Biology subjects

Misra, H. S.

Publications and source records attributed to Misra, H. S..

4 recordsLinked to original sources

bioRxiv2020

Topoisomerase IB interacts with genome segregation proteins and is involved in multipartite genome maintenance in Deinococcus radiodurans

Deinococcus radiodurans, an extremophile, resistant to many abiotic stresses including ionizing radiation, has 2 type I topoisomerases (drTopo IA and drTopo IB) and one type II topoisomerase (DNA gyrase). The role of drTopo IB in guanine quadruplex DNA (G4 DNA) metabolism was shown in vitro. Here we report that D. radiodurans cells lacking drTopo IB ({Delta}topoIB) show sensitivity to G4 DNA binding drug (NMM) under normal growth conditions. The activity of G4 motif containing promoters like mutL and recQ was reduced in the presence of NMM in mutant cells. In mutant cells, the percentage of anucleated cells was more while the ploidy numbers of genome elements were less as compared to wild type. Protein-protein interaction studies showed that drTopo IB interacts with genome segregation and DNA replication initiation (DnaA) proteins. The typical patterns of cellular localization of GFP-PprA were affected in the mutant cells. Microscopic examination of D. radiodurans cells expressing drTopo IB-RFP showed its localization on nucleoid forming a streak parallel to the old division septum and perpendicular to newly formed septum. These results together suggest the role of drTopo IB in genome maintenance in this bacterium.

microbiology↗

bioRxiv2020

DivIVA is essential in Deinococcus radiodurans and its C terminal domain regulates new septum orientation during cell division

FtsZ assembly at mid cell position in rod shaped bacteria is regulated by gradient of MinCDE complex across the poles. In round shaped bacteria, which lack predefined poles and the next plane of cell division is perpendicular to previous plane, the determination of site for FtsZ assembly is intriguing. Deinococcus radiodurans a coccus shaped bacterium, is characterized for its extraordinary resistance to DNA damage. Here we report that DivIVA a putative component of Min system in this bacterium (drDivIVA) interacts with cognate cell division and genome segregation proteins. The deletion of full length drDivIVA was found to be indispensable while its C-terminal deletion ({Delta}divIVAC) was dispensable but produced distinguishable phenotypes like slow growth, altered plane for new septum formation and angular septum. Both wild type and mutant showed FtsZ foci formation and their gamma radiation responses were nearly identical. But unlike in wild type, the FtsZ localization in mutant cells was found to be away from orthogonal axis with respect to plane of previous septum. Notably, DivIVA-RFP localizes to membrane during cell division and then perpendicular to previous plane of cell division. In trans expression of drDivIVA in {Delta}divIVAC background could restore the wild type pattern of septum formation perpendicular to previous septum. These results suggested that DivIVA is an essential protein in D. radiodurans and the C-terminal domain that contributes to its interaction with MinC determines the plane of new septum formation, possibly by controlling MinC oscillation through orthogonal axis in the cells.

microbiology↗

bioRxiv2020

Overlapping oriC and centromere-like functions in secondary genome replicons determine their maintenance independent of chromosome I in Deinococcus radiodurans

The Deinococcus radiodurans multipartite genome system (MGS) consists of chromosome I (ChrI) and secondary genome elements; Chr II and megaplasmid (MP). The sequences upstream to parAB operons in Chr II (cisII) and MP (cisMP) helped an E. coli plasmid maintenance in D. radiodurans and showed sequence specific interactions with DnaA and ParBs. The cells devoid of cisII ({Delta}cisII) or cisMP ({Delta}cisMP) showed reduced {gamma} radiation resistance and copy number of Chr II and MP. Fluorescent Reporter-Operator System (FROS) developed for ChrI, ChrII and MP in {Delta}cisII or {Delta}cisMP mutants showed no change in wild type pattern of Chr I localization. However, the relative copy numbers of Chr II and MP had reduced while anucleate cells had increased in mutants. These results suggested that cisII and cisMP elements contain both ori and centromere-like functions, and like other MGS bacteria, the Chr I and secondary genome are maintained independently in D. radiodurans.

cell biology↗

bioRxiv2020

PprA interacts with replication proteins and affects their physicochemical properties required for replication initiation in Deinococcus radiodurans

The deletion mutant of pprA, a gene encoding pleiotropic functions in radioresistant bacterium Deinococcus radiodurans, showed an increased genomic content and ploidy in chromosome I and chromosome II. We identified oriC in chromosome I (oriCI) and demonstrated the sequence specific interaction of deinococcal DnaA (drDnaA) with oriCI. drDnaA and drDnaB showed ATPase activity while drDnaB catalyzed 5'[->]3' dsDNA helicase activity. These proteins showed both homotypic and heterotypic interactions. The roles of C-terminal domain of drDnaA in oriCI binding and its stimulation of ATPase activity were demonstrated. Notably, PprA showed ~2 times higher affinity to drDnaA as compared to drDnaB and attenuated both homotypic and heterotypic interactions of these proteins. Interestingly, the ATPase activity of drDnaA but not drDnaB was inhibited in presence of PprA. These results suggested that PprA influences the physicochemical properties of drDnaA and drDnaB that are required for initiation of DNA replication at oriCI site in this bacterium.

molecular biology↗