I. Molecular underpinnings of a Tdp2 deficiency in resolution of Topoisomerase 2 DNA-protein crosslinks Normally, the eukaryotic type II topoisomerases (Top2α and Top2β) regulate DNA topology by employing a dsDNA cleavage and religation cycle involving transient formation of Top2-DNA cleavage-complexes (Top2cc). Top2 catalytic intermediates are characterized by the topoisomerase covalently linked to the DNA 5′-terminus by an active site tyrosine residue. However, aberrant DNA structure or targeted chemotherapeutic disruption of the Top2 reaction can generate Top2cc, protein-DNA crosslinks that block transcription and/or collapse DNA replication forks. Tyrosyl-DNA phosphodiesterase (Tdp2) directly reverses Topoisomerase 2 (Top2) DNA-protein crosslinks characterized by 5-phosphotyrosyl-DNA and -RNA linkages triggered by Top2 engagement of DNA damage or poisoning by anticancer Top2 drugs. Tdp2 deficiencies are linked to neurological disease and cellular sensitivity to Top2 poisons, but the molecular mechanisms of Tdp2 processing of DNA damage, and the underpinnings of Tdp2 inactivation remain incompletely defined. Our integrated results from biochemical studies, X-ray crystal structures of ligand-free Tdp2 and Tdp2 bound to abasic DNA damage, and QM/MM calculations support a single Mg2+ -ion assisted Tdp2 phosphotyrosyl phosphodiesterase reaction mechanism. Further, a Tdp2 single nucleotide polymorphism impacting the Mg2+ binding site stunts metamorphic Tdp2 active site assembly upon DNA substrate binding, ablates Tdp2 catalytic activity and Tdp2 driven non-homologous end joining (NHEJ) dependent repair of phosphotyrosyl-adducted DNA ends in vitro, and confers etoposide hyper-sensitivity to mammalian cells. Altogether, our results provide new key insights into the mechanism of Tdp2 action in chemotherapeutic drug resistance, and reveal that Tdp2 variation in the population impacts Tdp2 functions in the resolution of Top2 DNA-protein crosslinks. II. Mechanisms of DNA end processing by Ctp1Ctip/Sae2/Mre11/Rad50/Nbs1 DNA double strand breaks (DSBs) generated by clastogen exposures including ionizing radiation and topoisomerase poisons can sever entire chromosomes, thereby contributing to genomic instability and carcinogenesis. Error-free DSB repair of adducted DNA strand breaks by homologous recombination (HR) is initiated by the Mre11/Rad50/Nbs1 (MRN) complex. Ctp1CtIP/Sae2 collaborates with the Mre11-Rad50-Nbs1 (MRN) nuclease to modulate end processing, but the functional roles for Ctp1 remain unclear. We have established that Ctp1 harbors DNA-binding and bridging activities, but is not a nuclease. Our Ctp1 X-ray structures, small angle X-ray scattering (SAXS), and biophysical analysis define the salient features of Ctp1 architecture: an N-terminal interlocking tetrameric helical dimer of dimers domain (THDD), and an extended central intrinsically disordered region (IDR) linked to conserved C-terminal DNA-binding RHR motifs. The THDD, IDR and RHR regions are all required to support Ctp1 DNA bridging activity in vitro, and THDD or RHR disruption confers sensitivity of fission yeast to DNA damaging agents. Together, our results establish functional roles for tetrameric Ctp1 in the binding and coordination of DSB repair intermediates, and suggest that disruption of CtIP DNA binding activity by truncating mutations underlies CtIP-linked neurodegenerative Seckel and Jawad syndromes.
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