Structure of the PCNA unloader Elg1-RFC.

During DNA replication, the proliferating cell nuclear antigen (PCNA) clamps are loaded onto primed sites for each Okazaki fragment synthesis by the AAA+ heteropentamer replication factor C (RFC). PCNA encircling duplex DNA is quite stable and is removed from DNA by the dedicated clamp unloader Elg1-RFC. Here, we show the cryo-EM structure of Elg1-RFC in various states with PCNA. The structures reveal essential features of Elg1-RFC that explain how it is dedicated to PCNA unloading. Specifically, Elg1 contains two external loops that block opening of the Elg1-RFC complex for DNA binding, and an "Elg1 plug" domain that fills the central DNA binding chamber, thereby reinforcing the exclusive PCNA unloading activity of Elg1-RFC. Elg1-RFC was capable of unloading PCNA using non-hydrolyzable AMP-PNP. Both RFC and Elg1-RFC could remove PCNA from covalently closed circular DNA, indicating that PCNA unloading occurs by a mechanism that is distinct from PCNA loading. Implications for the PCNA unloading mechanism are discussed.

Annexin A1 conveys neuroprotective function via inhibiting oxidative stress in diffuse axonal injury of rats.

Diffuse axonal injury (DAI) is a critical pathological facet of traumatic brain injury (TBI). Oxidative stress plays a significant role in the progress of DAI. Annexin A1 (AnxA1) has been demonstrated to benefit from recovery of neurofunctional outcomes after TBI. However, whether AnxA1 exhibits neuronal protective function by modulating oxidative stress in DAI remains unknown. Expression of AnxA1 was evaluated via real-time PCR and western blotting in rat brainstem after DAI. The neurological effect of AnxA1 following DAI through quantification of modified neurologic severity score (mNSS) was compared between wild-type and AnxA1-knockout rats. Brain edema and neuronal apoptosis, as well as expression of oxidative factors and inflammatory cytokines, were analyzed between wild-type and AnxA1 deficiency rats after DAI. Furthermore, mNSS, oxidative and inflammatory cytokines were assayed after timely administration of recombinant AnxA1 for DAI rats. In the brainstem of DAI, the expression of AnxA1 remarkably increased. Ablation of AnxA1 increased the mNSS score and brain water content of rats after DAI. Neuron apoptosis in the brainstem after DAI was exaggerated by AnxA1 deficiency. In addition, AnxA1 deficiency significantly upregulated the level of oxidative and inflammatory factors in the brainstem of DAI rats. Moreover, mNSS decreased by AnxA1 treatment in rats following DAI. Expression of oxidative and inflammatory molecules in rat brainstem subjected to DAI inhibited by AnxA1 administration. AnxA1 exhibited neuronal protective function in the progression of DAI mainly dependent on suppressing oxidative stress and inflammation.

Structures of 9-1-1 DNA checkpoint clamp loading at gaps from start to finish and ramification on biology.

Rad24-RFC (replication factor C) loads the 9-1-1 checkpoint clamp onto the recessed 5' ends by binding a 5' DNA at an external surface site and threading the 3' single-stranded DNA (ssDNA) into 9-1-1. We find here that Rad24-RFC loads 9-1-1 onto DNA gaps in preference to a recessed 5' end, thus presumably leaving 9-1-1 on duplex 3' ss/double-stranded DNA (dsDNA) after Rad24-RFC ejects from DNA. We captured five Rad24-RFC-9-1-1 loading intermediates using a 10-nt gap DNA. We also determined the structure of Rad24-RFC-9-1-1 using a 5-nt gap DNA. The structures reveal that Rad24-RFC is unable to melt DNA ends and that a Rad24 loop limits the dsDNA length in the chamber. These observations explain Rad24-RFC's preference for a preexisting gap of over 5-nt ssDNA and suggest a direct role of the 9-1-1 in gap repair with various TLS (trans-lesion synthesis) polymerases in addition to signaling the ATR kinase.

Structures of 9-1-1 DNA checkpoint clamp loading at gaps from start to finish and ramification to biology.

Recent structural studies show the Rad24-RFC loads the 9-1-1 checkpoint clamp onto a recessed 5' end by binding the 5' DNA on Rad24 at an external surface site and threading the 3' ssDNA into the well-established internal chamber and into 9-1-1. We find here that Rad24-RFC loads 9-1-1 onto DNA gaps in preference to a recessed 5' DNA end, thus presumably leaving 9-1-1 on a 3' ss/ds DNA after Rad24-RFC ejects from the 5' gap end and may explain reports of 9-1-1 directly functioning in DNA repair with various TLS polymerases, in addition to signaling the ATR kinase. To gain a deeper understanding of 9-1-1 loading at gaps we report high-resolution structures of Rad24-RFC during loading of 9-1-1 onto 10-nt and 5-nt gapped DNAs. At a 10-nt gap we captured five Rad24-RFC-9-1-1 loading intermediates in which the 9-1-1 DNA entry gate varies from fully open to fully closed around DNA using ATPγS, supporting the emerging view that ATP hydrolysis is not needed for clamp opening/closing, but instead for dissociation of the loader from the clamp encircling DNA. The structure of Rad24-RFC-9-1-1 at a 5-nt gap shows a 180° axially rotated 3'-dsDNA which orients the template strand to bridge the 3'- and 5'- junctions with a minimum 5-nt ssDNA. The structures reveal a unique loop on Rad24 that limits the length of dsDNA in the inner chamber, and inability to melt DNA ends unlike RFC, thereby explaining Rad24-RFC's preference for a preexisting ssDNA gap and suggesting a direct role in gap repair in addition to its checkpoint role.

Cryo-EM structures reveal that RFC recognizes both the 3'- and 5'-DNA ends to load PCNA onto gaps for DNA repair.

RFC uses ATP to assemble PCNA onto primed sites for replicative DNA polymerases δ and ε. The RFC pentamer forms a central chamber that binds 3' ss/ds DNA junctions to load PCNA onto DNA during replication. We show here five structures that identify a second DNA binding site in RFC that binds a 5' duplex. This 5' DNA site is located between the N-terminal BRCT domain and AAA+ module of the large Rfc1 subunit. Our structures reveal ideal binding to a 7-nt gap, which includes 2 bp unwound by the clamp loader. Biochemical studies show enhanced binding to 5 and 10 nt gaps, consistent with the structural results. Because both 3' and 5' ends are present at a ssDNA gap, we propose that the 5' site facilitates RFC's PCNA loading activity at a DNA damage-induced gap to recruit gap-filling polymerases. These findings are consistent with genetic studies showing that base excision repair of gaps greater than 1 base requires PCNA and involves the 5' DNA binding domain of Rfc1. We further observe that a 5' end facilitates PCNA loading at an RPA coated 30-nt gap, suggesting a potential role of the RFC 5'-DNA site in lagging strand DNA synthesis.

DNA is loaded through the 9-1-1 DNA checkpoint clamp in the opposite direction of the PCNA clamp.

The 9-1-1 DNA checkpoint clamp is loaded onto 5'-recessed DNA to activate the DNA damage checkpoint that arrests the cell cycle. The 9-1-1 clamp is a heterotrimeric ring that is loaded in Saccharomyces cerevisiae by Rad24-RFC (hRAD17-RFC), an alternate clamp loader in which Rad24 replaces Rfc1 in the RFC1-5 clamp loader of proliferating cell nuclear antigen (PCNA). The 9-1-1 clamp loading mechanism has been a mystery, because, unlike RFC, which loads PCNA onto a 3'-recessed junction, Rad24-RFC loads the 9-1-1 ring onto a 5'-recessed DNA junction. Here we report two cryo-EM structures of Rad24-RFC-DNA with a closed or 27-Å open 9-1-1 clamp. The structures reveal a completely unexpected mechanism by which a clamp can be loaded onto DNA. Unlike RFC, which encircles DNA, Rad24 binds 5'-DNA on its surface, not inside the loader, and threads the 3' ssDNA overhang into the 9-1-1 clamp from above the ring.

Role of the conserved E2 residue G259 in classical swine fever virus production and replication.

The E2 glycoprotein of classical swine fever virus (CSFV) plays multiple roles in the viral life cycle. The chimeric live attenuated C strain with the E2 substitution of bovine viral diarrhea virus (BVDV) is a promising marker vaccine candidate. In this study, the recombinant chimeric CSFV/bE2 cDNA clone harboring heterologous E2 (bE2) of BVDV was constructed by genetic approaches. Recombinant infectious virus rCSFV/bE2 (P11) was recovered by 11 serial passages of transfected PK15 cells. Viral genome sequencing showed that a glutamic acid to glycine mutation (E260G) at position 260 of the bE2 was observed in rCSFV/bE2 P11. Alignment of amino acid sequences displayed that the glycine was one of three conserved residues in pestivirus E2. When the glutamic acid to glycine substitution (E260G) was introduced into chimeric CSFV/bE2 cDNA clone, the high-titer infectious rCSFV/bE2E260G was rescued. The glycine to glutamic acid substitution at corresponding position in CSFV E2 resulted in significantly decreased rCSFV/E2G259E production. We further identified that the conserved E2 residue G259 played a critical role in the release and binding activity of CSFV and that the E2 residues G259 and V111 modulated synergistically infectious virus production and replication.

Annexin A1 Promotes Reparative Angiogenesis and Ameliorates Neuronal Injury in Ischemic Retinopathy.

PURPOSE: Retinal ischemia is the main reason for vision threatening. Inflammation and aberrant angiogenesis play an important role in the pathogenesis of ischemia. Annexin A1 (ANXA1) is an endogenous protein modulating anti-inflammatory processes, and its therapeutic potential has been reported in a range of inflammatory diseases. However, the effect of ANXA1 on ischemic retinal injury has not been examined. METHODS: Expression of ANXA1 was assessed by real-time PCR and western blotting, and location of ANXA1 was evaluated by immunofluorescence staining in retina of OIR. The activation of ANXA1 was assayed in HRECs after hypoxia stimuli. The effect of ANXA1 on vascularization of OIR mouse through quantification vaso-obliteration (VO) and neovascularization (NV), as well as expression of relevant angiogenic factors and inflammatory cytokines was compared between wild type and ANXA1 deficiency mice. We also investigated the effect of ANXA1 on retinal neuronal degeneration as measured by electroretinography (ERG) and OCT. RESULTS: In retinas of OIR, the expression of ANXA1 significantly increased and located in inner retinal layers. ANXA1 was induced in HRECs after hypoxic stimuli. Furthermore, ANXA1 deficiency increased pro-angiogenic and pro-inflammatory cytokines. Ablation of ANXA1 suppressed aortic outgrowth and retinal reparative revascularization and promoted pathological NV to exacerbate retinal dysfunction after ischemia injury. CONCLUSIONS: ANXA1 inhibits angiogenic and inhibits pro-inflammatory cytokines and promotes reparative angiogenesis, thus exhibits neuronal protective function in ischemic retinopathy.

Decreased Methane Emissions Associated with Methanogenic and Methanotrophic Communities in a Pig Manure Windrow Composting System under Calcium Superphosphate Amendment.

With the rapid growth of livestock breeding, manure composting has evolved to be an important source of atmospheric methane (CH4) which accelerates global warming. Calcium superphosphate (CaSSP), as a commonly used fertilizer, was proposed to be effective in reducing CH4 emissions from manure composting, but the intrinsic biological mechanism remains unknown. Methanogens and methanotrophs both play a key role in mediating CH4 fluxes, therefore we hypothesized that the CaSSP-mediated reduction in CH4 emissions was attributed to the shift of methanogens and methanotrophs, which was regulated by physicochemical parameter changes. To test this hypothesis, a 60-day pig manure windrow composting experiment was conducted to investigate the response of CH4 emissions to CaSSP amendment, with a close linkage to methanogenic and methanotrophic communities. Results showed that CaSSP amendment significantly reduced CH4 emissions by 49.5% compared with the control over the whole composting period. The decreased mcrA gene (encodes the α-subunit of methyl-coenzyme M reductase) abundance in response to CaSSP amendment suggested that the CH4 emissions were reduced primarily due to the suppressed microbial CH4 production. Illumina MiSeq sequencing analysis showed that the overall distribution pattern of methanogenic and methanotrophic communities were significantly affected by CaSSP amendment. Particularly, the relative abundance of Methanosarcina that is known to be a dominant group for CH4 production, significantly decreased by up to 25.3% accompanied with CaSSP addition. Only Type I methanotrophs was detected in our study and Methylocaldum was the dominant methanotrophs in this composting system; in detail, CaSSP amendment increased the relative abundance of OTUs belong to Methylocaldum and Methylobacter. Moreover, the increased SO42- concentration and decreased pH acted as two key factors influencing the methanogenic and methanotrophic composition, with the former has a negative effect on methanogenesis growth and can later promote CH4 oxidation at a low level. This study deepens our understanding of the interaction between abiotic factors, function microbiota and greenhouse gas (GHG) emissions, as well as provides implication for practically reducing composting GHG emissions.

A positively charged surface patch on the pestivirus NS3 protease module plays an important role in modulating NS3 helicase activity and virus production.

Pestivirus nonstructural protein 3 (NS3) is a multifunctional protein with protease and helicase activities that are essential for virus replication. In this study, we used a combination of biochemical and genetic approaches to investigate the relationship between a positively charged patch on the protease module and NS3 function. The surface patch is composed of four basic residues, R50, K74 and K94 in the NS3 protease domain and H24 in the structurally integrated cofactor NS4APCS. Single-residue or simultaneous four-residue substitutions in the patch to alanine or aspartic acid had little effect on ATPase activity. However, single substitutions of R50, K94 or H24 or a simultaneous four-residue substitution resulted in apparent changes in the helicase activity and RNA-binding ability of NS3. When these mutations were introduced into a classical swine fever virus (CSFV) cDNA clone, a single substitution at K94 or a simultaneous four-residue substitution (Qua_A or Qua_D) impaired the production of infectious virus. Furthermore, the replication efficiency of the CSFV variants was partially correlated with the helicase activity of NS3 in vitro. Our results suggest that the conserved positively charged patch on NS3 plays an important role in modulating the NS3 helicase activity in vitro and CSFV production.

Water skating: How polymerase sliding clamps move on DNA.

Polymerase sliding clamps are ring-shaped proteins that encircle duplex DNA and hold polymerases to DNA for high processivity during synthesis. The crystal structure of clamp-DNA complex reveals that the DNA is highly tilted through the clamp with extensive interaction with the clamp inner surface. In contrast to the tilted clamp-DNA interaction without DNA polymerases, recent structures of replicative polymerases of bacteria, eukaryotes, and archaea that are bound to the clamp and DNA show that the polymerase positions DNA straight through the clamp without direct protein-DNA contacts. Instead, the clamp-to-DNA interaction is mediated by one or two layers of water. Hence, clamps 'water skate' on DNA during function with replicative polymerases from all domains of life, providing a nearly frictionless bearing for fast and processive DNA synthesis.

Structure of eukaryotic DNA polymerase δ bound to the PCNA clamp while encircling DNA.

The DNA polymerase (Pol) δ of Saccharomyces cerevisiae (S.c.) is composed of the catalytic subunit Pol3 along with two regulatory subunits, Pol31 and Pol32. Pol δ binds to proliferating cell nuclear antigen (PCNA) and functions in genome replication, repair, and recombination. Unique among DNA polymerases, the Pol3 catalytic subunit contains a 4Fe-4S cluster that may sense the cellular redox state. Here we report the 3.2-Šcryo-EM structure of S.c. Pol δ in complex with primed DNA, an incoming ddTTP, and the PCNA clamp. Unexpectedly, Pol δ binds only one subunit of the PCNA trimer. This singular yet extensive interaction holds DNA such that the 2-nm-wide DNA threads through the center of the 3-nm interior channel of the clamp without directly contacting the protein. Thus, a water-mediated clamp and DNA interface enables the PCNA clamp to "waterskate" along the duplex with minimum drag. Pol31 and Pol32 are positioned off to the side of the catalytic Pol3-PCNA-DNA axis. We show here that Pol31-Pol32 binds single-stranded DNA that we propose underlies polymerase recycling during lagging strand synthesis, in analogy to Escherichia coli replicase. Interestingly, the 4Fe-4S cluster in the C-terminal CysB domain of Pol3 forms the central interface to Pol31-Pol32, and this strategic location may explain the regulation of the oxidation state on Pol δ activity, possibly useful during cellular oxidative stress. Importantly, human cancer and other disease mutations map to nearly every domain of Pol3, suggesting that all aspects of Pol δ replication are important to human health and disease.

Uncoupling of Protease trans-Cleavage and Helicase Activities in Pestivirus NS3.

The nonstructural protein NS3 from the Flaviviridae family is a multifunctional protein that contains an N-terminal protease and a C-terminal helicase, playing essential roles in viral polyprotein processing and genome replication. Here we report a full-length crystal structure of the classical swine fever virus (CSFV) NS3 in complex with its NS4A protease cofactor segment (PCS) at a 2.35-Å resolution. The structure reveals a previously unidentified ∼2,200-Å2 intramolecular protease-helicase interface comprising three clusters of interactions, representing a "closed" global conformation related to the NS3-NS4A cis-cleavage event. Although this conformation is incompatible with protease trans-cleavage, it appears to be functionally important and beneficial to the helicase activity, as the mutations designed to perturb this conformation impaired both the helicase activities in vitro and virus production in vivo Our work reveals important features of protease-helicase coordination in pestivirus NS3 and provides a key basis for how different conformational states may explicitly contribute to certain functions of this natural protease-helicase fusion protein.IMPORTANCE Many RNA viruses encode helicases to aid their RNA genome replication and transcription by unwinding structured RNA. Being naturally fused to a protease participating in viral polyprotein processing, the NS3 helicases encoded by the Flaviviridae family viruses are unique. Therefore, how these two enzyme modules coordinate in a single polypeptide is of particular interest. Here we report a previously unidentified conformation of pestivirus NS3 in complex with its NS4A protease cofactor segment (PCS). This conformational state is related to the protease cis-cleavage event and is optimal for the function of helicase. This work provides an important basis to understand how different enzymatic activities of NS3 may be achieved by the coordination between the protease and helicase through different conformational states.

Protection of FK506 against neuronal apoptosis and axonal injury following experimental diffuse axonal injury.

Diffuse axonal injury (DAI) is the most common and significant pathological features of traumatic brain injury (TBI). However, there are still no effective drugs to combat the formation and progression of DAI in affected individuals. FK506, also known as tacrolimus, is an immunosuppressive drug, which is widely used in transplantation medicine for the reduction of allograft rejection. Previous studies have identified that FK506 may play an important role in the nerve protective effect of the central nervous system. In the present study, apoptosis of neuronal cells was observed following the induction of experimental DAI. The results demonstrated that it was closely related with the upregulation of death­associated protein kinase 1 (DAPK1). It was hypothesized that FK506 may inhibit the activity of DAPK1 by inhibiting calcineurin activity, which may be primarily involved in anti­apoptosis following DAI induction. Through researching the expression of nerve regeneration associated proteins (NF­H and GAP­43) following DAI, the present study provides novel data to suggest that FK506 promotes axon formation and nerve regeneration following experimental DAI. Therefore, FK506 may be a potent therapeutic for inhibiting nerve injury, as well as promoting the nerve regeneration following DAI.

The N-terminus of classical swine fever virus (CSFV) nonstructural protein 2 modulates viral genome RNA replication.

Pestivirus nonstructural protein 2 (NS2) is a multifunctional, hydrophobic protein with an important but poorly understood role in viral RNA replication and infectious virus production. In the present study, based on sequence analysis, we mutated several representative conserved residues within the N-terminus of NS2 of classical swine fever virus (CSFV) and investigated how these mutations affected viral RNA replication and infectious virus production. Our results demonstrated that the mutation of two aspartic acids, NS2/D60A or NS2/D60K and NS2/D78K, in the N-terminus of NS2 abolished infectious virus production and that the substitution of arginine for alanine at position 100 (NS2/R100A) resulted in significantly decreased viral titer. The serial passage of cells containing viral genomic RNA molecules generated the revertants NS2/A60D, NS2/K60D and NS2/K78D, leading to the recovery of infectious virus. In the context of the NS2/R100A mutant, the NS2/I90L mutation compensated for infectious virus production. The regulatory roles of the indicated amino acid residues were identified to occur at the viral RNA replication level. These results revealed a novel function for the NS2 N-terminus of CSFV in modulating viral RNA replication.

Blood pressure reverse-dipping is associated with early formation of carotid plaque in senior hypertensive patients.

Nocturnal variations in blood pressure (BP) were associated with carotid intima-media thickness. However, the precise relationship between circadian variations of BP and carotid plaques remains unknown. Therefore, the prognostic value of reverse-dipper pattern of BP for carotid plaque was investigated. In this cross-sectional study, a total of 524 hypertensive patients were recruited and evaluated with ambulatory BP monitoring between April 2012 and June 2013. Carotid plaque was classified into Grade 0 (normal or no observable plaque), Grade 1 (mild stenosis, 1%-24% narrowing), and Grade 2 (moderate stenosis, ≥25% narrowing). Multinomial logistic regression was applied to analyze the relationship between different degrees of carotid plaque and ambulatory BP monitoring results. Reverse-dipper pattern of BP was more common in older patients, smokers, and those with elevated fasting glucose. The incidences of coronary artery disease, lacunar infarction, and diabetes were also higher among hypertensive with reverse-dipper pattern. Multinomial logistic regression analysis showed that reverse dipper (odds ratio [OR] 2.500; 95% confidence interval [CI] 1.320-4.736; P = 0.005), age (OR 1.089; 95% CI 1.067-1.111; P < 0.001), smoke (OR 1.625; 95% CI 1.009-2.617; P = 0.046), and diabetes (OR 1.759; 95% CI 1.093-2.830; P = 0.020) were significantly different between mild carotid plaque and normal. Our results also suggested that mild carotid plaque was closely related to reverse-dipper pattern of BP (2.308; 95% CI 1.223-4.355; P = 0.010). Reverse-dipper pattern of BP may be a risk factor for carotid atherosclerosis and play a crucial role in the early formation of carotid plaque.