Bound protein was eluted with a step gradient of 2 column volumes

Bound protein was eluted with a step gradient of 2 column volumes of the elution buffer containing 40, 60, 80, 100, 140, 180, 220 and 250 mM imidazole. Fractions containing purified protein were pooled and dialysed against 25 mM Tris-HCl, pH 7.5, 300 mM NaCl and 10% glycerol. Assay for base excision of 8oxoG opposite C, A, G or T Duplex DNA substrates containing a single 8oxoG opposite of C, A, G or T were generated by 32P 5′ end-labelling of oligonucleotides, using T4 polynucleotide kinase (New England Biolabs, MA) followed by annealing to a complementary oligonucleotide

[20]. The oligonucleotide sequences of the DNA substrates are listed in Table PD0332991 mw 2. DNA glycosylase reactions were performed

by mixing purified protein with 10–50 fmol DNA substrate Selleck LY2109761 in a total volume of 10 μl. The enzyme activities were assayed in the reaction buffer previously described [20] and incubated at 37°C for 30 min. E. coli Fpg (New England Biolabs, MA) was included as a positive control. Products of the reactions were separated by 20% denaturing PAGE and visualized by phosphorimaging. The assay was performed in triplicate. Assay for formamidopyrimidine (faPy) DNA glycosylase activity N-[H3]-N-methyl-N’-nitrosourea (MNU; 1.5 Ci mmol-1) was used to prepare poly(dG-dC) DNA (12,000 dpm mg-1) [21]. DNA glycosylase activity was assayed by mixing purified protein with substrate in a reaction buffer containing 70 mM 3-(N-morpholino) propane sulfonic acid, pH 7.5, 1 mM EDTA, 1 Selleckchem Forskolin mM dithiothreitol (DTT) and 5% glycerol for 30 min at 37°C. Removal of bases was measured in a total reaction volume of 50 μl containing 14 μg of DNA substrate and 500 ng of purified meningococcal protein or 160 U of E. coli Fpg (New England Biolabs, MA). The assay was repeated 5 times. Screening for phase variation

by use of a universal rate of switching (UROS) cassette To promote efficient natural transformation, a 12-mer DNA uptake sequence was inserted into plasmid pARR2107 containing a Universal Rate of Switching (UROS) cassette (kind gift from H. L. Alexander, Emory University School of Medicine, Atlanta, GA) [22], creating plasmid pUD. Mc strain Z1099 (kind gift from D. A. Caugant, Norwegian Institute of Public Health, Oslo, Norway) was transformed with pUD and named NmZ1099_UROS. The mutS and fpg genes of NmZ1099_UROS were inactivated by insertion of a kanamycin resistance cassette as described by Davidsen et al., 2007 [9] in two separate genetic transformations creating strains NmZ1099_UROSΔmutS and NmZ1099_UROSΔfpg. The CUDC-907 mononucleotide tract of 8 G residues preceding the spectinomycin resistance gene of the UROS cassette was confirmed as an intact 8-mer by PCR and sequencing (by using the primers listed in Table 2) in all three strains before switching frequency/phase variation was assessed.

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