Evidence for preferential mismatch repair of lagging strand DNA replication errors in yeast

Duplex DNA is replicated in the 5'-3' direction by coordinated copying of leading and lagging strand templates with somewhat different proteins and mechanics, providing the potential for differences in the fidelity of replication of the two strands. We previously showed that in Saccharomyces cerevisiae, active replication origins establish a strand bias in the rate of base substitutions resulting from replication of unrepaired 8-oxo-guanine (GO) in DNA. Lower mutagenesis was associated with replicating lagging strand templates. Here, we test the hypothesis that this bias is due to more efficient repair of lagging stand mismatches by measuring mutation rates in ogg1 strains with a reporter allele in two orientations at loci on opposite sides of a replication origin on chromosome III. We compare a MMR-proficient strain to strains deleted for the MMR genes MSH2, MSH6, MLH1, or EXOI. Loss of MMR reduces the strand bias by preferentially increasing mutagenesis for lagging strand replication. We conclude that GO-A mismatches generated during lagging strand replication are more efficiently repaired. This is consistent with the hypothesis that 5' ends of Okazaki fragments and PCNA, present at high density during lagging strand replication, are used as strand discrimination signals for mismatch repair in vivo.     

Sequential Posttranslational Modifications Program FEN1 Degradation during Cell-Cycle Progression

We propose that cell-cycle-dependent timing of FEN1 nuclease activity is essential for cell-cycle progression and the maintenance of genome stability. After DNA replication is complete at the exit point of the S phase, removal of excess FEN1 may be crucial. Here, we report a mechanism that controls the programmed degradation of FEN1 via a sequential cascade of posttranslational modifications. We found that FEN1 phosphorylation stimulated its SUMOylation, which in turn stimulated its ubiquitination and ultimately led to its degradation via the proteasome pathway. Mutations or inhibitors that blocked the modification at any step in this pathway suppressed FEN1 degradation. Critically, the presence of SUMOylation- or ubiquitination-defective, nondegradable FEN1 mutant protein caused accumulation of Cyclin B, delays in the G1 and G2/M phases, and polyploidy. These findings may represent a newly identified regulatory mechanism used by cells to ensure precise cell-cycle progression and to prevent transformation.     

Molecular dynamics simulation of shear- and stretch-induced dissociation of P-selectin/PSGL-1 complex

By mediating the tethering and rolling of leukocytes on vascular surfaces, the interactions between P-selectin and the P-selectin glycoprotein ligand 1 (PSGL-1) play crucial roles during inflammation cascade. Tensile stretch produced by rolling leukocytes and shear stress exerted by blood flow constitute the two types of mechanical forces that act on the P-selectin/PSGL-1 bond. These forces modulate not only dissociation kinetics of this bond, but also the leukocyte adhesion dynamics. However, the respective contribution of the two forces to bond dissociation and to the corresponding microstructural bases remains unclear. To mimic the mechanical microenvironment, we developed two molecular dynamics approaches; namely, an approach involving the shear flow field with a controlled velocity gradient, and the track dragging approach with a defined trajectory. With each approach or with both combined, we investigate the microstructural evolution and dissociation kinetics of the P-LE/SGP-3 construct, which is the smallest functional unit of the P-selectin/PSGL-1 complex. The results demonstrate that both shear flow and tensile stretch play important roles in the collapse of the construct and that, before bond dissociation, the former causes more destruction of domains within the construct than the latter. Dissociation of the P-LE/SGP-3 construct features intramolecular destruction of the epidermal-growth-factor (EGF) domain and the breaking of hydrogen-bond clusters at the P-selectin-lectin/EGF interface. Thus, to better understand how mechanics impacts the dissociation kinetics of the P-selectin/PSGL-1 complex, we propose herein two approaches to mimic its physiological mechanical environment.     

Conformational consequences of protein glycosylation: preparation of O-mannosyl serine and threonine building blocks, and their incorporation into glycopeptide sequences derived from alpha-dystroglycan

With the goal to investigate the structural impact of O-mannosyl glycosylation on alpha-dystroglycan, a glycoprotein that has an important role in the extracellular organization of muscle, glycopeptides derived from its mucin-like sequence have been prepared by solid-phase peptide synthesis. Two approaches have been explored to obtain needed mannosylated serine and threonine building blocks. With the alpha-carboxyl group unprotected, and with tetraaceto-1-fluoro-alpha-D-mannose as the sugar donor, the desired alpha-O-mannosyl-Fmoc-Ser/Thr formed, along with mannosyl ester isomers and the species with mannose attached to both hydroxyl and carboxyl functions. Relevant mechanistic questions and stability issues were elucidated. Alternatively, building blocks were made with the alpha-carboxyl protected/activated as the pentafluorophenyl (Pfp) ester. Glycopeptides synthesized herein contained 5-9 residues, and featured one, two, and four consecutive Ser and/or Thr residues O-glycosylated with mannose. Circular dichroism (CD) spectra for Man-containing glycopeptides recorded in water show them to be not well ordered. For one of the alpha-dystroglycan-derived sequences, the comparative conformational consequences of glycosylation by either Man or GalNAc have been examined by CD and NMR, with both methods showing a more organized structure when GalNAc is present.     

Measuring binding kinetics of surface-bound molecules using the surface plasmon resonance technique

Anandamide (N-arachidonoylethanolamine, AEA) is an endocannabinoid present in human plasma that is associated with several physiological functions and disease states. Significant variability in AEA plasma concentrations has been reported between studies, because quantification of AEA is fraught with methodological difficulties. A rapid, highly sensitive, robust, specific, and highly reproducible ultra high performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method is described here for the analysis of AEA in human plasma. This fully validated method using octa-deuterated AEA (AEA-d8) as an internal standard represents an improvement over previous analyses in terms of run time (4 min), limit of detection (0.055 fmol on column, 18.75 fmol/ml plasma), precision (relative standard deviations of 3.7, 3.9, and 4.8% for 1.66, 6.65, and 133 fmol on column), and accuracy (97.5–104.5%). AEA analysis was linear over the range 0.23 to 19 nM (1.66 to 133 fmol on column). To demonstrate the usefulness of this method for the measurement of AEA levels in clinical samples, plasma samples obtained from female volunteers at different stages of the menstrual cycle and pregnant women were assayed. Plasma AEA concentrations were significantly (P = 0.0078) lower in the luteal phase of the menstrual cycle compared to the follicular phase. In pregnancy, the concentrations were lowest in the first and second trimesters with levels comparable to those observed in the luteal phase of the menstrual cycle and modestly increased in the third trimester. The highest plasma AEA levels were observed in women in active labour, and these were significantly (P = 0.0147) higher than those observed in women at term but not in active labour. Postmenopausal women had AEA concentrations comparable to levels observed during the luteal phase of premenopausal women and were significantly (P = 0.0389) lower than AEA plasma concentrations obtained during the follicular phase. The sensitivity and precision of the validated method described here suggests that this method is suitable for the analysis of AEA in clinical samples and may be utilised for the investigation of biomatrices containing limited amounts of AEA.     

Cutting edge: FimH adhesin of type 1 fimbriae is a novel TLR4 ligand

Several TLR ligands of bacterial origin induce innate immune responses. Although FimH, the adhesin portion of type 1 fimbria, plays an important role in the pathogenicity of some gram-negative bacteria, its ability to stimulate the innate immune system via TLR signaling remains unclear. In this study we report that FimH induces potent innate responses in a MyD88-dependent fashion. The FimH-induced innate activity was restricted to cells expressing TLR4. In addition, FimH was able to bind directly to TLR4. More importantly, cells unresponsive to LPS were responsive to FimH and the presence or absence of MD-2 and CD14 had no effect on FimH activity. Our data suggest that TLR4 is a functional receptor for the adhesin portion of bacterial type 1 fimbria.     

Nucleolar localization and dynamic roles of flap endonuclease 1 in ribosomal DNA replication and damage repair

Despite the wealth of information available on the biochemical functions and our recent findings of its roles in genome stability and cancer avoidance of the structure-specific flap endonuclease 1 (FEN1), its cellular compartmentalization and dynamics corresponding to its involvement in various DNA metabolic pathways are not yet elucidated. Several years ago, we demonstrated that FEN1 migrates into the nucleus in response to DNA damage and under certain cell cycle conditions. In the current paper, we found that FEN1 is superaccumulated in the nucleolus and plays a role in the resolution of stalled DNA replication forks formed at the sites of natural replication fork barriers. In response to UV irradiation and upon phosphorylation, FEN1 migrates to nuclear plasma to participate in the resolution of UV cross-links on DNA, most likely employing its concerted action of exonuclease and gap-dependent endonuclease activities. Based on yeast complementation experiments, the mutation of Ser(187)Asp, mimicking constant phosphorylation, excludes FEN1 from nucleolar accumulation. The replacement of Ser(187) by Ala, eliminating the only phosphorylation site, retains FEN1 in nucleoli. Both of the mutations cause UV sensitivity, impair cellular UV damage repair capacity, and decline overall cellular survivorship.     

C57BL／6J-HBV转基因小鼠血清生化指标与性别年龄的关系

目的观察C57BL/6J-HBV乙型肝炎病毒转基因小鼠血清总胆红素（T-BIL）、丙氨酸氨基转移酶（ALT）、天冬氨酸氨基转移酶（AST）、总蛋白（TP）和白蛋白（ALB）与性别和年龄的关系,以及与遗传背景相同的C57BL／6J小鼠的差异。方法选取8周龄和24周龄的C57BL／6J-HBV转基因小鼠及C57BL／6J小鼠的血清测定T-BIL、ALT、AST、TP和ALB值。结果C57BL／6J-HBV转基因小鼠与同周龄同性别的C57BL／6J小鼠相比,T-BIL、ALT、AST、TP和ALB均存在显著差异（P〈0．05）;24周龄的C57BL／6J-HBV转基因小鼠ALT和AST与其8周龄鼠相比均存在显著差异（P〈0．05）。结论C57BL／6J-HBV转基因小鼠T-BIL、ALT、AST、TP和ALB值显著高于C57BL／6J小鼠;且C57BL／6J-HBV转基因小鼠ALT和AST值与年龄有关,与性别无关。