Biology subjects

Baral, J.

Publications and source records attributed to Baral, J..

3 recordsLinked to original sources

bioRxiv2025

Bringing the Ends together: Cryo-EM structures of mycobacterial Ku in complex with DNA define its role in NHEJ synapsis

Non-homologous end joining (NHEJ) is the sole pathway for repairing double-strand breaks in Mycobacterium tuberculosis during dormancy, relying on mycobacterial Ku (mKu) and ligase D, with mKu as the rate-limiting factor. Despite its essential role, the lack of structural information on prokaryotic Ku has hindered understanding of the molecular mechanisms underlying bacterial two-component NHEJ machinery. Here, we present the first cryo-EM structures of mKu in DNA-bound and higher-order super-complex forms, revealing a Ku-mediated DNA synapsis mechanism unique to prokaryotes. Integrating cryo-EM with hydrogen-deuterium exchange mass spectrometry, we define key mKu-mKu dimerization, DNA-binding, and synapsis interactions essential for efficient NHEJ, bridging structure with function. Structure-guided in silico mutagenesis, coupled with electrophoretic mobility shift assays, identifies residues essential for DNA binding and synaptic assembly, which are crucial for NHEJ. Forster resonance energy transfer confirms DNA-dependent mKu oligomerization in solution, while live-cell imaging captures its spatiotemporal dynamics during DSB repair. These findings provide fundamental insights into the architecture and function of prokaryotic NHEJ, positioning mKu as a potential therapeutic target against tuberculosis and offering a framework for understanding DNA repair across bacterial species.

biochemistry↗

bioRxiv2023

In silico and in vitro characterization of the mycobacterial protein Ku to unravel its role in non-homologous end-joining DNA repair

Non-homologous end-joining DNA repair is essential for the survival and sustenance of M. tuberculosis (Mtb) in the dormant stage of its life cycle. The ability of Mtb to sustain itself in the inactive form has been reported to be the critical factor for its resilience over the years. To unravel one of the salient features of the Mtbs arsenal, we exploited in silico and in vitro tools to characterize the DNA binding properties of mycobacterial protein Ku (mKu) and its role in mycobacterial NHEJ. Here, we report the strong affinity of mKu for linear dsDNA exhibiting positive cooperativity for dsDNAs ({zeta}40bp). Molecular dynamics complemented with in vitro experiments showed that the DNA binding of mKu provides stability to both mKu homodimer and the DNA. Furthermore, mKu end-capping of DNA was seen to protect the DNA termini against nucleolytic degradation by exonuclease. The DNA-mKu association formed higher-order oligomers probably due to the lodgement of two DNA molecules at opposite ends of the mKu homodimer. The ability of mKu to form continuous filament-like structures with DNA indicated its potential role in mycobacterial NHEJ synapsis.

biochemistry↗

bioRxiv2021

Spatially Interacting Phosphorylation Sites and Mutations in Cancer

Advances in mass-spectrometry have generated increasingly large-scale proteomics datasets containing tens of thousands of phosphorylation sites (phosphosites) that require prioritization. We develop a bioinformatics tool called HotPho and systematically discover 3D co-clustering of phosphosites and cancer mutations on protein structures. HotPho identifies 474 such hybrid clusters containing 1,255 co-clustering phosphosites, including RET p.S904/Y928, the conserved HRAS/KRAS p.Y96, and IDH1 p.Y139/IDH2 p.Y179 that are adjacent to recurrent mutations on protein structures not found by linear proximity approaches. Hybrid clusters, enriched in histone and kinase domains, frequently include expression-associated mutations experimentally shown as activating and conferring genetic dependency. Approximately 300 co-clustering phosphosites are verified in patient samples of 5 cancer types or previously implicated in cancer, including CTNNB1 p.S29/Y30, EGFR p.S720, MAPK1 p.S142, and PTPN12 p.S275. In summary, systematic 3D clustering analysis highlights nearly 3,000 likely functional mutations and over 1,000 cancer phosphosites for downstream investigation and evaluation of potential clinical relevance.

bioinformatics↗