Structural Biology of Genome Maintenance and DNA repair

National Institute Of Environmental Health Sciences

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Abstract

Progress 2021-2022 Molecular Basis for Processing of Topoisomerase 1-Triggered DNA damage by Apn2/APE2 Topoisomerase 1 (Top1) incises DNA containing ribonucleotides to generate complex DNA lesions that are resolved by APE2 (Apn2 in yeast). How Apn2 engages and processes this DNA damage is unknown. We now report X-ray crystal structures and biochemical analysis of Apn2-DNA complexes to demonstrate how Apn2 frays and cleaves 3 DNA termini via a wedging mechanism that facilitates 1-6 nucleotide endonucleolytic cleavages. APN2 deletion and DNA wedge mutant Saccharomyces cerevisiae strains display a novel mutator phenotype, cell growth defects and sensitivity to genotoxic stress in a Ribonucleotide Excision Repair (RER)-defective background harboring a high density of Top1-incised ribonucleotides. Our data implicate a wedge-and-cut mechanism underpinning the broad-specificity Apn2 nuclease activity that mitigates mutagenic and genome instability phenotypes caused by Top1 incision at genomic ribonucleotides incorporated by DNA polymerase epsilon. Structures of PolG2 DNA complexes PolG2 is the homodimeric accessory subunit within the polymerase gamma (PolG) heterotrimer responsible for replicating mitochondrial DNA. PolG2 enhances the ability of the PolG catalytic subunit to bind dsDNA and promotes processivity of the PolG holoenzyme. While it is known that PolG2 itself binds DNA, the nature and function of this interaction has been unclear as existing PolG-PolG2-DNA complex structures show that PolG2 stabilizes PolG-DNA interactions, rather than directly binding DNA. To better understand PolG2 DNA-binding functions, we determined the crystal structures of PolG2 bound to dsDNA both as a dimer, and as a hexameric PolG2 scaffold that assembles an extensive DNA-protein interface. Our structures uncover four PolG2 DNA-binding regions and provide an explanation for molecular defects associated with PolG2 disease variants L475DfsX2 and R369G. Data from biochemical analysis indicates that the conserved PolG2 DNA-binding surfaces are critical for supporting DNA binding in vitro. Overall, X-ray structural, and biochemical data indicate that PolG2 DNA-binding has both PolG-dependent and -independent functions. Discovery and Structural Basis of the Selectivity of Potent Cyclic Peptide Inhibitors of MAGE-A4. MAGE proteins are cancer testis antigens (CTAs) that are characterized by highly conserved MAGE homology domains (MHDs) and are increasingly being found to play pivotal roles in promoting aggressive cancer types. MAGE-A4, in particular, increases DNA damage tolerance and chemoresistance in a variety of cancers by stabilizing the E3-ligase RAD18 and promoting trans-lesion synthesis (TLS). Inhibition of the MAGE-A4:RAD18 axis could sensitize cancer cells to chemotherapeutics like platinating agents. We use an mRNA display of thioether cyclized peptides to identify a series of potent and highly selective macrocyclic inhibitors of the MAGE-A4:RAD18 interaction. Co-crystal structure indicates that these inhibitors bind in a pocket that is conserved across MHDs but take advantage of A4-specific residues to achieve high isoform selectivity. Cumulatively, our data represent the first reported inhibitor of the MAGE-A4:RAD18 interaction and establish biochemical tools and structural insights for the future development of MAGE-A4-targeted cellular probes.