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Biology subjects

Sung, P.

Publications and source records attributed to Sung, P..

11 recordsLinked to original sources
bioRxiv2026

BRCA1 promotes homologous recombination through separable DNA and RAD51 binding activities within its disordered region.

The breast and ovarian tumor suppressor BRCA1 heterodimerizes with BARD1 to promote DNA double-strand break repair by homologous recombination (HR) and to protect stressed DNA replication forks against nuclease attack. The large, intrinsically disordered central region of BRCA1 harbors binding sites for DNA and multiple repair factors, but its lack of stable structure has hindered mechanistic dissection of these activities. Using biochemical mapping and NMR spectroscopy, we delineate the DNA binding and RAD51 interaction interfaces within this region and construct separation-of-function mutants that selectively ablate each activity. Both DNA binding and RAD51 interaction are required for BRCA1-BARD1 to promote RAD51-mediated DNA strand invasion, and DNA binding also contributes to BLM-DNA2 end resection. These findings provide mechanistic insights into how individual ligand binding activities within BRCA1 contribute to genome maintenance.

biochemistry↗
bioRxiv2026

High-throughput mapping of 6,888 RAD51D variants identifies distinct biochemical functions needed for homologous recombination and olaparib response

The tumor suppressor RAD51D is essential for homologous recombination (HR). Pathogenic variants in RAD51D are associated with breast and ovarian cancers. However, most clinical missense variants are of unknown significance. We performed a multiplex assay of variant effect to test 6,888 RAD51D coding variants for loss-of-function. The resulting variant-to-function map perfectly separates known pathogenic and benign variants and is validated by orthogonal HR and biochemical assays across 70 clinical variants. Our screen shows that variants in the DNA-binding or ATPase core most severely compromise HR, and we identify the RAD51D-RAD51C interface within the BCDX2 complex as essential for regulating its ATPase activity. We hypothesize that, paradoxically, the primary function of RAD51D is to slow the ATPase activity of BCDX2, thereby allowing sufficient time and space for RAD51 filament assembly. Together, we identify hotspots of deleterious RAD51D variants and uncover the mechanisms by which variants compromise its biochemical functions. HighlightsO_LIUsed a multiplexed assay of functional effect (MAVE) to assess the functionality via olaparib sensitivity of 6,888 RAD51D coding variants, which can be used for variant classification C_LIO_LIProvided cellular functional analysis for 70 clinically-identified breast and ovarian cancer RAD51D variants C_LIO_LIIdentified key regions and enzymatic activities of RAD51D critical for its function in the BCDX2 and the X3CDX2 complexes C_LIO_LIDetermined mechanism of RAD51D-mediated regulation of BCDX2 ATPase activity C_LI

cancer biology↗
bioRxiv2025

Phosphoregulation of RAD51AP1 function in homology-directed repair

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.

cell biology↗
bioRxiv2022

TLK1-mediated RAD54 phosphorylation spatio-temporally regulates Homologous Recombination Repair

Environmental agents like ionizing radiation (IR) and chemotherapeutic drugs can cause severe damage to the DNA, often in the form of double-strand breaks (DSBs). Remaining unrepaired, DSBs can lead to chromosomal rearrangements, and cell death. One major error-free pathway to repair DSBs is homologous recombination repair (HRR). Tousled-like kinase 1 (TLK1), a Ser/Thr kinase that regulates the DNA damage checkpoint, has been found to interact with RAD54, a central DNA translocase in HRR. To determine how TLK1 regulates RAD54, we inhibited or depleted TLK1 and tested how this impacts HRR in human cells using a ISce-I-GR-DsRed fused reporter endonuclease. Our results show that TLK1 phosphorylates RAD54 at three threonines (T41, T59, and T700), two of which are located within its N-terminal domain (NTD) and one is located within its C-terminal domain (CTD). Phosphorylation at both T41 and T59 supports HRR and protects cells from DNA DSB damage. In contrast, phosphorylation of T700 leads to impaired HRR and engenders no protection to cells from cytotoxicity and rather results in repair delay. Further, our work enlightens the effect of RAD54-T700 (RAD54-CTD) phosphorylation by TLK1 in mammalian system and reveals a new site of interaction with RAD51.

molecular biology↗
bioRxiv2021

RAD51AP1 compensates for the loss of RAD54 in homology-directed DNA repair

Homologous recombination (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 sub-pathways. In this study, we describe a novel synthetic interaction between RAD51AP1 and RAD54, two structurally unrelated proteins that function downstream of the RAD51 recombinase in HR. We show that deletion of both RAD51AP1 and RAD54 synergistically sensitizes human cancer cell lines to treatment with a Poly(adenosine 5 s-diphosphate-ribose) polymerase inhibitor, to the DNA inter-strand crosslinking agent mitomycin C, and to hydroxyurea, which stalls the progression of DNA replication forks. We infer that HR-directed anti-cancer treatment modalities shall consider this intra-pathway functional overlap, and we hypothesize that in cancerous cells the simultaneous inactivation of both RAD54 and RAD51AP1 will accentuate tumor kill.

cell biology↗
bioRxiv2021

FMRP bridges R-loops and DHX9 through direct interactions

Mutations in, or deficiency of, FMRP is responsible for the Fragile X syndrome (FXS), the most common cause for inherited intellectual disability. FMRP is a nucleocytoplasmic protein, primarily characterized as a translation repressor with poorly understood nuclear function(s). We recently uncovered a genome protective role of FMRP. We reported that FXS patient-derived cells lacking FMRP sustain higher level of DNA double-strand breaks than normal cells, a phenotype further exacerbated by DNA replication stress. The stress-induced DSBs occur at sequences prone to form R-loops, which are co-transcriptional RNA:DNA hybrids that have been associated with genome instability. Concordantly, we showed that FXS cells accumulate R-loops under replication stress. Moreover, expression of FMRP and not a mutant deficient in binding nucleic acids and known to cause FXS, FMRPI304N, reduced R-loop-associated DSBs. These observations demonstrated that FMRP promotes genome integrity by preventing R-loop accumulation and chromosome breakage. Here, we explore the mechanism through which FMRP prevents R-loop accumulation in an isogenically controlled CRISPR KO of FMR1 (gene encoding for FMRP) in HEK293T cells. We demonstrate for the first time that FMRP directly binds R-loops. We show that FMRP interacts with DHX9, an RNA helicase that unwinds both double strand RNA and RNA:DNA hybrids and regulates R-loop formation through modulating these activities. This interaction is reduced with FMRPI304N, suggesting that FMRP regulation of R-loop is mediated through DHX9. Interestingly, we show that FMRP inhibits DHX9 helicase activity on RNA:DNA hybrids. Moreover, DHX9 binds chromatin containing R-loops more efficiently in the absence of a functional FMRP. These results suggest an antagonistic relationship between FMRP and DHX9 at the chromatin, where FMRP prevents R-loop formation by suppressing DHX9. Our study sheds new light on our understanding of the genome functions of FMRP.

biochemistry↗
bioRxiv2020

Rad51 paralog complex Rad55-Rad57 acts as a molecular chaperone during homologous recombination

Homologous recombination (HR) is essential for the maintenance of genome integrity. Rad51 paralogs fulfill a conserved, but undefined role in HR, and their mutations are associated with increased cancer risk in humans. Here, we use single-molecule imaging to reveal that the Saccharomyces cerevisiae Rad51 paralog complex Rad55-Rad57 promotes the assembly of Rad51 recombinase filaments through transient interactions, providing evidence that it acts as a classical molecular chaperone. Srs2 is an ATP-dependent anti-recombinase that downregulates HR by actively dismantling Rad51 filaments. Contrary to the current model, we find that Rad55- Rad57 does not physically block the movement of Srs2. Instead, Rad55-Rad57 promotes rapid re- assembly of Rad51 filaments after their disruption by Srs2. Our findings support a model in which Rad51 is in flux between free and ssDNA-bound states, the rate of which is dynamically controlled though the opposing actions of Rad55-Rad57 and Srs2.

biophysics↗
bioRxiv2018

The innate immune DNA sensor cGAS is a negative regulator of DNA repair hence promotes genome instability and cell death

Stringent regulation of DNA repair is essential for organismal integrity, but the mechanisms are not fully understood. Cyclic cGMP-AMP synthase (cGAS), the DNA sensor that alerts the innate immune system to the presence of foreign or damaged self-DNA in the cytoplasm is critical for the outcome of infections, inflammatory diseases and cancer. Besides this cytoplasmic function as an innate immune sensor, whether cGAS fulfills other biological roles remains unknown. Here we report that cGAS has a distinct role in the nucleus: it inhibits homologous recombination DNA repair (HR) thereby promoting genome instability and associated micronuclear generation and mitotic death. We show that cGAS-mediated inhibition of HR requires its DNA binding and oligomerization but not its catalytic activity or the downstream innate immune signaling events. Mechanistically, we show that cGAS impede RAD51-mediated DNA strand invasion, a key step in HR. These results uncover a new function of cGAS relevant for understanding its involvement in genome instability- associated disorders.

cell biology↗
bioRxiv2017

Importance of homo-dimerization of Fanconi-associated nuclease 1 in DNA flap cleavage

Fanconi-associated nuclease 1 (FAN1) removes interstrand DNA crosslinks (ICLs) through its DNA flap endonuclease and exonuclease activities. Crystal structures of human and bacterial FAN1 bound to a DNA flap have been solved. The Pseudomonas aeruginosa bacterial FAN1 and human FAN1 (hFAN1) missing a flexible loop are monomeric, while intact hFAN1 is homo-dimeric in structure. Importantly, the monomeric and dimeric forms of FAN1 exhibit very different DNA binding modes. Here, we interrogate the functional differences between monomeric and dimeric forms of FAN1 and provide an explanation for the discrepancy in oligomeric state between the two hFAN1 structures. Specifically, we show that the flexible loop in question is needed for hFAN1 dimerization. While monomeric and dimeric bacterial or human FAN1 proteins cleave a short 5 flap strand with similar efficiency, optimal cleavage of a long 5 flap strand is contingent upon protein dimerization. Our study therefore furnishes biochemical evidence for a role of hFAN1 homodimerization in biological processes that involve 5 DNA Flap cleavage.

biochemistry↗
bioRxiv2017

Inhibition of DNA2 nuclease as a therapeutic strategy targeting replication stress in cancer cells.

Replication stress is a characteristic feature of cancer cells, which is resulted from sustained proliferative signaling induced by activation of oncogenes or loss of tumor suppressors. In cancer cells, oncogene-induced replication stress manifests as replication-associated lesions, predominantly double-strand DNA breaks (DSBs). An essential mechanism utilized by cells to repair replication-associated DSBs is homologous recombination (HR). In order to overcome replication stress and survive, cancer cells often require enhanced HR repair capacity. Therefore, the key link between HR repair and cellular tolerance to replication-associated DSBs provides us with a mechanistic rationale for exploiting synthetic lethality between HR repair inhibition and replication stress. Our studies showed that DNA2 nuclease is an evolutionarily conserved essential component of HR repair machinery. Here we demonstrate that DNA2 is indeed overexpressed in pancreatic cancers, one of the deadliest and more aggressive forms of human cancers, where mutations in the KRAS are present in 90%-95% of cases. In addition, depletion of DNA2 significantly reduces pancreatic cancer cell survival and xenograft tumor growth, suggesting the therapeutic potential of DNA2 inhibition. Finally, we develop a robust high-throughput biochemistry assay to screen for inhibitors of the DNA2 nuclease activity. The top inhibitors were shown to be efficacious against both yeast Dna2 and human DNA2. Treatment of cancer cells with DNA2 inhibitors recapitulates phenotypes observed upon DNA2 depletion, including decreased DNA end resection and attenuation of HR repair. Similar to genetic ablation of DNA2, chemical inhibition of DNA2 selectively attenuates the growth of various cancer cells with oncogene-induced replication stress. Taken together, our findings open a new avenue to develop a new class of anti-cancer drugs by targeting druggable nuclease DNA2. We 4, 16. In propose DNA2 inhibition as new strategy in cancer therapy by targeting replication stress, a molecular property of cancer cells that is acquired as a result of oncogene activation instead of targeting undruggable oncoprotein itself such as KRAS.

molecular biology↗