Survival of M13 and MS2 phages and MMS-induced mutagenesis in <i>E. coli alkB⁻</i> strain.

<p>The survival of MMS (A, C) or CAA (B) treated M13 (A,B) or MS2 (C) phage in the <i>E. coli alkB</i>⁻ strains harboring pVB1x plasmids expressing cyanobacterial AlkB homologs (“empty” vector served as control). Panel D presents the frequency of MMS-induced Arg⁺ revertants in the same strains. Mean values are from at least 4 independent experiments with standard deviation, asterisk indicates statistically significant difference compared to strain with pVB1x plasmid on the basis of Student t-test (p<0.05, two-sided, implication of different variances).</p

CLANS clustering of 1943 ALKBH proteins.

<p>CLANS clustering of 1943 ALKBH proteins.</p

Subcellular localization of GFP-tagged AlkB homologs.

<p><i>A. thaliana</i> protoplasts from cell suspension culture were transfected with constructs expressing the indicated proteins in N- and C- terminal fusion with GFP and visualized by confocal laser-scanning microscopy. AlkB homologs represent 6 types of subcellular localization. The image for AtALKBH1D homolog localization is merged with the red autofluorescence of chlorophyll (orange color comes from overlay of GFP and chlorophyll fluorescence). For comparison GFP fluorescence alone (pSAT6-eGFP) was also analyzed. N - nucleus, Nu - nucleous, Ch - chloroplasts, V - vacuole. For more details see Supplementary Data.</p

CLANS clustering of 1943 ALKBH proteins.

<p>Particular ALKBH groups (A) or taxons (B) are color coded. The groups numbered 9–16 are novel ALKBH family members described in this paper. Color codes are explained in the legend in the upper right corner of (B).</p

Relocalization of <i>A. thaliana</i> nucleo-cytoplasmic AlkB homologs after LMB inhibition of nuclear export.

<p>Protoplasts transfected with GFP-AlkB homologs were incubated with LMB for 4 h and analyzed for GFP fluorescence using confocal laser-scanning microscopy. As a positive control plasmid expressing GFP-NLS-CHS-NES was used. All of the protoplasts transfected with AtALKBH6 and AtALKBH9C(l), and 30% of those transfected with AtALKBH8B changed their localization to exclusively nuclear after LMB treatment. In the case of AtALKBH9A and AtALKBH10B the signal in the nucleus was more intense after incubation with LMB, and LMB did not inhibit AtALKBH1A, AtALKBH8A, AtALKBH9C, AtALKBH6s and AtTRM9 export. The scans demonstrate the main localization of presented homologs.</p

The <i>Cyanobacteria</i> strains with indicated <i>alkB</i> homologs (GI numbers according to NCBI database).

<p>The <i>Cyanobacteria</i> strains with indicated <i>alkB</i> homologs (GI numbers according to NCBI database).</p

Relocalization of <i>A. thaliana</i> AlkB homologs after MMS treatment.

<p><i>A. thaliana</i> suspension culture protoplast transiently transfected with GFP-fused AlkB homologs were incubated with 10 mM MMS for 30 min and analyzed using confocal laser-scanning microscopy. While the localization of AtALKBH1A, AtALKBH1B, AtALKBH8B, AtALKBH9C homologs and AtTRM9 protein did not change during the MMS-treatment, the localization of AtALKBH1C, AtALKBH6 (s), AtALKBH9A and AtALKBH9C (l) shifted to more nuclear; AtALKBH6 localized exclusively in the nucleus. In the case of the AtALKBH8A, both cytoplasmatic and nuclear localizations were observed. The scan demonstrate the main localization of presented homologs.</p

The time course of repair of the studied adducts at the pH optimal for each substrate.

<p>Comparison of repair efficiency between EcAlkB at 37°C and PpAlkB at 30 and 37°C. Reactions for each substrate (0.5 nmol) were performed in parallel and differ only in the protein added. The adducts and the conditions applied for their repair were as follows: 3meC, pH 7.5, 100 µM Fe(II), 5 pmol of the studied enzyme (A); HPC pH 7.5, 100 µM Fe(II), 10 pmol of the studied enzyme (B); HEC pH 5.8, 1 mM Fe(II), 10 pmol of the studied enzyme (C); εA pH 5.0 for PpAlkB and 4.6 for EcAlkB, 3 mM Fe(II) 40 pmol of the studied enzyme (D); and εC pH 5.0 for PpAlkB and 4.6 for EcAlkB, 5 mM Fe(II), 80 pmol of the studied enzyme (E).</p

Sensitivity of <i>P. putida</i> AlkA-, AlkB-, and Ada-deficient mutants to alkylating chemicals MMS and MNNG.

<p>The serial dilution drop test of <i>P</i>. <i>putida</i> mutants: 1 – wild-type, 2 – alkB‾, 3 – alkA‾, 4 – ada‾, 5 – alkB‾ada‾, 6 – alkA‾ada‾, 7 – alkA‾alkB‾, 8 – alkA‾alkB‾ada‾ on the medium containing the indicated concentrations of MMS (A) or MNNG (B). The vertical arrow shows the direction of dilution: from 10^-1 to 10^-8.</p

Frequency of MMS (A, B) or MNNG (C, D)-induced Rif^R mutations in <i>P. putida</i> (A, C) and <i>E. coli</i> (B, D) strains.

<p>Frequency of MMS (A, B) or MNNG (C, D)-induced Rif^R mutations in <i>P. putida</i> (A, C) and <i>E. coli</i> (B, D) strains.</p