The ubiquitin ligase RFWD3 is required for translesion DNA synthesis.

Lesions on DNA uncouple DNA synthesis from the replisome, generating stretches of unreplicated single-stranded DNA (ssDNA) behind the replication fork. These ssDNA gaps need to be filled in to complete DNA duplication. Gap-filling synthesis involves either translesion DNA synthesis (TLS) or template switching (TS). Controlling these processes, ubiquitylated PCNA recruits many proteins that dictate pathway choice, but the enzymes regulating PCNA ubiquitylation in vertebrates remain poorly defined. Here we report that the E3 ubiquitin ligase RFWD3 promotes ubiquitylation of proteins on ssDNA. The absence of RFWD3 leads to a profound defect in recruitment of key repair and signaling factors to damaged chromatin. As a result, PCNA ubiquitylation is inhibited without RFWD3, and TLS across different DNA lesions is drastically impaired. We propose that RFWD3 is an essential coordinator of the response to ssDNA gaps, where it promotes ubiquitylation to drive recruitment of effectors of PCNA ubiquitylation and DNA damage bypass.

Rethinking the X- + CH3Y [X = OH, SH, CN, NH2, PH2; Y = F, Cl, Br, I] SN2 reactions.

Moving beyond the textbook mechanisms of bimolecular nucleophilic substitution (SN2) reactions, we characterize several novel stationary points and pathways for the reactions of X- [X = OH, SH, CN, NH2, PH2] nucleophiles with CH3Y [Y = F, Cl, Br, I] molecules using the high-level explicitly-correlated CCSD(T)-F12b method with the aug-cc-pVnZ(-PP) [n = D, T, Q] basis sets. Besides the not-always-existing traditional pre- and post-reaction ion-dipole complexes, X-H3CY and XCH3Y-, and the Walden-inversion transition state, [X-CH3-Y]-, we find hydrogen-bonded X-HCH2Y (X = OH, CN, NH2; Y ≠ F) and front-side H3CYX- (Y ≠ F) complexes in the entrance and hydrogen-bonded XH2CHY- (X = SH, CN, PH2) and H3CXY- (X = OH, SH, NH2) complexes in the exit channels depending on the nucleophile and leaving group as indicated in parentheses. Retention pathways via either a high-energy front-side attack barrier, XYCH3-, or a novel double-inversion transition state, XHCH2Y-, having lower energy for X = OH, CN, and NH2 and becoming submerged (barrier-less) for X = OH and Y = I as well as X = NH2 and Y = Cl, Br, and I, are also investigated.

Chemical Approach to Biological Safety: Molecular-Level Control of an Integrated Zinc Finger Nuclease.

Application of artificial nucleases (ANs) in genome editing is still hindered by their cytotoxicity related to off-target cleavages. This problem can be targeted by regulation of the nuclease domain. Here, we provide an experimental survey of computationally designed integrated zinc finger nucleases, constructed by linking the inactivated catalytic centre and the allosteric activator sequence of the colicin E7 nuclease domain to the two opposite termini of a zinc finger array. DNA specificity and metal binding were confirmed by electrophoretic mobility shift assays, synchrotron radiation circular dichroism spectroscopy, and nano-electrospray ionisation mass spectrometry. In situ intramolecular activation of the nuclease domain was observed, resulting in specific cleavage of DNA with moderate activity. This study represents a new approach to AN design through integrated nucleases consisting of three (regulator, DNA-binding, and nuclease) units, rather than simple chimera. The optimisation of such ANs could lead to safe gene editing enzymes.

Benchmark ab Initio Characterization of the Inversion and Retention Pathways of the OH- + CH3Y [Y = F, Cl, Br, I] SN2 Reactions.

We study the Walden-inversion, front-side attack retention, and double-inversion retention pathways of the OH- + CH3Y [Y = F, Cl, Br, I] SN2 reactions using high-level ab initio methods. Benchmark stationary-point structures and frequencies are computed at the CCSD(T)-F12b/aug-cc-pVTZ level of theory and the best technically feasible relative energies are determined on the basis of CCSD(T)-F12b/aug-cc-pVQZ computations complemented with post-CCSD(T) correlation effects at the CCSDT(Q)/aug-cc-pVDZ level, core correlation corrections at the CCSD(T)/aug-cc-pwCVTZ level, scalar relativistic effects using effective core potentials for Br and I, and zero-point energy corrections at the CCSD(T)-F12b/aug-cc-pVTZ level. Walden inversion proceeds via hydrogen-bonded HO-···HCH2Y (Cl, Br, I) complex → hydrogen-bonded HO-···HCH2Y (Cl, Br, I) transition state → ion-dipole HO-···H3CY (F, Cl, Br) complex → Walden-inversion [HO-CH3-Y]- (F, Cl, Br) transition state → hydrogen-bonded CH3OH···Y- (F, Cl, Br, I) complex, where the Y-dependent existence of the submerged stationary points is indicated in parentheses. Front-side HO-···YCH3 (Cl, Br, I) complexes are also found and HO-···ICH3 is a deeper minimum than HO-···HCH2I. Front-side attacks go over high barriers of 42.8 (F), 28.7 (Cl), 22.4 (Br), and 17.2 (I) kcal/mol, well above the double-inversion barrier heights of 16.7 (F), 3.4 (Cl), 1.1 (Br), and -3.7 (I) kcal/mol.

PARP1-dependent DNA-protein crosslink repair.

DNA-protein crosslinks (DPCs) are toxic lesions that inhibit DNA related processes. Post-translational modifications (PTMs), including SUMOylation and ubiquitylation, play a central role in DPC resolution, but whether other PTMs are also involved remains elusive. Here, we identify a DPC repair pathway orchestrated by poly-ADP-ribosylation (PARylation). Using Xenopus egg extracts, we show that DPCs on single-stranded DNA gaps can be targeted for degradation via a replication-independent mechanism. During this process, DPCs are initially PARylated by PARP1 and subsequently ubiquitylated and degraded by the proteasome. Notably, PARP1-mediated DPC resolution is required for resolving topoisomerase 1-DNA cleavage complexes (TOP1ccs) induced by camptothecin. Using the Flp-nick system, we further reveal that in the absence of PARP1 activity, the TOP1cc-like lesion persists and induces replisome disassembly when encountered by a DNA replication fork. In summary, our work uncovers a PARP1-mediated DPC repair pathway that may underlie the synergistic toxicity between TOP1 poisons and PARP inhibitors.

Profiling ubiquitin signalling with UBIMAX reveals DNA damage- and SCFß-Trcp1-dependent ubiquitylation of the actin-organizing protein Dbn1.

Ubiquitin widely modifies proteins, thereby regulating most cellular functions. The complexity of ubiquitin signalling necessitates unbiased methods enabling global detection of dynamic protein ubiquitylation. Here, we describe UBIMAX (UBiquitin target Identification by Mass spectrometry in Xenopus egg extracts), which enriches ubiquitin-conjugated proteins and quantifies regulation of protein ubiquitylation under precise and adaptable conditions. We benchmark UBIMAX by investigating DNA double-strand break-responsive ubiquitylation events, identifying previously known targets and revealing the actin-organizing protein Dbn1 as a major target of DNA damage-induced ubiquitylation. We find that Dbn1 is targeted for proteasomal degradation by the SCFß-Trcp1 ubiquitin ligase, in a conserved mechanism driven by ATM-mediated phosphorylation of a previously uncharacterized ß-Trcp1 degron containing an SQ motif. We further show that this degron is sufficient to induce DNA damage-dependent protein degradation of a model substrate. Collectively, we demonstrate UBIMAX's ability to identify targets of stimulus-regulated ubiquitylation and reveal an SCFß-Trcp1-mediated ubiquitylation mechanism controlled directly by the apical DNA damage response kinases.