Browsing by Author "Kumari, Poonam"

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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.
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    Holliday junction resolution by At-HIGLE: an SLX1 lineage endonuclease from Arabidopsis thaliana with a novel in-built regulatory mechanism
    (Oxford University Press, 2022) Verma, Prabha; Kumari, Poonam; Negi, Shreya; Yadav, Gitanjali; Gaur, Vineet
    Holliday junction is the key homologous recombination intermediate, resolved by structure-selective endonucleases (SSEs). SLX1 is the most promiscuous SSE of the GIY-YIG nuclease superfamily. In fungi and animals, SLX1 nuclease activity relies on a non-enzymatic partner, SLX4, but no SLX1-SLX4 like complex has ever been characterized in plants. Plants exhibit specialized DNA repair and recombination machinery. Based on sequence similarity with the GIY-YIG nuclease domain of SLX1 proteins from fungi and animals, At-HIGLE was identified to be a possible SLX1 like nuclease from plants. Here, we elucidated the crystal structure of the At-HIGLE nuclease domain from Arabidopsis thaliana, establishing it as a member of the SLX1-lineage of the GIY-YIG superfamily with structural changes in DNA interacting regions. We show that At-HIGLE can process branched-DNA molecules without an SLX4 like protein. Unlike fungal SLX1, At-HIGLE exists as a catalytically active homodimer capable of generating two coordinated nicks during HJ resolution. Truncating the extended C-terminal region of At-HIGLE increases its catalytic activity, changes the nicking pattern, and monomerizes At-HIGLE. Overall, we elucidated the first structure of a plant SLX1-lineage protein, showed its HJ resolving activity independent of any regulatory protein, and identified an in-built novel regulatory mechanism engaging its C-terminal region.