Tyrosyl-DNA phosphodiesterase 1 (TDP1) and SPRTN protease repair histone 3 and topoisomerase 1 DNA-protein crosslinks in vivo.

DNA-protein crosslinks (DPCs) are frequent and damaging DNA lesions that affect all DNA transactions, which in turn can lead to the formation of double-strand breaks, genomic instability and cell death. At the organismal level, impaired DPC repair (DPCR) is associated with cancer, ageing and neurodegeneration. Despite the severe consequences of DPCs, little is known about the processes underlying repair pathways at the organism level. SPRTN is a protease that removes most cellular DPCs during replication, whereas tyrosyl-DNA phosphodiesterase 1 repairs one of the most abundant enzymatic DPCs, topoisomerase 1-DPC (TOP1-DPC). How these two enzymes repair DPCs at the organism level is currently unknown. We perform phylogenetic, syntenic, structural and expression analysis to compare tyrosyl-DNA phosphodiesterase 1 (TDP1) orthologues between human, mouse and zebrafish. Using the zebrafish animal model and human cells, we demonstrate that TDP1 and SPRTN repair endogenous, camptothecin- and formaldehyde-induced DPCs, including histone H3- and TOP1-DPCs. We show that resolution of H3-DNA crosslinks depends on upstream proteolysis by SPRTN and subsequent peptide removal by TDP1 in RPE1 cells and zebrafish embryos, whereas SPRTN and TDP1 function in different pathways in the repair of endogenous TOP1-DPCs and total DPCs. Furthermore, we have found increased TDP2 expression in TDP1-deficient cells and embryos. Understanding the role of TDP1 in DPCR at the cellular and organismal levels could provide an impetus for the development of new drugs and combination therapies with TOP1-DPC inducing drugs.

Importance of protein intrinsic conformational dynamics and transient nature of non-covalent interactions in ligand binding affinity.

We have recently identified BEN1 as a protein interactor of seryl-tRNA synthetase (SerRS) from model plant Arabidopsis thaliana. BEN1 contains an NADP+ binding domain and possesses acidic N-terminal extension essential for interaction with A. thaliana SerRS. This extension, specific for BEN1 homologues from Brassicaceae family, is solvent-exposed and distant to the nucleotide-binding site. We prepared a truncated BEN1 variant ΔN17BEN1 lacking the first 17 amino acid of this N-terminal extension as well as full-length BEN1 to investigate how the truncation affects the binding affinity towards coenzyme NADP+. By performing microscale thermophoresis (MST) experiments we have shown that both BEN1 variants bind the NADP+ cofactor, however, truncated BEN1 showed 34-fold higher affinity towards NADP+ indicating that its core protein structure is not just preserved but it binds NADP+ even stronger. To further corroborate the obtained results, we opted for a computational approach based on classical molecular dynamics simulations of both complexes. Our results have shown that both truncated and intact BEN1 variants form the same number of interactions with the NADP+ cofactor; however, it was the interaction occupancy that was affected. Namely, three independent MD simulations showed that the ΔN17BEN1 variant in complex with NADP+ has significantly higher interaction occupancy thus binds NADP+ with more than one order of magnitude higher affinity. Contrary to our expectations, the truncation of this distant region that does not communicate with the nucleotide-binding site didn't result in the gain of interaction but affected the intrinsic conformational dynamics which in turn fine-tuned the binding affinity by increasing the interaction occupancy and strength of the key conserved cation-π interaction between Arg69 and adenine of NADP+ and hydrogen bond between Ser244 and phosphate of NADP+.