Biology subjects

Huang, T. T.

Publications and source records attributed to Huang, T. T..

6 recordsLinked to original sources

bioRxiv2022

Translesion DNA synthesis polymerase kappa functions in the nucleolar stress response

DNA polymerase kappa (Pol{kappa}) has multiple cellular roles such as translesion DNA synthesis, replication of repetitive sequences and nucleotide excision repair. However, the mechanisms regulating Pol{kappa}s cellular activities are unknown. Since all polymerases insert the canonical deoxynucleotide triphosphates, it is difficult to determine which polymerase is active at a particular genomic site. To counter this difficulty, we utilized the selective Pol{kappa} substrate, N2-(4-ethynylbenyl)-2-deoxyguanosine (EBndG), as bait to capture proteins associated with Pol{kappa} synthesized DNA. Here we show that, Pol{kappa} is active in the nucleolus and Pol{kappa} synthesized DNA are enriched with nucleolar proteins. Exposure of cells to benzo[a]pyrene diol epoxide (BPDE) induced hallmarks of nucleolar stress, increased Pol{kappa} stability and nucleolar activity. Other agents that induce nucleolar stress such as mitomycin C, cisplatin and actinomycin D also increased Pol{kappa}s nucleolar activity. The Pol{kappa} activity was cell-cycle independent and dependent on PCNA ubiquitination. In addition, we identified that the expression and activity of Pol{kappa} is regulated by the polycomb complex protein Ring Finger Protein 2 (RNF2) and by poly(ADP)-ribose polymerase 1 (PARP1) catalyzed PARylation. This study provides insight into the novel role of Pol{kappa} in ribosomal RNA synthesis, in maintaining ribosomal DNA integrity after DNA damage thus protecting the cells from nucleolar stress.

molecular biology↗

bioRxiv2020

Activity profiling of SARS-CoV-2-PLpro protease provides structural framework for anti-COVID-19 drug design

In December 2019, the first cases of a novel coronavirus infection causing COVID-19 were diagnosed in Wuhan, China. Viral Papain-Like cysteine protease (PLpro, NSP3) is essential for SARS-CoV-2 replication and represents a promising target for the development of antiviral drugs. Here, we used a combinatorial substrate library containing natural and a wide variety of nonproteinogenic amino acids and performed comprehensive activity profiling of SARS-CoV-2-PLpro. On the scaffold of best hits from positional scanning we designed optimal fluorogenic substrates and irreversible inhibitors with a high degree of selectivity for SARS PLpro variants versus other proteases. We determined crystal structures of two of these inhibitors (VIR250 and VIR251) in complex with SARS-CoV-2-PLpro which reveals their inhibitory mechanisms and provides a structural basis for the observed substrate specificity profiles. Lastly, we demonstrate that SARS-CoV-2-PLpro harbors deISGylating activities similar to SARS-CoV-1-PLpro but its ability to hydrolyze K48-linked Ub chains is diminished, which our sequence and structure analysis provides a basis for. Altogether this work has revealed the molecular rules governing PLpro substrate specificity and provides a framework for development of inhibitors with potential therapeutic value or drug repositioning.

biochemistry↗

bioRxiv2020

Fused in sarcoma regulates DNA replication timing and progression

Fused in sarcoma (FUS) encodes a low complexity RNA-binding protein with diverse roles in transcriptional activation and RNA processing. While oncogenic fusions of FUS and transcription factor DNA-binding domains are associated with soft tissue sarcomas, dominant mutations in FUS cause amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). FUS has also been implicated in DNA double-strand break repair (DSBR) and genome maintenance. However, the underlying mechanisms are unknown. Here we employed quantitative proteomics, transcriptomics, and DNA copy number analysis (Sort-Seq), in conjunction with FUS-/- cells to ascertain roles of FUS in genome protection. FUS-/- cells exhibited alterations in the recruitment and retention of DSBR factors BRCA1 and 53BP1 but were not overtly sensitive to genotoxins. By contrast, FUS-deficient cells had reduced proliferative potential that correlated with reduced replication fork speed, diminished loading of pre-replication complexes, and attenuated expression of S-phase associated genes. FUS interacted with lagging strand DNA synthesis factors and other replisome components, but did not translocate with active replication forks. Using a Sort-Seq workflow, we show that FUS contributes to genome-wide control of DNA replication timing and is essential for the early replication of transcriptionally active DNA. These findings illuminate new roles for FUS in DNA replication initiation and timing that may contribute to genome instability and functional defects in cells harboring disease-associated FUS fusions.

genomics↗

bioRxiv2020

Engineered unnatural ubiquitin for optimal detection of deubiquitinating enzymes

Deubiquitinating enzymes (DUBs) are responsible for removing ubiquitin (Ub) from its protein conjugates. DUBs have been implicated as attractive therapeutic targets in the treatment of viral diseases, neurodegenerative disorders and cancer. The lack of selective chemical tools for the exploration of these enzymes significantly impairs the determination of their roles in both normal and pathological states. Commercially available fluorogenic substrates are based on the C-terminal Ub motif or contain Ub coupled to a fluorophore (Z-LRGG-AMC, Ub-AMC); therefore, these substrates suffer from lack of selectivity. By using a hybrid combinatorial substrate library (HyCoSuL) and a defined P2 library containing a wide variety of nonproteinogenic amino acids, we established a full substrate specificity profile for two DUBs--MERS PLpro and human UCH-L3. Based on these results, we designed and synthesized Ub-based substrates and activity-based probes (ABPs) containing selected unnatural amino acids located in the C-terminal Ub motif. Biochemical analysis and cell-based experiments confirmed the activity and selectivity of engineered Ub-based substrates and probes. Using this approach, we propose that for any protease that recognizes Ub and Ub-like substrates, a highly active and selective unnatural substrate or probe can be engineered.

biochemistry↗

bioRxiv2019

Topoisomerase 2-dependent nuclear DNA damage shapes extracellular growth factor responses through AKT phosphorylation dynamics to control virus latency

The mTOR pathway integrates both extracellular and intracellular signals and serves as a central regulator of cell metabolism, growth, survival and stress responses. Neurotropic viruses, such as herpes simplex virus-1 (HSV-1), also rely on cellular AKT-mTORC1 signaling to achieve viral latency. Here, we define a novel genotoxic response whereby spatially separated signals initiated by extracellular neurotrophic factors and nuclear DNA damage are integrated by the AKT-mTORC1 pathway. We demonstrate that endogenous DNA double-strand breaks (DSBs) mediated by Topoisomerase 2{beta}-DNA cleavage complex (TOP2{beta}cc) intermediates are required to achieve AKT-mTORC1 signaling and maintain HSV-1 latency in neurons. Suppression of host DNA repair pathways that remove TOP2{beta}cc trigger HSV-1 reactivation. Moreover, perturbation of AKT phosphorylation dynamics by downregulating the PHLPP1 phosphatase led to AKT mis-localization and disruption of DSB-induced HSV-1 reactivation. Thus, the cellular genome integrity and environmental inputs are consolidated and co-opted by a latent virus to balance lifelong infection with transmission.

molecular biology↗

bioRxiv2018

Transcription drives DNA replication initiation and termination in human cells

The locations of active DNA replication origins in the human genome, and the determinants of origin activation, remain controversial. Additionally, neither the predominant sites of replication termination nor the impact of transcription on replication-fork mobility have been defined. We demonstrate that replication initiation occurs preferentially in the immediate vicinity of the transcription start site of genes occupied by high levels of RNA polymerase II, ensuring co-directional replication of the most highly transcribed genes. Further, we demonstrate that dormant replication origin firing represents the global activation of pre-existing origins. We also show that DNA replication naturally terminates at the polyadenylation site of transcribed genes. During replication stress, termination is redistributed to gene bodies, generating a global reorientation of replication relative to transcription. Our analysis provides a unified model for the coupling of transcription with replication initiation and termination in human cells.

molecular biology↗