Biology subjects

Rajpurohit, Y. S.

Publications and source records attributed to Rajpurohit, Y. S..

5 recordsLinked to original sources

bioRxiv2026

Operon-driven antagonism and novel modulation of DNA damage sensor STPK specific PP2C phosphatase in Deinococcus radiodurans

This study identifies a novel operon-driven signaling module in Deinococcus radiodurans. The operon includes a von Willebrand A domain protein (DRA0331), a Ser/Thr protein kinase (DRA0332), a canonical FHA-domain protein (DRA0333), and a PP2C-type phosphatase (DRA0334). DRA0334 shows Mn{superscript 2}{square}-dependent phosphatase activity and has a unique dual-domain structure that combines a Kinase-Interacting FHA (KI-FHA) domain with a PP2C catalytic domain. Functional assays show that FHA-domain protein, DRA0333 boosts the phosphorylation of STPKs like RqkA and DR1243 while operonic partner PP2C-type phosphatase, DRA0334 counteracts this through targeted dephosphorylation, establishing a phospho-regulatory antagonism. Notably, the KI-FHA domain of the DRA0334 phosphatase competitively interacts with the FHA domain to modulate the radiation-responsive RqkA kinase, thereby maintaining kinase-phosphatase balance. This KI-FHA domain also imparts substrate specificity and enables feedback regulation. Additionally, DRA0334 modular variants confirm separate roles of catalytic and docking modules, and STRING analyses link DRA0334 functions to DNA repair and stress recovery. Collectively, the findings suggest an operonic connection between DRA0333 and DRA0334, indicating that the KI-FHA and FHA domains may act as phospho-docking switches. These switches can regulate both kinase and phosphatase activities in a push-pull regulatory mechanism within the phosphorylation-dephosphorylation cycle of signal transduction, depending on their association with the type of catalytic domain.

molecular biology↗

bioRxiv2024

Surviving the Storm: Exploring the Role of Natural Transformation in Nutrition and DNA Repair of Stressed Deinococcus radiodurans

Deinococcus radiodurans, a natural transformation (NT) enabled bacterium renowned for its exceptional radiation resistance, employs unique DNA repair and oxidative stress mitigation mechanisms as a strategic response to DNA damage. This study excavate into the intricate roles of NT machinery in the stressed D. radiodurans, focusing on the genes comEA, comEC, endA, pilT and dprA, which are instrumental in the uptake and processing of extracellular DNA (eDNA). Our data reveals that NT not only supports the nutritional needs of D. radiodurans under stress but also have roles in DNA repair. The study findings establish that NT-specific proteins (ComEA, ComEC, and EndA) might contribute to support the nutritional requirements in unstressed and heavily DNA-damaged cells while DprA contribute differently and in a context-dependent manner to navigating through the DNA damage storm. Thus, this dual functionality of NT-specific genes is proposed to be one of factor in D. radiodurans remarkable ability to survive and thrive in environments characterized by high levels of DNA-damaging agents. Author SummaryDeinococcus radiodurans, a bacterium known for its extraordinary radiation resistance. This study explores the roles of natural transformation (NT) machinery in the radiation-resistant bacterium Deinococcus radiodurans, focusing on the genes comEA, comEC, endA, pilT, and dprA. These genes are crucial for the uptake and processing of extracellular DNA (eDNA) and contribute to the bacterium nutritional needs and DNA repair under stress. The findings suggest that the NT-specific proteins ComEA, ComEC, and EndA may help meet the nutritional needs of unstressed and heavily DNA-damaged cells, whereas DprA plays a distinct role that varies depending on the context in aiding cells to cope with DNA damage. The functionality of NT genes is proposed to enhance D. radiodurans survival in environments with high levels of DNA-damaging agents.

molecular biology↗

bioRxiv2023

Natural transformation specific DprA coordinate DNA double strand break repair pathways in heavily irradiated D. radiodurans

Deinococcus radiodurans exhibits remarkable survival under extreme conditions, including ionizing radiation, desiccation, and various DNA-damaging agents. It employs unique repair mechanisms, such as single-strand annealing (SSA) and extended synthesis-dependent strand annealing (ESDSA), to efficiently restore damaged DNA fragments. In this study, we investigate the regulatory role of the NT-specific protein DprA in DNA repair pathways following acute gamma radiation exposure. Our findings demonstrate that the absence of DprA leads to rapid repair of gamma radiation-induced DNA double-strand breaks (DSBs), with diminished involvement of the ESDSA pathway. Furthermore, our data suggest that the SSA pathway becomes the primary mechanism for DNA DSB repair in the absence of DprA. Overall, our results highlight the regulatory function of DprA in modulating the choice between SSA and ESDSA pathways for DNA repair in the radiation-resistant bacterium D. radioduransx.

cell biology↗

bioRxiv2022

Characterization of DNA processing protein A (DprA) of the radiation-resistant bacterium Deinococcus radiodurans

The uptake of environmental DNA (eDNA) by certain bacteria and its integration into their genome creates genetic diversity and new phenotypes. A DNA processing protein A (DprA) is part of a multiprotein complex and facilitate natural transformation (NT) phenotype in most bacteria. The Deinococcus radiodurans, an extremely radioresistant bacterium, is efficient in N T, and its genome encodes nearly all the components of the natural competence complex. Here, we have characterized the DprA of this bacterium (DrDprA) for the known characteristics of DprA proteins in other bacteria and the mechanisms underlying its roles in the transformation of eDNA into the bacterial genome. DrDprA is found to be a unique domain organization implicating some unique functions compared with DprA of other bacteria. In vitro studies showed that the purified recombinant DrDprA binds to both ssDNA and dsDNA with nearly equal affinity and protects ssDNA from nucleolytic degradation. DrDprA showed a strong interaction with DrRecA indicating its role in RecA catalyzed functions in vivo. Mutational studies identified amino acid residues responsible for its oligomerization, interaction with DrRecA, and DNA binding characteristics of DrDprA. Further, we demonstrated that both oligomerization and DNA binding properties of DrDprA are integral to its support in DrRecA catalyzed strand exchange reaction (SER) in vitro. These results suggested that DrDprA is largely structurally conserved with DprA homologs but showed some unique structure-function features like additional domain, the same affinity to ss/ds DNA and both oligomerization and DNA binding domains collectively contribute to its support in DrRecA functions.

molecular biology↗

bioRxiv2020

WD 40 domain of RqkA regulates its kinase activity and role in extraordinary radioresistance in Deinococcus radiodurans

RqkA, a DNA damage responsive Serine / Threonine kinase is characterized for its role in DNA repair and cell division in D. radiodurans. It has a unique combination of a kinase domain at N-terminus and a WD40 type domain at C-terminus joined through a linker. WD40 domain is comprised of eight {beta} propeller repeats held together via "tryptophan-docking motifs" and forming a typical velcro closure structure. RqkA mutants lacking the WD40 region (hereafter referred to as WD mutant) could not complement RqkA loss in {gamma} radiation resistance in D. radiodurans and lacked {gamma} radiation mediated activation of kinase activity in vivo. WD mutants failed to phosphorylate its cognate substrate (e.g. DrRecA) in surrogate E. coli cells. Further, unlike wild type enzyme, the kinase activity of its WD40 mutants was not stimulated by Pyrroloquinoline quinine (PQQ) indicating the role of the WD motifs in PQQ interaction and stimulation of its kinase activity. Together, results highlighted the importance of the WD40 domain in the regulation of RqkA kinase signaling functions in vivo and thus the role of WD40 domain in the regulation of any STPK is the first time demonstrated in bacteria. ImportanceThis study highlights the importance of the WD40 domain in activity regulation and signaling activity of bacterial serine/ threonine kinase for the first time in the bacterial response to gamma radiation and DNA damage.

molecular biology↗