Biology subjects

Dhingra, N.

Publications and source records attributed to Dhingra, N..

3 recordsLinked to original sources

bioRxiv2025

Integrative Chemical Genetics Platform Identifies Condensate Modulators Linked to Neurological Disorders

Aberrant biomolecular condensates are implicated in neurological disorders including ALS, frontotemporal dementia, and DYT1 dystonia, yet approaches to systematically identify their modulators remain limited. Here we establish MLF2 as a versatile condensate biomarker and develop CondenScreen, an integrated high-content screening and bioinformatic pipeline enabling identification of condensate modulators across chemical and genetic space. Screening 1,760 FDA-approved compounds in a cellular DYT1 dystonia model, we identify drugs that alter aberrant condensate properties, validating the platform for condensate-targeted drug discovery. In parallel, a genome-wide CRISPR/Cas9 screen links condensate accumulation to microcephaly genes and more than 10 additional neurodevelopmental disorders. Machine learning and confocal imaging resolves distinct condensate phenotypes: loss of microcephaly-associated ZNF335 results in nucleoplasmic condensates, whereas RNF26 deletion produces nuclear envelope condensates that phenocopy hallmarks of torsin deficiency. Our study provides a scalable resource for identifying corrective modulators of condensates and establishes a link between nuclear condensate accumulation and neurodevelopmental disorders.

cell biology↗

bioRxiv2024

Srs2 binding to PCNA and its sumoylation contribute to RPA antagonism during the DNA damage response

Activation of the DNA damage checkpoint upon genotoxin treatment induces a multitude of cellular changes to cope with genome stress. After prolonged genotoxin treatment, the checkpoint can be downregulated to allow cell cycle and growth resumption. In yeast, downregulation of the DNA damage checkpoint requires the Srs2 DNA helicase, which removes the ssDNA binding complex RPA and the associated Mec1 checkpoint kinase from DNA, thus dampening Mec1-mediated checkpoint. However, it is unclear whether the anti-checkpoint role of Srs2 is temporally and spatially regulated to allow timely checkpoint termination while preventing superfluous RPA removal. Here we address this question by examining regulatory elements of Srs2, such as its phosphorylation, sumoylation, and protein-interaction sites. Our genetic analyses and checkpoint level assessment suggest that the RPA countering role of Srs2 is promoted by Srs2 binding to PCNA, which recruits Srs2 to a subset of ssDNA containing regions. RPA antagonism is further fostered by Srs2 sumoylation, which we found depending on the Srs2-PCNA interaction and Mec1, and peaking after Mec1 activity reaches maximal levels. These data suggest that Srs2 recruitment to PCNA proximal ssDNA-RPA filaments followed by its sumoylation can promote checkpoint recovery, whereas Srs2 action is minimized at regions with no proximal PCNA to permit RPA-mediated ssDNA protection.

molecular biology↗

bioRxiv2022

Rtt105 configurationally staples RPA and blocks facilitated exchange and interactions with RPA-interacting proteins

Replication Protein A (RPA) binds to single-stranded DNA (ssDNA) and recruits over three dozen RPA-interacting proteins (RIPs) to coordinate multiple aspects of DNA metabolism including DNA replication, repair, and recombination. Rtt105 is a molecular chaperone that regulates nuclear localization of RPA. Whether and how Rtt105 regulates the activities of RPA is poorly understood. Here, we show that Rtt105 binds to multiple DNA binding and protein-interaction domains of RPA and configurationally staples the complex. In the absence of ssDNA, Rtt105 inhibits RPA binding to Rad52, thus preventing spurious binding to RPA-interacting proteins (RIPs). When ssDNA is available, Rtt105 promotes formation of high-density RPA nucleoprotein filaments and dissociates during this process. Free Rtt105 further stabilizes the RPA-ssDNA filaments by inhibiting RPA facilitated exchange. Collectively, our data suggest that Rtt105 sequesters free RPA in the nucleus to prevent untimely RIP interaction, while stabilizing RPA-ssDNA filaments at DNA lesion sites.

biochemistry↗