Browsing by Author "Rai, Praveen"

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    Branched DNA processing by a thermostable CAS-Cas4 from Thermococcus onnurineus: expanding biochemical landscape of nuclease activity
    (Elsevier B.V., 2025) Jain, Muskan; Pattnayak, Asish Kumar; Aggarwal, Sakshi; Rai, Praveen; Kavya, J.; Chandrayan, Sanjeev; Goel, Manisha; Gaur, Vineet
    The adaptive immune function of CRISPR-Cas systems in bacteria and archaea is mediated through CRISPR-Associated Proteins (Cas). The adaptation module, typically involving Cas1, Cas2, and Cas4, helps integrate viral “spacer” sequences into the host genome. Cas4 proteins are classified into two types based on neighboring genes: CAS-Cas4, flanked by other cas genes, and Solo-Cas4, which exist independently. While CAS-Cas4 proteins are implicated in adaptation, they remain biochemically uncharacterized in archaea, unlike archaeal Solo-Cas4 proteins. This study biochemically characterizes TON_0321, a CAS-Cas4 protein from the Type IV-C CRISPR cassette of Thermococcus onnurineus. TON_0321 exhibits 5′ to 3′ exonuclease activity and unique structure-dependent endonuclease activity, shedding light on CAS-Cas4 functional diversity. A distinct spatial organization of the catalytic site, angled with the positively charged patch on the protein surface, enables TON_0321 to recognize branching points in DNA substrates. Furthermore, this spatial arrangement facilitates cleavage 2 to 3 nucleotides away from the branch point in the 5′ direction, demonstrating structure-specific endonuclease activity.
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    Erasing methylation marks on DNA by plant-specific DEMETER family DNA glycosylases
    (Springer Nature Publishing AG, 2025) Rai, Praveen; Kumari, Poonam; Gaur, Vineet
    Plants, being sessile and incapable of evading the continually fluctuating environment, display enormous plasticity in responding to constantly changing environmental challenges through assimilating changes in the genome (resulting in sequence polymorphism) and epigenome (resulting in gene expression alterations). DNA methylation is one of the reversible epigenetic modifications imparting a swift tunability to the genome through a complex interplay of methylation and demethylation processes. Plants have diligently repurposed the base excision repair machinery to actively erase methylation marks on DNA by evolving a specific family of HhH DNA glycosylases collectively called the DEMETER family. The DML family comprises four types of HhH DNA glycosylases: DEMETER, REPRESSOR OF SILENCING 1, and the paralogs DML2 and DML3 (DEMETER-LIKE proteins 2 and 3). The DML family enzymes are bifunctional glycosylases with a bipartite glycosylase domain and a C-terminal domain crucial for the catalytic activity. Removal of methylation proceeds through a classical mechanism of DNA bending and base flipping. DML family DNA glycosylases play roles in maintaining genome stability, plant development, and various biotic and abiotic stresses. This review aims to provide a comprehensive summary of the current understanding of the molecular mechanisms and biological functions of the DEMETER family DNA glycosylases in plants.