The ASCC2 CUE domain in the ALKBH3-ASCC DNA repair complex recognizes adjacent ubiquitins in K63-linked polyubiquitin

Alkylation of DNA and RNA is a potentially toxic lesion that can result in mutations and even cell death. In response to alkylation damage, K63-linked polyubiquitin chains are assembled that localize the Alpha-ketoglutarate-dependent dioxygenase alkB homolog 3-Activating Signal Cointegrator 1 Complex Subunit (ASCC) repair complex to damage sites in the nucleus. The protein ASCC2, a subunit of the ASCC complex, selectively binds K63-linked polyubiquitin chains via its coupling of ubiquitin conjugation to ER degradation (CUE) domain. The basis for polyubiquitin-binding specificity was unclear, because CUE domains in other proteins typically bind a single ubiquitin and do not discriminate among different polyubiquitin linkage types. We report here that the ASCC2 CUE domain selectively binds K63-linked diubiquitin by contacting both the distal and proximal ubiquitin. The ASCC2 CUE domain binds the distal ubiquitin in a manner similar to that reported for other CUE domains bound to a single ubiquitin, whereas the contacts with the proximal ubiquitin are unique to ASCC2. Residues in the N-terminal portion of the ASCC2 α1 helix contribute to the binding interaction with the proximal ubiquitin of K63-linked diubiquitin. Mutation of residues within the N-terminal portion of the ASCC2 α1 helix decreases ASCC2 recruitment in response to DNA alkylation, supporting the functional significance of these interactions during the alkylation damage response. Our study reveals the versatility of CUE domains in ubiquitin recognition

Polθ promotes the repair of 5\u27-DNA-protein crosslinks by microhomology-mediated end-joining

DNA polymerase θ (Polθ) confers resistance to chemotherapy agents that cause DNA-protein crosslinks (DPCs) at double-strand breaks (DSBs), such as topoisomerase inhibitors. This suggests Polθ might facilitate DPC repair by microhomology-mediated end-joining (MMEJ). Here, we investigate Polθ repair of DSBs carrying DPCs by monitoring MMEJ in Xenopus egg extracts. MMEJ in extracts is dependent on Polθ, exhibits the MMEJ repair signature, and efficiently repairs 5\u27 terminal DPCs independently of non-homologous end-joining and the replisome. We demonstrate that Polθ promotes the repair of 5\u27 terminal DPCs in mammalian cells by using an MMEJ reporter and find that Polθ confers resistance to formaldehyde in addition to topoisomerase inhibitors. Dual deficiency in Polθ and tyrosyl-DNA phosphodiesterase 2 (TDP2) causes severe cellular sensitivity to etoposide, which demonstrates MMEJ as an independent DPC repair pathway. These studies recapitulate MMEJ in vitro and elucidate how Polθ confers resistance to etoposide

Molecular basis of microhomology-mediated end-joining by purified full-length Polθ

DNA polymerase θ (Polθ) is a unique polymerase-helicase fusion protein that promotes microhomology-mediated end-joining (MMEJ) of DNA double-strand breaks (DSBs). How full-length human Polθ performs MMEJ at the molecular level remains unknown. Using a biochemical approach, we find that the helicase is essential for Polθ MMEJ of long ssDNA overhangs which model resected DSBs. Remarkably, Polθ MMEJ of ssDNA overhangs requires polymerase-helicase attachment, but not the disordered central domain, and occurs independently of helicase ATPase activity. Using single-particle microscopy and biophysical methods, we find that polymerase-helicase attachment promotes multimeric gel-like Polθ complexes that facilitate DNA accumulation, DNA synapsis, and MMEJ. We further find that the central domain regulates Polθ multimerization and governs its DNA substrate requirements for MMEJ. These studies identify unexpected functions for the helicase and central domain and demonstrate the importance of polymerase-helicase tethering in MMEJ and the structural organization of Polθ

Polθ reverse transcribes RNA and promotes RNA-templated DNA repair

Genome-embedded ribonucleotides arrest replicative DNA polymerases (Pols) and cause DNA breaks. Whether mammalian DNA repair Pols efficiently use template ribonucleotides and promote RNA-templated DNA repair synthesis remains unknown. We find that human Polθ reverse transcribes RNA, similar to retroviral reverse transcriptases (RTs). Polθ exhibits a significantly higher velocity and fidelity of deoxyribonucleotide incorporation on RNA versus DNA. The 3.2-Å crystal structure of Polθ on a DNA/RNA primer-template with bound deoxyribonucleotide reveals that the enzyme undergoes a major structural transformation within the thumb subdomain to accommodate A-form DNA/RNA and forms multiple hydrogen bonds with template ribose 2\u27-hydroxyl groups like retroviral RTs. Last, we find that Polθ promotes RNA-templated DNA repair in mammalian cells. These findings suggest that Polθ was selected to accommodate template ribonucleotides during DNA repair

Publisher Correction: Molecular basis of microhomology-mediated end-joining by purified full-length Polθ (Nature Communications, (2019), 10, 1, (4423), 10.1038/s41467-019-12272-9)

The original version of this Article contained errors in Figure 6. In panel o, the labels incorrectly stated ‘Poleθ’ and “Poleθ + DNA” and should be labelled “Polθ” and “Polθ + DNA”. In the result section, in the sub-section entitled “Polθ Promotes MMEJ of Long ssDNA”, the sentence “Importantly, the ability of Polθ- pol to perform MMEJ on short (≤12 nt) ssDNA (Fig. 1p, left; Supplementary Fig. 3D and 3E), and short (≤15 nt) overhangs, demonstrates it performs interstrand pairing without Polθ-hel”. should read as follow: “Importantly, the ability of Polθ-pol to perform MMEJ on short (≤12 nt) ssDNA (Fig. 1p, left; Supplementary Fig. 3D and 3E), and short (≤15 nt) overhangs, demonstrates that it performs interstrand pairing without Polθ-hel”. In the sub-section entitled “Preventing Intrastrand Pairing Stimulates MMEJ by Polθ-Pol”, the sentence “We predicted that preventing base-pairing opportunities between 3' terminal bases and bases upstream along long the 5' region of long ssDNA substrates would suppress intrastrand pairing and enable interstrand pairing by Polθ-pol (Fig. 3c)”. should read as follows: “We predicted that preventing base-pairing opportunities between 3' terminal bases and bases upstream along the 5' region of long ssDNA substrates would suppress intrastrand pairing and enable interstrand pairing by Polθ-pol (Fig. 3c)”. In the method section, in the “Proteins” sub-section the sentence “Polθ-pol, Polθ-hel and RPA were purified as described”. should read as follows: “Polθ-pol and Polθ-hel were purified as described”. These corrections have now been included in the HTML and pdf of the article. Additionally, a technical problem during the publication process resulted in loss of image quality in Figs. 1, 3 and 4. This has now been corrected in both the PDF and HTML versions of the Article

Midrapidity antiproton-to-proton ratio in pp collisons root s=0.9 and 7 TeV measured by the ALICE experiment

The ratio of the yields of antiprotons to protons in pp collisions has been measured by the ALICE experiment at root s = 0.9 and 7 TeV during the initial running periods of the Large Hadron Collider. The measurement covers the transverse momentum interval 0.45 < p(t) < 1.05 GeV/c and rapidity vertical bar y vertical bar < 0.5. The ratio is measured to be R-vertical bar y vertical bar<0.5 = 0.957 +/- 0.006(stat) +/- 0.0014(syst) at 0.9 Tev and R-vertical bar y vertical bar<0.5 = 0.991 +/- 0.005 +/- 0.014(syst) at 7 TeV and it is independent of both rapidity and transverse momentum. The results are consistent with the conventional model of baryon-number transport and set stringent limits on any additional contributions to baryon-number transfer over very large rapidity intervals in pp collisions

Centrality dependence of the charged-particle multiplicity density at mid-rapidity in Pb-Pb collisions at sNN\sqrt{s_{NN}} = 2.76 TeV

The centrality dependence of the charged-particle multiplicity density at mid-rapidity in Pb-Pb collisions at $\sqrt{s_{NN}}$ = 2.76 TeV is presented. The charged-particle density normalized per participating nucleon pair increases by about a factor 2 from peripheral (70-80%) to central (0-5%) collisions. The centrality dependence is found to be similar to that observed at lower collision energies. The data are compared with models based on different mechanisms for particle production in nuclear collisions.The centrality dependence of the charged-particle multiplicity density at mid-rapidity in Pb-Pb collisions at $\sqrt{s_{\rm NN}}$ = 2.76 TeV is presented. The charged-particle density normalized per participating nucleon pair increases by about a factor 2 from peripheral (70-80%) to central (0-5%) collisions. The centrality dependence is found to be similar to that observed at lower collision energies. The data are compared with models based on different mechanisms for particle production in nuclear collisions