PAXX and its paralogues synergistically direct DNA polymerase λ activity in DNA repair

PAXX is a recently identified component of the nonhomologous end joining (NHEJ) DNA repair pathway. The molecular mechanisms of PAXX action remain largely unclear. Here we characterise the interactomes of PAXX and its paralogs, XLF and XRCC4, to show that these factors share the ability to interact with DNA polymerase λ (Pol λ), stimulate its activity and are required for recruitment of Pol λ to laser-induced DNA damage sites. Stimulation of Pol λ activity by XRCC4 paralogs requires a direct interaction between the SP/8 kDa domain of Pol λ and their N-terminal head domains to facilitate recognition of the 5′ end of substrate gaps. Furthermore, PAXX and XLF collaborate with Pol λ to promote joining of incompatible DNA ends and are redundant in supporting Pol λ function in vivo. Our findings identify Pol λ as a novel downstream effector of PAXX function and show XRCC4 paralogs act in synergy to regulate polymerase activity in NHEJ.This work was supported by the Medical Research Council (MRC) UK

NR4A Nuclear Receptors Target Poly-ADP-Ribosylated DNA-PKcs Protein to Promote DNA Repair.

Although poly-ADP-ribosylation (PARylation) of DNA repair factors had been well documented, its role in the repair of DNA double-strand breaks (DSBs) is poorly understood. NR4A nuclear orphan receptors were previously linked to DSB repair; however, their function in the process remains elusive. Classically, NR4As function as transcription factors using a specialized tandem zinc-finger DNA-binding domain (DBD) for target gene induction. Here, we show that NR4A DBD is bi-functional and can bind poly-ADP-ribose (PAR) through a pocket localized in the second zinc finger. Separation-of-function mutants demonstrate that NR4A PAR binding, while dispensable for transcriptional activity, facilitates repair of radiation-induced DNA double-strand breaks in G1. Moreover, we define DNA-PKcs protein as a prominent target of ionizing radiation-induced PARylation. Mechanistically, NR4As function by directly targeting poly-ADP-ribosylated DNA-PKcs to facilitate its autophosphorylation-promoting DNA-PK kinase assembly at DNA lesions. Selective targeting of the PAR-binding pocket of NR4A presents an opportunity for cancer therapy

Dynamic self-assembly of DNA minor groove-binding ligand DB921 into nanotubes triggered by an alkali halide.

We describe a novel self-assembling supramolecular nanotube system formed by a heterocyclic cationic molecule which was originally designed for its potential as an antiparasitic and DNA sequence recognition agent. Our structural characterisation work indicates that the nanotubes form via a hierarchical assembly mechanism that can be triggered and tuned by well-defined concentrations of simple alkali halide salts in water. The nanotubes assembled in NaCl have inner and outer diameters of ca. 22 nm and 26 nm respectively, with lengths that reach into several microns. Our results suggest the tubes consist of DB921 molecules stacked along the direction of the nanotube long axis. The tubes are stabilised by face-to-face π-π stacking and ionic interactions between the charged amidinium groups of the ligand and the negative halide ions. The assembly process of the nanotubes was followed using small-angle X-ray and neutron scattering, transmission electron microscopy and ultraviolet/visible spectroscopy. Our data demonstrate that assembly occurs through the formation of intermediate ribbon-like structures that in turn form helices that tighten and compact to form the final stable filament. This assembly process was tested using different alkali-metal salts, showing a strong preference for chloride or bromide anions and with little dependency on the type of cation. Our data further demonstrates the existence of a critical anion concentration above which the rate of self-assembly is greatly enhanced

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Pre-validation of the WHO organ dysfunction based criteria for identification of maternal near miss

<p>Abstract</p> <p>Background</p> <p>To evaluate the performance of the WHO criteria for defining maternal near miss and identifying deaths among cases of severe maternal morbidity (SMM) admitted for intensive care.</p> <p>Method</p> <p>Between October 2002 and September 2007, 673 women with SMM were admitted, and among them 18 died. Variables used for the definition of maternal near miss according to WHO criteria and for the SOFA score were retrospectively evaluated. The identification of at least one of the WHO criteria in women who did not die defined the case as a near miss. Organ failure was evaluated through the maximum SOFA score above 2 for each one of the six components of the score, being considered the gold standard for the diagnosis of maternal near miss. The aggregated score (Total Maximum SOFA score) was calculated using the worst result of the maximum SOFA score. Sensitivity, specificity, positive and negative predictive values of these WHO criteria for predicting maternal death and also for identifying cases of organ failure were estimated.</p> <p>Results</p> <p>The WHO criteria identified 194 cases of maternal near miss and all the 18 deaths. The most prevalent criteria among cases of maternal deaths were the use of vasoactive drug and the use of mechanical ventilation (≥1 h). For the prediction of maternal deaths, sensitivity was 100% and specificity 70.4%. These criteria identified 119 of the 120 cases of organ failure by the maximum SOFA score (Sensitivity 99.2%) among 194 case of maternal near miss (61.34%). There was disagreement in 76 cases, one organ failure without any WHO criteria and 75 cases with no failure but with WHO criteria. The Total Maximum SOFA score had a good performance (area under the curve of 0.897) for prediction of cases of maternal near miss according to the WHO criteria.</p> <p>Conclusions</p> <p>The WHO criteria for maternal near miss showed to be able to identify all cases of death and almost all cases of organ failure. Therefore they allow evaluation of the severity of the complication and consequently enable clinicians to build a plan of care or to provide an early transfer for appropriate reference centers.</p

PARP14 is a PARP with both ADP-ribosyl transferase and hydrolase activities

This is the final version. Available on open access from the American Association for the Advancement of Science via the DOI in this recordData availability: The MS proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE (92) partner repository with the dataset identifier PXD043452.PARP14 is a mono-ADP-ribosyl transferase involved in the control of immunity, transcription, and DNA replication stress management. However, little is known about the ADP-ribosylation activity of PARP14, including its substrate specificity or how PARP14-dependent ADP-ribosylation is reversed. We show that PARP14 is a dual-function enzyme with both ADP-ribosyl transferase and hydrolase activity acting on both protein and nucleic acid substrates. In particular, we show that the PARP14 macrodomain 1 is an active ADP-ribosyl hydrolase. We also demonstrate hydrolytic activity for the first macrodomain of PARP9. We reveal that expression of a PARP14 mutant with the inactivated macrodomain 1 results in a marked increase in mono(ADP-ribosyl)ation of proteins in human cells, including PARP14 itself and antiviral PARP13, and displays specific cellular phenotypes. Moreover, we demonstrate that the closely related hydrolytically active macrodomain of SARS2 Nsp3, Mac1, efficiently reverses PARP14 ADP-ribosylation in vitro and in cells, supporting the evolution of viral macrodomains to counteract PARP14-mediated antiviral response.Biotechnology and Biological Sciences Research Council (BBSRC)Wellcome TrustOxford University Challenge Seed FundEdward Penley Abraham Research FundOvarian Cancer Research AllianceResearch Council of NorwaySwedish Research CouncilMedical Research Council (MRC)Novo Nordisk Foundation Center for Protein ResearchDanish Council of Independent ResearchDanish Cancer Societ