Human DNA polymerase kappa encircles DNA: implications for mismatch extension and lesion bypass

Human DNA polymerase kappa (Pol kappa) is a proficient extender of mispaired primer termini on undamaged DNAs and is implicated in the extension step of lesion bypass. We present here the structure of Pol kappa catalytic core in ternary complex with DNA and an incoming nucleotide. The structure reveals encirclement of the DNA by a unique "N-clasp" at the N terminus of Pol kappa, which augments the conventional right-handed grip on the DNA by the palm, fingers, and thumb domains and the PAD and provides additional thermodynamic stability. The structure also reveals an active-site cleft that is constrained by the close apposition of the N-clasp and the fingers domain, and therefore can accommodate only a single Watson-Crick base pair. Together, DNA encirclement and other structural features help explain Pol kappa's ability to extend mismatches and to promote replication through various minor groove DNA lesions, by extending from the nucleotide incorporated opposite the lesion by another polymerase.     

Yeast Rad5 protein required for postreplication repair has a DNA helicase activity specific for replication fork regression

Lesions in the template DNA strand block the progression of the replication fork. In the yeast Saccharomyces cerevisiae, replication through DNA lesions is mediated by different Rad6-Rad18-dependent means, which include translesion synthesis and a Rad5-dependent postreplicational repair pathway that repairs the discontinuities that form in the DNA synthesized from damaged templates. Although translesion synthesis is well characterized, little is known about the mechanisms that modulate Rad5-dependent postreplicational repair. Here we show that yeast Rad5 has a DNA helicase activity that is specialized for replication fork regression. On model replication fork structures, Rad5 concertedly unwinds and anneals the nascent and the parental strands without exposing extended single-stranded regions. These observations provide insight into the mechanism of postreplicational repair in which Rad5 action promotes template switching for error-free damage bypass.     

Wing morphology and flight development in the short-nosed fruit bat Cynopterus sphinx

Postnatal changes in wing morphology, flight development and aerodynamics were studied in captive free-flying short-nosed fruit bats, Cynopterus sphinx. Pups were reluctant to move until 25 days of age and started fluttering at the mean age of 40 days. The wingspan and wing area increased linearly until 45 days of age by which time the young bats exhibited clumsy flight with gentle turns. At birth, C. sphinx had less-developed handwings compared to armwings; however, the handwing developed faster than the armwing during the postnatal period. Young bats achieved sustained flight at 55 days of age. Wing loading decreased linearly until 35 days of age and thereafter increased to a maximum of 12.82 Nm(-2) at 125 days of age. The logistic equation fitted the postnatal changes in wingspan and wing area better than the Gompertz and von Bertalanffy equations. The predicted minimum power speed (V(mp)) and maximum range speed (V(mr)) decreased until the onset of flight and thereafter the V(mp) and V(mr) increased linearly and approached 96.2% and 96.4%, respectively, of the speed of postpartum females at the age of 125 days. The requirement of minimum flight power (P(mp)) and maximum range power (P(mr)) increased until 85 days of age and thereafter stabilised. The minimum theoretical radius of banked turn (r(min)) decreased until 35 days of age and thereafter increased linearly and attained 86.5% of the r(min) of postpartum females at the age of 125 days.     

Brevibacterium frigoritolerans a novel entomopathogen of Anomala dimidiata and Holotrichia longipennis (Scarabaeidae: Coleoptera)

We describe the isolation, biochemical characterization, phylogenetic analysis, and pathogenecity of a novel entomopathogenic bacterium Brevibacterium frigoritolerans to first instar larvae of Anomala dimidiata and Holotrichia longipennis. The almost full length 16S rRNA sequence of the bacterium has 99% identity with the type strain of B. frigoritolerans, while phylogenetic analysis revealed that the isolate formed a tightly linked branch with the type strain of B. frigoritolerans. Under in vitro bioassay conditions, the isolate infected and caused 89±5.4 and 74±7.7% mortality, in first instar larvae of A. dimidiata and H. longipennis, respectively. The infected larvae exhibited bacteremia like symptoms and mortality occurred between the second and fifth weeks after inoculation. This is an early report on the entomopathogenic potential of the hitherto lesser-known bacterium Brevibacterium frigoritolerans.     

Structure based prediction of functional sites with potential inhibitors to Nudix enzymes from disease causing microbes

The functional sites were predicted for Nudix enzymes from pathogenic microorganisms such as Streprococcus pneumonia (2B06) and Enterococcus faecalis (2AZW). Their structures are already determined, however, no data is reported about their functional sites, substrates and inhibitors. Therefore, we report prediction of functional sites in these Nudix enzymes via Geometric Invariant (GI) technique (Construct different geometries of peptides which remain unchanged). The GI method enumerated 2B06: RA57, EA58, EA61, EA62 and 2AZW: RA62, EA63, EA66, EA67 as putative functional sites in these Nudix enzymes. In addition, the substrate was predicted via Molecular docking (Docking of substrates against whole structure of Nudix enzymes). The substrate ADP-Ribose was docked with the Nudix enzymes, 2B06 (Docking energy -15.68 Kcal/mol) and 2AZW (Docking energy -10.86 Kcal/mol) with the higher affinity and the lower docking energy as compared to other substrates. The residues EA62 in 2B06 and RA62 in 2AZW make hydrogen bonds with the ADP-ribose. Furthermore, we screened 51 inhibitor compounds against structures of 2B06 and 2AZW. The inhibitor compounds AMPCPR and CID14258187 were docked well as compared to other compounds. The compound CID14258187 was also in agreement with Lipinski rule of 5 for drug likeness properties. Therefore, our findings of functional sites, substrates and inhibitors for these Nudix enzymes may help in structure based drug designing against Streprococcus pneumonia and Enterococcus faecalis.     

Prognostic significance of cyclooxygenase-2 and response to chemotherapy in invasive ductal breast carcinoma patients by real time surface plasmon resonance analysis

Cyclooxygenase-2 (COX-2), an inducible enzyme, has been implicated in the progression and angiogenesis of breast cancer. The aim of the study is to quantify the concentration of COX-2 and its association with clinico-pathological parameters and response to treatment in patients with invasive ductal carcinoma receiving both neo-adjuvant and adjuvant chemotherapy. The level of COX-2 was estimated using a novel biosensor-based surface plasmon resonance technique in serum of 84 patients with breast cancer (48 patients of neo-adjuvant chemotherapy and 36 patients of adjuvant chemotherapy) and 40 age- and gender-matched normal individuals. A significant increase in COX-2 level was observed in patients compared with normal individuals (p>0.0001). The COX-2 level in serum was found to be significantly higher in patients with lymph node involvement (p<0.0061). 68% (33/48) of the patients receiving neo-adjuvant chemotherapy showed significantly (p<0.0025) reduced COX-2 levels. This study shows significant decrease of COX-2 level in patients with breast cancer treated with both neo-adjuvant and adjuvant chemotherapy. Estimation of COX-2 level in serum may serve as a tumor biomarker in patients with breast cancer.     

G(12/13) signaling pathways substitute for integrin αIIbβ3-signaling for thromboxane generation in platelets

Background

We have previously shown that ADP-induced TXA₂ generation requires signaling from αIIbβ3 integrin in platelets. Here we observed that, unlike ADP, protease-activated receptor (PAR)-mediated TXA₂ generation occurs independently of αIIbβ3. PAR agonists, but not ADP, activate G_12/13 signaling pathways. Hence, we evaluated the role of these pathways in TXA₂ generation.

Principal Findings

Inhibition of ADP-induced thromboxane generation by fibrinogen receptor antagonist SC57101 was rescued by co-stimulation of G_12/13 pathways with YFLLRNP. This observation suggested an existence of a common signaling effector downstream of integrins and G_12/13 pathways. Hence, we evaluated role of three potential tyrosine kinases; c-Src, Syk and FAK (Focal Adhesion Kinase) that are known to be activated by integrins. c-Src and Syk kinase did not play a role in ADP-induced functional responses in platelets. Selective activation of G_12/13 pathways resulted in the activation of FAK, in the absence of integrin signaling. Interestingly, αIIbβ3-mediated FAK activation occurred in a Src family kinase (SFK)-independent manner whereas G_12/13 pathway caused FAK activation in a SFK and RhoA-dependent manner. A FAK selective inhibitor TAE-226, blocked TXA₂ generation. However, in comparison to WT mice, Pf4-Cre/Fak-Floxed mice did not show any difference in platelet TXA₂ generation.

Conclusions

Therefore, we conclude that differential activation of FAK occurs downstream of Integrins and G_12/13 pathways. However, the common effector molecule, possibly a tyrosine kinase downstream of integrins and G_12/13 pathways contributing to TXA₂ generation in platelets remains elusive.