduced with increased viral prevalence and that this could be mitigated by using 4-sample pools. We also present a strategy for, and modeling data supporting, the creation of a third paired pool as an additional strategy to employ under high viral prevalence.

DNA binding and RAD51 engagement by the BRCA2 C-terminus orchestrate DNA repair and replication fork preservation.

The tumor suppressor BRCA2 participates in DNA double-strand break repair by RAD51-dependent homologous recombination and protects stressed DNA replication forks from nucleolytic attack. We demonstrate that the C-terminal Recombinase Binding (CTRB) region of BRCA2, encoded by gene exon 27, harbors a DNA binding activity. CTRB alone stimulates the DNA strand exchange activity of RAD51 and permits the utilization of RPA-coated ssDNA by RAD51 for strand exchange. Moreover, CTRB functionally synergizes with the Oligonucleotide Binding fold containing DNA binding domain and BRC4 repeat of BRCA2 in RPA-RAD51 exchange on ssDNA. Importantly, we show that the DNA binding and RAD51 interaction attributes of the CTRB are crucial for homologous recombination and protection of replication forks against MRE11-mediated attrition. Our findings shed light on the role of the CTRB region in genome repair, reveal remarkable functional plasticity of BRCA2, and help explain why deletion of Brca2 exon 27 impacts upon embryonic lethality.

RAD51AP1 and RAD54L Can Underpin Two Distinct RAD51-Dependent Routes of DNA Damage Repair via Homologous Recombination.

Homologous recombination DNA repair (HR) is a complex DNA damage repair pathway and an attractive target of inhibition in anti-cancer therapy. To help guide the development of efficient HR inhibitors, it is critical to identify compensatory HR sub-pathways. In this study, we describe a novel synthetic interaction between RAD51AP1 and RAD54L, two structurally unrelated proteins that function downstream of the RAD51 recombinase in HR. We show that concomitant deletion of RAD51AP1 and RAD54L further sensitizes human cancer cell lines to treatment with olaparib, a Poly (adenosine 5'-diphosphate-ribose) polymerase inhibitor, to the DNA inter-strand crosslinking agent mitomycin C, and to hydroxyurea, which induces DNA replication stress. We also show that the RAD54L paralog RAD54B compensates for RAD54L deficiency, although, surprisingly, less extensively than RAD51AP1. These results, for the first time, delineate RAD51AP1- and RAD54L-dependent sub-pathways and will guide the development of inhibitors that target HR stimulators of strand invasion.

RAD51AP1 mediates RAD51 activity through nucleosome interaction.

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.

NUCKS1 promotes RAD54 activity in homologous recombination DNA repair.

NUCKS1 (nuclear ubiquitous casein kinase and cyclin-dependent kinase substrate 1) is a chromatin-associated, vertebrate-specific, and multifunctional protein with a role in DNA damage signaling and repair. Previously, we have shown that NUCKS1 helps maintain homologous recombination (HR) DNA repair in human cells and functions as a tumor suppressor in mice. However, the mechanisms by which NUCKS1 positively impacts these processes had remained unclear. Here, we show that NUCKS1 physically and functionally interacts with the DNA motor protein RAD54. Upon exposure of human cells to DNA-damaging agents, NUCKS1 controls the resolution of RAD54 foci. In unperturbed cells, NUCKS1 prevents RAD54's inappropriate engagement with RAD51AP1. In vitro, NUCKS1 stimulates the ATPase activity of RAD54 and the RAD51-RAD54-mediated strand invasion step during displacement loop formation. Taken together, our data demonstrate that the NUCKS1 protein is an important new regulator of the spatiotemporal events in HR.

Antitumor Reactive T-Cell Responses Are Enhanced In Vivo by DAMP Prothymosin Alpha and Its C-Terminal Decapeptide.

Prothymosin α (proTα) and its C-terminal decapeptide proTα(100-109) were shown to pleiotropically enhance innate and adaptive immune responses. Their activities have been broadly studied in vitro, focusing primarily on the restoration of the deficient immunoreactivity of cancer patients' leukocytes. Previously, we showed that proTα and proTα(100-109) act as danger-associated molecular patterns (DAMPs), ligate Toll-like receptor-4, signal through TRIF- and MyD88-dependent pathways, promote the maturation of dendritic cells and elicit T-helper type 1 (Th1) immune responses in vitro, leading to the optimal priming of tumor antigen-reactive T-cell functions. Herein, we assessed their activity in a preclinical melanoma model. Immunocompetent mice bearing B16.F1 tumors were treated with two cycles of proTα or proTα(100-109) together with a B16.F1-derived peptide vaccine. Coadministration of proTα or proTα(100-109) and the peptide vaccine suppressed melanoma-cell proliferation, as evidenced by reduced tumor-growth rates. Higher melanoma infiltration by CD3+ T cells was observed, whereas ex vivo analysis of mouse total spleen cells verified the in vivo induction of melanoma-reactive cytotoxic responses. Additionally, increased levels of proinflammatory and Th1-type cytokines were detected in mouse serum. We propose that, in the presence of tumor antigens, DAMPs proTα and proTα(100-109) induce Th1-biased immune responses in vivo. Their adjuvant ability to orchestrate antitumor immunoreactivities can eventually be exploited therapeutically in humans.