Structural Biology of Genome Maintenance and DNA repair

National Institute Of Environmental Health Sciences

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Abstract

Progress 2022-2023 1) Architecture of the Sen1 helicase: RNA-DNA Hybrid Resolution, Autoregulation, and Insights into SETX Inactivation in AOA2 The Senataxin (SETX, Sen1 in yeasts) RNA-DNA hybrid resolving helicase regulates multiple nuclear transactions including RNA processing and maturation, DNA replication, transcription, and DNA repair, but the molecular basis for Sen1 activities are ill-defined. We report cryo-EM reconstructions of Chaetomium thermophilum Sen1 that reveal an elongated inchworm-like architecture of the full-length enzyme. Sen1 is comprised of two structured halves, an amino terminal helical repeat containing Sen1 N-terminal (Sen1N) regulatory domain that is linked to its C-terminal RNA-DNA helicase motor core (Sen1Hel) via an intrinsically disordered tether. In an autoinhibited state, the Sen1Sen1N helical repeat controls substrate engagement and helicase activity by occluding the RNA-DNA substrate binding cleft through interaction with the Sen1Hel RecA1 -barrel, RecA1 core, and a Sen1-specific leucine-zipper-zipper. The X-ray structure of an activated Sen1 helicase core engaging single-stranded RNA and ADP-SO4 further shows how the enzyme encircles RNA and implicates a single nucleotide power stroke in the Sen1 RNA translocation mechanism. Human SETX Ataxia with Oculomotor Apraxia 2 (AOA2) mutations map to the RNA binding cleft and impair its RNA binding and helicase activities. Our data unveil dynamic protein-protein and protein-RNA interfaces underpinning helicase regulation and inactivation of SETX activity in neurodegenerative disease. 2)Structure-specific roles for PolG2-DNA complexes in maintenance and replication of mitochondrial DNA The homodimeric PolG2 accessory subunit of the mitochondrial DNA polymerase gamma enhances DNA binding and processive DNA synthesis by the PolG catalytic subunit. PolG2 also directly binds DNA, although the underlying molecular basis and functional significance are unknown. Here, data from Atomic Force Microscopy (AFM) and X-ray structures of PolG2-DNA complexes define dimeric and hexameric PolG2 DNA binding modes. Targeted disruption of PolG2 DNA-binding interfaces impairs processive DNA synthesis without diminishing Pol gamma subunit affinities. In addition, a structure-specific DNA-binding role for PolG2 oligomers is supported by X-ray structures and AFM showing that oligomeric PolG2 localizes to DNA crossings and targets forked DNA structures resembling the mitochondrial D-loop. Overall, data indicate that PolG2 DNA binding has both PolG-dependent and -independent functions in mitochondrial DNA replication and maintenance, which provide new insight into molecular defects associated with PolG2 disruption in mitochondrial disease. 3) Reviewing the structural basis for DNA end processing by the Mre11-Rad50-Nbs1 complex The Mre11-Rad50-Nbs1 (MRN) complex (Mre11-Rad50 or MR in bacteria and archaea) orchestrates DNA double strand break (DSB) repair by homologous recombination, an essential response to DSBs created by ionizing radiation, CRISPR-Cas9, programmed strand breaks during meiosis, and other sources. MRN is a multifunctional DSB first responder, and MRN activities include DNA end sensing and tethering, nucleolytic activities, and cell cycle signaling through the Ataxia telangiectasia (ATM) kinase. Extensive efforts probing the structure and function of MRN subcomplexes over the past 20+ years have provided important insights into its functions. Despite these advances, MRN has kept some of its key atomic secrets hidden. The molecular bases for MRN catalytic DNA processing activities have remained enigmatic in the absence of structures of full-length enzymes engaging relevant DNA substrates. We review recent tour de force advances reporting how Cryo-EM analysis of prokaryotic MR bound to nucleic acid, that resolve long-standing questions regarding the molecular underpinnings of the broadly specific MR nuclease activity. Additional imaging of a fully reconstituted Chaetomium thermophlium (Ct) MRN complex in its autoinhibited state unveils conservation of the MR core architecture, and the basis for eukaryotic Nbs1 engagement of the MR DNA processing head.