8-Oxoguanine incorporation into DNA repeats in vitro and mismatch recognition by MutSα

DNA 8-oxoguanine (8-oxoG) causes transversions and is also implicated in frameshifts. We previously identified the dNTP pool as a likely source of mutagenic DNA 8-oxoG and demonstrated that DNA mismatch repair prevented oxidation-related frameshifts in mononucleotide repeats. Here, we show that both Klenow fragment and DNA polymerase α can utilize 8-oxodGTP and incorporate the oxidized purine into model frameshift targets. Both polymerases incorporated 8-oxodGMP opposite C and A in repetitive DNA sequences and efficiently extended a terminal 8-oxoG. The human MutSα mismatch repair factor recognized DNA 8-oxoG efficiently in some contexts that resembled frameshift intermediates in the same C or A repeats. DNA 8-oxoG in other slipped/mispaired structures in the same repeats adopted configurations that prevented recognition by MutSα and by the OGG1 DNA glycosylase thereby rendering it invisible to DNA repair. These findings are consistent with a contribution of oxidative DNA damage to frameshifts. They also suggest how mismatch repair might reduce the burden of DNA 8-oxoG and prevent frameshift formation

Phosphorylation by CK2 regulates MUS81/EME1 in mitosis and after replication stress

The MUS81 complex is crucial for preserving genome stability through the resolution of branched DNA intermediates in mitosis. However, untimely activation of the MUS81 complex in S-phase is dangerous. Little is known about the regulation of the human MUS81 complex and how deregulated activation affects chromosome integrity. Here, we show that the CK2 kinase phosphorylates MUS81 at Serine 87 in late-G2/mitosis, and upon mild replication stress. Phosphorylated MUS81 interacts with SLX4, and this association promotes the function of the MUS81 complex. In line with a role in mitosis, phosphorylation at Serine 87 is suppressed in S-phase and is mainly detected in the MUS81 molecules associated with EME1. Loss of CK2-dependent MUS81 phosphorylation contributes modestly to chromosome integrity, however, expression of the phosphomimic form induces DSBs accumulation in S-phase, because of unscheduled targeting of HJ-like DNA intermediates, and generates a wide chromosome instability phenotype. Collectively, our findings describe a novel regulatory mechanism controlling the MUS81 complex function in human cells. Furthermore, they indicate that, genome stability depends mainly on the ability of cells to counteract targeting of branched intermediates by the MUS81/EME1 complex in S-phase, rather than on a correct MUS81 function in mitosis

Toward Supramolecular Nanozymes for the Photocatalytic Activation of Pt(IV) Anticancer Prodrugs

A supramolecular nanozyme for the photocatalytic conversion of a Pt(IV) anticancer complex to cisplatin is described herein. We employed 1.9 nm Au nanoparticles decorated with thiol ligands bearing a TACN (1,4,7-triazacyclononane) headgroup to encapsulate FMN (riboflavin-5'-phosphate). In the presence of an electron donor, flavin-loaded nanoparticles photocatalyzed the reductive activation of the prodrug cis,cis,trans-[Pt(NH3)(2)(Cl-2)(O2CCH2CH2COOH)(2)] to cisplatin, achieving turnover frequency values of 7.4 min(-1).We acknowledge financial support from the Spanish State Research Agency for the grants CTQ2016-80844-R, BIO201677367-R, PID2019-111649RB-I00 and PCI2018-092984. This work was performed under the Severo Ochoa Centres of Excellence and Maria de Maeztu Units of Excellence Program from the Spanish State Research Agency -Grant No. CEX2018-000867-S (DIPC) and MDM-2017-0720 (CIC biomaGUNE). We also thank Fondazione Cariparo (Ricerca Scientifica di Eccellenza Grant "SELECT''). A. M. has received funding from the European Union's Horizon 2020 Research and Innovation Programme under Marie SklodowskaCurie Actions grant no. 793702. We thank the Spanish MultiMetDrugs network (RED2018-102471-T) for fruitful discussion

Loss of MUTYH function in human cells leads to accumulation of oxidative damage and genetic instability

The DNA glycosylase MUTYH (mutY homolog (Escherichia coli)) counteracts the mutagenic effects of 8-oxo-7,8-dihydroguanine (8-oxodG) by removing adenine (A) misincorporated opposite the oxidized purine. Biallelic germline mutations in MUTYH cause the autosomal recessive MUTYH-associated adenomatous polyposis (MAP). Here we designed new tools to investigate the biochemical defects and biological consequences associated with different MUTYH mutations in human cells. To identify phenotype(s) associated with MUTYH mutations, lymphoblastoid cell lines (LCLs) were derived from seven MAP patients harboring missense as well as truncating mutations in MUTYH. These included homozygous p.Arg245His, p.Gly264TrpfsX7 or compound heterozygous variants (p.Gly396Asp/Arg245Cys, p.Gly396Asp/Tyr179Cys, p.Gly396Asp/Glu410GlyfsX43, p.Gly264TrpfsX7/Ala385ProfsX23 and p.Gly264TrpfsX7/Glu480del). DNA glycosylase assays of MAP LCL extracts confirmed that all these variants were defective in removing A from an 8-oxoG:A DNA substrate, but retained wild-type OGG1 activity. As a consequence of this defect, MAP LCLs accumulated DNA 8-oxodG in their genome and exhibited a fourfold increase in spontaneous mutagenesis at the PIG-A gene compared with LCLs from healthy donors. They were also hypermutable by KBrO3--a source of DNA 8-oxodG--indicating that the relatively modest spontaneous mutator phenotype associated with MUTYH loss can be significantly enhanced by conditions of oxidative stress. These observations identify accumulation of DNA 8-oxodG and a mutator phenotype as likely contributors to the pathogenesis of MUTYH variants

Engineering hybrid nanostructures for the photocatalytic activation of Pt(IV) prodrugs

242 p.In this thesis I studied the photocatalytic activation of platinum anticancer agents under blue lightirradiations. This reaction was promoted by the use of flavin molecules as catalyst. In the first part of thethesis, the reaction procedures for the functionalised riboflavin and the platinum complexes employed forthe studies of this thesis, are reported. Then, the photocatalytic activation of Pt(IV) prodrugs was firstinvestigated in a nanozyme model. I used interacting gold nanoparticles as scaffold for the system. Then,I moved the investigation of the photoactivation of the platinum complexes, employing in this caseengineered repeat proteins as templates. The use of protein-based hybrid was indeed adopted forcontrolling the delivery and the photoactivation of platinum prodrugs, mediated by riboflavin, in vitro.CICbiomaGUNE; DIP

Sequence-dependent DNA torsional rigidity: a tetranucleotide code

Using fluorescence polarization anisotropy (FPA), we measured the torsional constant of various DNA oligomers in different sequences and calculated the value for each of the 136 unique tetranucleotides. From these values, we obtained a "rigidity profile" for every double stranded DNA sequence. We tested the code in the analysis of DNA sequences able to form nucleosomes. More than 50% of the sequences studied showed a common 20 and/or 30 bp modulation of the torsional constant. Many other profiles of rigidity were observed in the remaining sequences and this variety in torsional constant modulation may be related to functional differences between nucleosomes. (C) 2004 Elsevier B.V. All rights reserved