Previous webinars > 2nd webinar 12/07/2021

2nd EDRA Webinar

Dec. 7^th, 2021, from 4.30 to 6 pm CET

Dr Wim Vermeulen - Nucleotide Excision Repair Group, Erasmus Mc, Dept. of Molecular Genetics, Rotterdam, NL

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Ubiquitin-mediated regulation of Nucleotide Excision Repair

DNA damage sensors DDB2 and XPC initiate global genome nucleotide excision repair (NER) to protect DNA from mutagenesis caused by helix-distorting lesions. XPC recognizes helical distortions by binding to unpaired ssDNA opposite DNA lesions. DDB2, as part of a DDB1-Cul4-RBX1 ubiquitin-ligase complex, binds directly to UV-induced lesions directly and facilitates efficient recognition by XPC. We have evidence that not only lesion binding but also timely DDB2 dissociation is required for DNA damage handover to XPC and swift progression of the multistep repair reaction. DNA-binding-induced DDB2 ubiquitylation and subsequent degradation of DDB2 itself to regulate its homeostasis to prevent excessive lesion (re)binding. Additionally, damage handover from DDB2 to XPC coincides with the arrival of the TFIIH complex, which further promotes DDB2 dissociation and the formation of a stable XPC-TFIIH damage verification complex. Our results reveal a reciprocal coordination between DNA damage recognition and verification within NER and illustrate that timely repair factor dissociation is vital for correct spatiotemporal control of a multistep repair process.

Short 15-min talks:

1.Marcin Suskiewicz - Centre de Biophysique Moléculaire, CNRS Orléans, France

Structural and mechanistic insights into PARP1 and PARP2 function and PARP inhibitor response

Poly(ADP-ribose) polymerase 1 (PARP1) and its close paralogue PARP2 are sensors of DNA damage in human cells that become activated upon binding to DNA breaks. Once activated, PARP1 and PARP2 use NAD+ to modify numerous substrates, including themselves and histones, with the post-translational modification called poly(ADP-ribosyl)ation, which then recruits DNA repair factors and facilitates repair by promoting chromatin decompaction. However, as PARP1 is the most abundant nuclear protein after histones with high affinity for DNA breaks, it tends to get trapped on damaged chromatin, inhibiting DNA repair and causing other toxic events, especially in cells with mutations in DNA repair machinery. By both blocking PARP1's and PARP2's positive role in DNA repair and enhancing PARP1 trapping, PARP inhibitors promote cell death and are particularly effective against cancer cells mutated in DNA repair factors BRCA1 or BRCA2. During my post-doctoral work performed in Ivan Ahel's group at Sir William Dunn School of Pathology at Oxford University over the last three years, I have investigated fundamental mechanistic questions regarding the function of PARP1 and PARP2 in DNA repair and inhibitor-induced PARP1 trapping. In this presentation, I will first describe our discovery that both PARP1 and PARP2 are incomplete enzymes that require complementation by an accessory factor, HPF1. We demonstrated with crystal and cryo-EM structures, as well as NMR, biochemical, and cellular studies, how HPF1 completes the PARP active site by providing an additional catalytic base, Glu284, which allows PARP1 and PARP2 to modify their typical physiological targets, protein serine residues. We also showed with Cryo-EM that the PARP2-HPF1 complex is able to bridge two double-strand DNA breaks, presumably for subsequent ligation. Finally, by using cell biology approaches, we demonstrated that PARP1 counteracts its own trapping through a negative feed-back loop, whereby PARP1 activation by DNA breaks leads to its automodification on three main serine residues and subsequent release from chromatin. As this self-regulatory process requires a PARP1-HPF1 complex, HPF1-deficient cells show enhanced PARP1 trapping and are hypersensitive to PARP inhibitors. I will continue my studies of DNA repair-associated post-translational modifications as a CNRS researcher in Bertrand Castaing's laboratory at the Centre de Biophysique Moléculaire, CNRS, UPR 4301, Orléans using French integrative structural biology infrastructure.

2. Maxence Vincent - Oxford University, UK

Cellular heterogeneity in DNA alkylation repair increases population genetic plasticity

DNA repair mechanisms fulfil a dual role, as they are essential for cell survival and genome maintenance. Here, we studied how cells regulate the interplay between DNA repair and mutation. We focused on the adaptive response that increases the resistance of Escherichia coli cells to DNA alkylation damage. Combination of single-molecule imaging and microfluidic-based single-cell microscopy showed that noise in the gene activation timing of the master regulator Ada is accurately propagated to generate a distinct subpopulation of cells in which all proteins of the adaptive response are essentially absent. Whereas genetic deletion of these proteins causes extreme sensitivity to alkylation stress, a temporary lack of expression is tolerated and increases genetic plasticity of the whole population. We demonstrated this by monitoring the dynamics of nascent DNA mismatches during alkylation stress as well as the frequency of fixed mutations that are generated by the distinct subpopulations of the adaptive response. We propose that stochastic modulation of DNA repair capacity by the adaptive response creates a viable hypermutable subpopulation of cells that acts as a source of genetic diversity in a clonal population.