Checkpoint Kinase ATR Promotes Nucleotide Excision Repair of UV-Induced DNA Damage via Physical Interaction Withxeroderma Pigmentosum Group A

In response to DNA damage, eukaryotic cells activate a series of DNA damage-dependent pathways that serve to arrest cell cycle progression and remove DNA damage. Coordination of cell cycle arrest and damage repair is critical for maintenance of genomic stability. However, this process is still poorly understood. Nucleotide excision repair (NER) and the ATR-dependent cell cycle checkpoint are the major pathways responsible for repair of UV-induced DNA damage. Here we show that ATR physically interacts with the NER factor Xeroderma pigmentosum group A (XPA). Using a mass spectrometry-based protein footprinting method, we found that ATR interacts with a helixturn-helix motif in the minimal DNA-binding domain of XPA where anATRphosphorylation site (serine 196) is located.XPAdeficient cells complemented with XPA containing a point mutation of S196A displayed a reduced repair efficiency of cyclobutane pyrimidine dimers as compared with cells complemented with wild-type XPA, although no effect was observed for repair of (6-4) photoproducts. This suggests that the ATR-dependent phosphorylation of XPA may promote NER repair of persistentDNAdamage. In addition, a K188A point mutation of XPA that disrupts the ATR-XPA interaction inhibits the nuclear import of XPA after UV irradiation and, thus, significantly reduced DNA repair efficiency. By contrast, the S196A mutation has no effect on XPA nuclear translocation. Taken together, our results suggest that the ATR-XPA interaction mediated by the helix-turn-helix motif of XPA plays an important role in DNA-damage responses to promote cell survival and genomic stability after UV irradiation

The binary near-Earth asteroid (175706) 1996 FG3 - An observational constraint on its orbital evolution

Using our photometric observations taken between 1996 and 2013 and other published data, we derived properties of the binary near-Earth asteroid (175706) 1996 FG3 including new measurements constraining evolution of the mutual orbit with potential consequences for the entire binary asteroid population. We also refined previously determined values of parameters of both components, making 1996 FG3 one of the most well understood binary asteroid systems. We determined the orbital vector with a substantially greater accuracy than before and we also placed constraints on a stability of the orbit. Specifically, the ecliptic longitude and latitude of the orbital pole are 266{\deg} and -83{\deg}, respectively, with the mean radius of the uncertainty area of 4{\deg}, and the orbital period is 16.1508 +/- 0.0002 h (all quoted uncertainties correspond to 3sigma). We looked for a quadratic drift of the mean anomaly of the satellite and obtained a value of 0.04 +/- 0.20 deg/yr^2, i.e., consistent with zero. The drift is substantially lower than predicted by the pure binary YORP (BYORP) theory of McMahon and Scheeres (McMahon, J., Scheeres, D. [2010]. Icarus 209, 494-509) and it is consistent with the theory of an equilibrium between BYORP and tidal torques for synchronous binary asteroids as proposed by Jacobson and Scheeres (Jacobson, S.A., Scheeres, D. [2011]. ApJ Letters, 736, L19). Based on the assumption of equilibrium, we derived a ratio of the quality factor and tidal Love number of Q/k = 2.4 x 10^5 uncertain by a factor of five. We also derived a product of the rigidity and quality factor of mu Q = 1.3 x 10^7 Pa using the theory that assumes an elastic response of the asteroid material to the tidal forces. This very low value indicates that the primary of 1996 FG3 is a 'rubble pile', and it also calls for a re-thinking of the tidal energy dissipation in close asteroid binary systems.Comment: Many changes based on referees comment

YORP and Yarkovsky effects in asteroids (1685) Toro, (2100) Ra-Shalom, (3103) Eger, and (161989) Cacus

The rotation states of small asteroids are affected by a net torque arising from an anisotropic sunlight reflection and thermal radiation from the asteroids' surfaces. On long timescales, this so-called YORP effect can change asteroid spin directions and their rotation periods. We analyzed lightcurves of four selected near-Earth asteroids with the aim of detecting secular changes in their rotation rates that are caused by YORP. We use the lightcurve inversion method to model the observed lightcurves and include the change in the rotation rate $\mathrm{d} \omega / \mathrm{d} t$ as a free parameter of optimization. We collected more than 70 new lightcurves. For asteroids Toro and Cacus, we used thermal infrared data from the WISE spacecraft and estimated their size and thermal inertia. We also used the currently available optical and radar astrometry of Toro, Ra-Shalom, and Cacus to infer the Yarkovsky effect. We detected a YORP acceleration of $\mathrm{d}\omega / \mathrm{d} t = (1.9 \pm 0.3) \times 10^{-8}\,\mathrm{rad}\,\mathrm{d}^{-2}$ for asteroid Cacus. For Toro, we have a tentative ($2\sigma$) detection of YORP from a significant improvement of the lightcurve fit for a nonzero value of $\mathrm{d}\omega / \mathrm{d} t = 3.0 \times 10^{-9}\,\mathrm{rad}\,\mathrm{d}^{-2}$. For asteroid Eger, we confirmed the previously published YORP detection with more data and updated the YORP value to $(1.1 \pm 0.5) \times 10^{-8}\,\mathrm{rad}\,\mathrm{d}^{-2}$. We also updated the shape model of asteroid Ra-Shalom and put an upper limit for the change of the rotation rate to $|\mathrm{d}\omega / \mathrm{d} t| \lesssim 1.5 \times 10^{-8}\,\mathrm{rad}\,\mathrm{d}^{-2}$. Ra-Shalom has a greater than $3\sigma$ Yarkovsky detection with a theoretical value consistent with observations assuming its size and/or density is slightly larger than the nominally expected values

ATR Prevents Ca\u3csup\u3e2+\u3c/sup\u3e Overload-Induced Necrotic Cell Death Through Phosphorylation-Mediated Inactivation of PARP1 Without DNA Damage Signaling

Hyperactivation of PARP1 is known to be a major cause of necrotic cell death by depleting NAD+/ATP pools during Ca2+ overload which is associated with many ischemic diseases. However, little is known about how PARP1 hyperactivity is regulated during calcium overload. In this study we show that ATR kinase, well known for its role in DNA damage responses, suppresses ionomycin, glutamate, or quinolinic acid-induced necrotic death of cells including SH-SY5Y neuronal cells. We found that the inhibition of necrosis requires the kinase activity of ATR. Specifically, ATR binds to and phosphorylates PARP1 at Ser179 after the ionophore treatments. This site-specific phosphorylation inactivates PARP1, inhibiting ionophore-induced necrosis. Strikingly, all of this occurs in the absence of detectable DNA damage and signaling up to 8 hours after ionophore treatment. Furthermore, little AIF was released from mitochondria/cytoplasm for nuclear import, supporting the necrotic type of cell death in the early period of the treatments. Our results reveal a novel ATR-mediated anti-necrotic mechanism in the cellular stress response to calcium influx without DNA damage signaling

Allosteric HIV-1 integrase inhibitors lead to premature degradation of the viral RNA genome and integrase in target cells

Recent evidence indicates that inhibition of HIV-1 integrase (IN) binding to the viral RNA genome by allosteric integrase inhibitors (ALLINIs) or through mutations within IN yields aberrant particles in which the viral ribonucleoprotein complexes (vRNPs) are eccentrically localized outside the capsid lattice. These particles are noninfectious and are blocked at an early reverse transcription stage in target cells. However, the basis of this reverse transcription defect is unknown. Here, we show that the viral RNA genome and IN from ALLINI-treated virions are prematurely degraded in target cells, whereas reverse transcriptase remains active and stably associated with the capsid lattice. The aberrantly shaped cores in ALLINI-treated particles can efficiently saturate and be degraded by a restricting TRIM5 protein, indicating that they are still composed of capsid proteins arranged in a hexagonal lattice. Notably, the fates of viral core components follow a similar pattern in cells infected with eccentric particles generated by mutations within IN that inhibit its binding to the viral RNA genome. We propose that IN-RNA interactions allow packaging of both the viral RNA genome and IN within the protective capsid lattice to ensure subsequent reverse transcription and productive infection in target cells. Conversely, disruption of these interactions by ALLINIs or mutations in IN leads to premature degradation of both the viral RNA genome and IN, as well as the spatial separation of reverse transcriptase from the viral genome during early steps of infection. © 2017 American Society for Microbiology

Altering Murine Leukemia Virus Integration Through Disruption of the Integrase and BET Protein Family Interaction

We report alterations to the murine leukemia virus (MLV) integrase (IN) protein that successfully result in decreasing its integration frequency at transcription start sites and CpG islands, thereby reducing the potential for insertional activation. The host bromo and extraterminal (BET) proteins Brd2, 3 and 4 interact with the MLV IN protein primarily through the BET protein ET domain. Using solution NMR, protein interaction studies, and next generation sequencing, we showthat the C-terminal tail peptide region ofMLV IN is important for the interaction with BET proteins and that disruption of this interaction through truncation mutations affects the global targeting profile of MLV vectors. The use of the unstructured tails of gammaretroviral INs to direct association with complexes at active promoters parallels that used by histones and RNA polymerase II. Viruses bearingMLV IN C-terminal truncations can provide new avenues to improve the safety profile of gammaretroviral vectors for human gene therapy

Biochemical Characterization of a Structure-Specific Resolving Enzyme from Sulfolobus islandicus Rod-Shaped Virus 2

Sulfolobus islandicus rod shaped virus 2 (SIRV2) infects the archaeon Sulfolobus islandicus at extreme temperature (70°C–80°C) and acidity (pH 3). SIRV2 encodes a Holliday junction resolving enzyme (SIRV2 Hjr) that has been proposed as a key enzyme in SIRV2 genome replication. The molecular mechanism for SIRV2 Hjr four-way junction cleavage bias, minimal requirements for four-way junction cleavage, and substrate specificity were determined. SIRV2 Hjr cleaves four-way DNA junctions with a preference for cleavage of exchange strand pairs, in contrast to host-derived resolving enzymes, suggesting fundamental differences in substrate recognition and cleavage among closely related Sulfolobus resolving enzymes. Unlike other viral resolving enzymes, such as T4 endonuclease VII or T7 endonuclease I, that cleave branched DNA replication intermediates, SIRV2 Hjr cleavage is specific to four-way DNA junctions and inactive on other branched DNA molecules. In addition, a specific interaction was detected between SIRV2 Hjr and the SIRV2 virion body coat protein (SIRV2gp26). Based on this observation, a model is proposed linking SIRV2 Hjr genome resolution to viral particle assembly

The mechanism of H171T resistance reveals the importance of Nδ-protonated His171 for the binding of allosteric inhibitor BI-D to HIV-1 integrase

Background: Allosteric HIV-1 integrase (IN) inhibitors (ALLINIs) are an important new class of anti-HIV-1 agents. ALLINIs bind at the IN catalytic core domain (CCD) dimer interface occupying the principal binding pocket of its cellular cofactor LEDGF/p75. Consequently, ALLINIs inhibit HIV-1 IN interaction with LEDGF/p75 as well as promote aberrant IN multimerization. Selection of viral strains emerging under the inhibitor pressure has revealed mutations at the IN dimer interface near the inhibitor binding site. Results: We have investigated the effects of one of the most prevalent substitutions, H171T IN, selected under increasing pressure of ALLINI BI-D. Virus containing the H171T IN substitution exhibited an ~68-fold resistance to BI-D treatment in infected cells. These results correlated with ~84-fold reduced affinity for BI-D binding to recombinant H171T IN CCD protein compared to its wild type (WT) counterpart. However, the H171T IN substitution only modestly affected IN-LEDGF/p75 binding and allowed HIV-1 containing this substitution to replicate at near WT levels. The x-ray crystal structures of BI-D binding to WT and H171T IN CCD dimers coupled with binding free energy calculations revealed the importance of the Nδ- protonated imidazole group of His171 for hydrogen bonding to the BI-D tert-butoxy ether oxygen and establishing electrostatic interactions with the inhibitor carboxylic acid, whereas these interactions were compromised upon substitution to Thr171. Conclusions: Our findings reveal a distinct mechanism of resistance for the H171T IN mutation to ALLINI BI-D and indicate a previously undescribed role of the His171 side chain for binding the inhibitor. Electronic supplementary material The online version of this article (doi:10.1186/s12977-014-0100-1) contains supplementary material, which is available to authorized users

A New Class of Allosteric HIV-1 Integrase Inhibitors Identified by Crstallographic Fragment Screening of the Catalytic Core Domain

HIV-1 integrase (IN) is essential for virus replication and represents an important multifunctional therapeutic target. Recently discovered quinoline-based allosteric IN inhibitors (ALLINIs) potently impair HIV-1 replication and are currently in clinical trials. ALLINIs exhibit a multimodal mechanism of action by inducing aberrant IN multimerization during virion morphogenesis and by competing with IN for binding to its cognate cellular cofactor LEDGF/p75 during early steps of HIV-1 infection. However, quinoline-based ALLINIs impose a low genetic barrier for the evolution of resistant phenotypes, which highlights a need for discovery of second-generation inhibitors. Using crystallographic screening of a library of 971 fragments against the HIV-1 IN catalytic core domain (CCD) followed by a fragment expansion approach, we have identified thiophenecarboxylic acid derivatives that bind at the CCD-CCD dimer interface at the principal lens epithelium-derived growth factor (LEDGF)/p75 binding pocket. The most active derivative (5) inhibited LEDGF/p75-dependent HIV-1 IN activity in vitro with an IC50 of 72 μm and impaired HIV-1 infection of T cells at an EC50 of 36 μm. The identified lead compound, with a relatively small molecular weight (221 Da), provides an optimal building block for developing a new class of inhibitors. Furthermore, although structurally distinct thiophenecarboxylic acid derivatives target a similar pocket at the IN dimer interface as the quinoline-based ALLINIs, the lead compound, 5, inhibited IN mutants that confer resistance to quinoline-based compounds. Collectively, our findings provide a plausible path for structure-based development of second-generation ALLINIs