In:
Cancer Research, American Association for Cancer Research (AACR), Vol. 72, No. 8_Supplement ( 2012-04-15), p. 5678-5678
Abstract:
Tyrosyl-DNA phosphodiesterase I (Tdp1) is a conserved eukaryotic DNA repair enzyme involved in repair of 3′-DNA adducts, such as the 3′-phosphotyrosyl bond formed between DNA topoisomerase I (Top1) and DNA which is reversibly stabilized by camptothecins (CPTs), like the FDA approved analogs topotecan and irinotecan. Tdp1 is a member of the phospholipase D superfamily and contains paired catalytic histidine and lysine residues within two conserved HxK(x)4D motifs. Tdp1's two catalytic histidines function as a nucleophile (N-terminal His182) and as a general acid/base (C-terminal His432). Substitution of the latter in human Tdp1 to Arg (hTdp1His493Arg) contributes to the rare recessive neurodegenerative disease SCAN1. The hTdp1His493Arg mutant and its yeast analog (Tdp1His432Arg) increase cell sensitivity to CPTs, whereas mutation of this His to Asn produces a Top1-CPT-dependent lethality. Interestingly, substitution of His432 to Lys produces wild type-like biochemical and in cell activity. Our recent crystal structures revealed that Tdp1 and Tdp1His432Arg stabilize the phospho-His182, which is not observed in the Tdp1His432Asn structure. In vitro analysis showed that Tdp1His432Arg and Tdp1His432Lys but not Tdp1 or Tdp1His432Asn, formed a non-covalent interaction with the DNA while only the Tdp1His432Arg mutant retained a covalent protein-DNA bond. However, a band depletion assay showed that soluble Tdp1His432Asn and Top1 are depleted, suggesting Tdp1His432Asn remains in complex with Top1 on the DNA. The difference between our in vivo and in vitro observations could be the differences between substrates; full length Top1-genomic DNA complex vs. Tyr-oligonucleotide complex. While Tdp1His182Ala is reported to be biochemically inactive, introduction of His182Ala in Tdp1His432Asn could not suppress the observed lethality. Yet, Tdp1His182AlaHis432Asn gained a Top1-independent toxic phenotype. Substitution of His182 to Phe suppresses the His432Asn toxicity. Preliminary results revealed Tdp1His182Ala activity in vitro, leading us to posit that substitution of His182 to Ala would allow the adjacent conserved His181 to rotate into the active site and act as a nucleophile to resolve the Top1-DNA intermediate. Consistent with this model, mutating His181 to Ala suppressed the Top1-CPT dependent lethality of the H182A and H432N single and double mutants. However, the structure of the apo H182A mutants revealed His181 in its wild type position, and showed that Ala at position 182 introduces the necessary space for such rotation. We posit that this rotation only occurs infrequently and upon binding of the substrate, which we will investigate via NMR. Overall, our findings give insight into the catalytic mechanism and suggest that protein-protein interactions modulate Tdp1 catalytic activity, which supports Tdp1 as a novel drug target. Supported by the Alabama Drug Discovery Alliance. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5678. doi:1538-7445.AM2012-5678
Type of Medium:
Online Resource
ISSN:
0008-5472
,
1538-7445
DOI:
10.1158/1538-7445.AM2012-5678
Language:
English
Publisher:
American Association for Cancer Research (AACR)
Publication Date:
2012
detail.hit.zdb_id:
2036785-5
detail.hit.zdb_id:
1432-1
detail.hit.zdb_id:
410466-3
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