NMR study of repair mechanism of DNA photolyase by FAD-induced paramagnetic relaxation enhancement

Cyclobutane pyrimidine dimer (CPD) photolyases, which contain FAD as a cofactor, use light to repair CPDs. We performed structural analyses of the catalytic site of the Thermus thermophilus CPD photolyase-DNA complex, using FAD-induced paramagnetic relaxation enhancement (PRE). The distances between the tryptophan residues and the FAD calculated from the PRE agree well with those observed in the x-ray structure (with an error of <3 A). Subsequently, a single-stranded DNA containing 13C-labeled CPD was prepared, and the FAD-induced PRE of the NMR resonances from the CPD lesion in complex with the CPD photolyase was investigated. The distance between the FAD and the CPD calculated from the PRE is 16 +/- 3 A. The FAD-induced PRE was also observed in the CPD photolyase-double-stranded DNA complex. Based on these results, a model of the CPD photolyase-DNA complex was constructed, and the roles of Arg-201, Lys-240, Trp-247, and Trp-353 in the CPD-repair reaction are discussed.     

Palm mutants in DNA polymerases alpha and eta alter DNA replication fidelity and translesion activity

We isolated active mutants in Saccharomyces cerevisiae DNA polymerase alpha that were associated with a defect in error discrimination. Among them, L868F DNA polymerase alpha has a spontaneous error frequency of 3 in 100 nucleotides and 570-fold lower replication fidelity than wild-type (WT) polymerase alpha. In vivo, mutant DNA polymerases confer a mutator phenotype and are synergistic with msh2 or msh6, suggesting that DNA polymerase alpha-dependent replication errors are recognized and repaired by mismatch repair. In vitro, L868F DNA polymerase alpha catalyzes efficient bypass of a cis-syn cyclobutane pyrimidine dimer, extending the 3' T 26000-fold more efficiently than the WT. Phe34 is equivalent to residue Leu868 in translesion DNA polymerase eta, and the F34L mutant of S. cerevisiae DNA polymerase eta has reduced translesion DNA synthesis activity in vitro. These data suggest that high-fidelity DNA synthesis by DNA polymerase alpha is required for genomic stability in yeast. The data also suggest that the phenylalanine and leucine residues in translesion and replicative DNA polymerases, respectively, might have played a role in the functional evolution of these enzyme classes.     

Depletion of vesicle-tethering factor p115 causes mini-stacked Golgi fragments with delayed protein transport

Depletion of p115 with small interfering RNA caused fragmentation of the Golgi apparatus, resulting in dispersed distribution of stacked short cisternae and a vesicular structure (mini-stacked Golgi). The mini-stacked Golgi with cis- and trans-organization is functional in protein transport and glycosylation, although secretion is considerably retarded in p115 knockdown cells. The fragmented Golgi was further disrupted by treatment with breferdin A and reassembled into the mini-stacked Golgi by removal of the drug, as observed in control cells. In addition, p115 knockdown cells maintained retrograde transport from the Golgi to the endoplasmic reticulum, although the rate was not as efficient as in control cells. While no alternation of microtubule networks was found in p115 knockdown cells, the fragmented Golgi resembled those in cells treated with anti-microtubule drugs. The results suggest that p115 is involved in vesicular transport between endoplasmic reticulum and the Golgi, along with microtubule networks.     

Ramelteon (TAK-375) accelerates reentrainment of circadian rhythm after a phase advance of the light-dark cycle in rats

In vivo pharmacological effects of ramelteon (TAK-375), a novel, highly MT1/MT2-selective receptor agonist, were studied in rats to determine ramelteon's ability to reentrain the circadian rhythm after an abrupt phase advance. Experiments were also conducted to assess the potential cognitive side effects of ramelteon and its potential to become a drug of abuse. After an abrupt 8-h phase shift, ramelteon (0.1 and 1 mg/kg, p.o.) and melatonin (10 mg/kg, p.o.) accelerated reentrainment of running wheel activity rhythm to the new lightdark cycle. Ramelteon (3-30 mg/kg, p.o.) and melatonin (10-100 mg/kg, p.o.) did not affect learning or memory in rats tested by the water maze task and the delayed match to position task, although diazepam and triazolam impaired both of the tasks. Neither ramelteon (3-30 mg/kg, p.o.) nor melatonin (10-100 mg/kg, p.o.) demonstrated a rewarding property in the conditioned place-preference test, implying that MT1/MT2 receptor agonists have no abuse potential. In contrast, benzodiazepines and morphine showed rewarding properties in this test. The authors' results suggest that ramelteon may be useful for treatment of circadian rhythm sleep disorders without adverse effects typically associated with benzodiazepine use, such as learning and memory impairment, and drug dependence.     

DDB1-DDB2 (xeroderma pigmentosum group E) protein complex recognizes a cyclobutane pyrimidine dimer, mismatches, apurinic/apyrimidinic sites, and compound lesions in DNA

The DDB protein complex, comprising the subunits DDB1 and DDB2, binds tightly to UV light-irradiated DNA. Mutations in DDB2 are responsible for xeroderma pigmentosum group E, a disorder with defects in nucleotide excision repair of DNA. Both subunits are also components of a complex involved in ubiquitin-mediated proteolysis. Cellular defects in DDB2 disable repair of the major UV radiation photoproduct in DNA, a cyclobutane pyrimidine dimer, but no significant direct binding of DDB to this photoproduct in DNA has ever been demonstrated. Thus, it has been uncertain how DDB could play a specific role in DNA repair of such damage. We investigated DDB function using highly purified proteins. Co-purified DDB1-DDB2 or DDB reconstituted with individual DDB1 and DDB2 subunits binds to damaged DNA as a ternary complex. We found that DDB can indeed recognize a cyclobutane pyrimidine dimer in DNA with an affinity (K(app)a) 6-fold higher than that of nondamaged DNA. The DDB1-DDB2 complex also bound with high specificity to a UV radiation-induced (6-4) photoproduct and to an apurinic site in DNA. Unexpectedly, DDB also bound avidly to DNA containing a 2- or 3-bp mismatch (and does not bind well to DNA containing larger mismatches). These data indicate that DDB does not detect lesions per se. It instead recognizes other structural features of damaged DNA, acting as a sensor that probes DNA for a subset of conformational changes. Lesions recognized may include those arising when translesion polymerases such as POLH incorporate bases across from DNA lesions caused by UV radiation.     

Radical Prostatectomy versus External Beam Radiotherapy for cT1-4N0M0 Prostate Cancer: Comparison of Patient Outcomes Including Mortality

Background

Although radical prostatectomy (RP) and external beam radiotherapy (EBRT) have been considered as comparable treatments for localized prostate cancer (PC), it is controversial which treatment is better. The present study aimed to compare outcomes, including mortality, of RP and EBRT for localized PC.

Methods

We retrospectively analyzed 891 patients with cT1-4N0M0 PC who underwent either RP (n = 569) or EBRT (n = 322) with curative intent at our single institution between 2005 and 2012. Of the EBRT patients, 302 (93.8%) underwent intensity-modulated radiotherapy. Primary endpoints were overall survival (OS) and cancer-specific survival (CSS). Related to these, other-cause mortality (OCM) was also calculated. Biochemical recurrence-free survival was assessed as a secondary endpoint. Cox proportional hazards model was used for multivariate analysis.

Results

Median follow-up durations were 53 and 45 months, and median ages were 66 and 70 years (P <0.0001), in the RP and EBRT groups, respectively. As a whole, significantly better prognoses of the RP group than the EBRT group were observed for both OS and CSS, although OCM was significantly higher in the EBRT group. There was no death from PC in men with low and intermediate D’Amico risks, except one with intermediate-risk in the EBRT group. In high-risk patients, significantly more patients died from PC in the EBRT group than the RP group. Multivariate analysis demonstrated the RP group to be an independent prognostic factor for better CSS. On the other hand, the EBRT group had a significantly longer biochemical recurrence-free survival than the RP group.

Conclusions

Mortality outcomes of both RP and EBRT were generally favorable in low and intermediate risk patients. Improvement of CSS in high risk patients was seen in patients receiving RP over those receiving EBRT.     

Activin A is essential for Feeder‐free culture of human induced pluripotent stem cells

Feeder‐free culture of human induced pluripotent stem (hiPS) cells is necessary for their clinical application to avoid adverse effects of foreign proteins. hiPS cells were cultured with combinations of activin (A), CHIR99021 (C), basic fibroblast growth factor (F), and leukemia inhibitory factor (L) under feeder‐free conditions. Culture was terminated after 12 passages or when the cell morphology changed from pluripotency. Pluripotency was analyzed by alkaline phosphatase (ALP) staining and immunostaining with antibodies to Oct3/4, Nanog, SSEA4, and TRA‐1‐60. SB431542 (SB), an activin inhibitor, was added to the culture, and the morphology of the cells was observed. hiPS cells cultured with A, AC, and ACL after 12 passages were positive for ALP staining. Oct3/4 was positive in hiPS cells cultured with A, AC, and ACL. hiPS cells were positive for Nanog when cultured with A and AC; however, Nanog signal was weaker in cells cultured with ACL. SSEA4 was positive in hiPS cells cultured with A and AC but almost negative in those cultured with ACL. hiPS cells were positive for TRA‐1‐60 when cultured with A, AC, and ACL. hiPS cells lose their undifferentiated morphology at six passages when cultured with A + SB, five passages with AC + SB, and nine passages with ACL. We conclude that feeder‐free culture of hiPS cells requires A or AC to maintain pluripotency. J. Cell. Biochem. 114: 584–588, 2013. © 2012 Wiley Periodicals, Inc.     

Inhibition of chymotrypsin- and subtilisin-like serine proteases with Tk-serpin from hyperthermophilic archaeon Thermococcus kodakaraensis

A serpin homologue (Tk-serpin) from the hyperthermophilic archaeon Thermococcus kodakaraensis was overproduced in E. coli, purified, and characterized. Tk-serpin irreversibly inhibits Tk-subtilisin (TKS) from the same organism with the second-order association rate constants (k(ass)) of 5.2×103 M?1 s?1 at 40°C and 3.1×10? M?1 s?1 at 80°C, indicating that Tk-serpin inhibits TKS more strongly at 80°C than at 40°C. It also irreversibly inhibits chymotrypsin, subtilisin Carlsberg, and proteinase K at 40°C with the k(ass) values comparable to that for TKS at 80°C. Casein zymography showed that Tk-serpin inhibits these proteases by forming a SDS-resistant complex, which is typical to inhibitory serpins. The ratio of moles of Tk-serpin needed to inhibit 1 mol of protease (stoichiometry of inhibition, SI) varies from 40 to 80 at 20°C, but decreases to the minimum values of 3-7 as the temperature increases. The inhibitory activities of Tk-serpin for these proteases increase as the stabilities of these proteases decrease, suggesting that a flexibility of the active-site of protease is one of the determinants for susceptibility of protease to inhibition by Tk-serpin. This report showed for the first time that Tk-serpin inhibits both chymotrypsin- and subtilisin-like serine proteases and its inhibitory activity increases as the temperature increases up to 100°C.     

Crystallization and Some Properties of Mannanase

A mannan-hydrolyzing enzyme produced by a certain strain of Bacillus subtilis was purified from the culture broth and isolated in a crystalline state by being treated with several ion-exchangers. The optimal pH of the enzyme was 6.0. The enzyme was stable in a pH region of 5.0 to 9.5 and at temperatures less than 55°C. The enzyme attacked only β-1,4-mannosidic linkages in the main chain of galactomannan of soybean seed coat, guar gum and coffee bean, and of glucomannan of konjak (Amorphophalus konjac). Investigation of the hydrolysis mode revealed that the enzyme attacked coffee bean galactomannan endowise to form mannobiose, mannotriose and mannotetraose. The action patterns on several mannohomooligomers prepared from a partial hydrolysate of coffee bean galactomannan were also investigated, indicating that the enzyme preferentially attacked the β-1,4-mannosidic linkages that were present apart three to four mannose residues from the non-reducing end of the mannose chain.