ALKBH1 deficient fibroblasts are not more sensitive to DNA modifying agents than hALKBH1 expressing cells.

<p>A. Expression of hALKBH1 in mouse Alkbh1^−/− fibroblasts. Lysates of indicated clones (20 µg) were analyzed by Western blot using a monoclonal anti-ALKBH1 antibody. The positions of two molecular mass markers are shown in kDa. B. Growth curves of Alkbh1^−/− fibroblasts and the same cells producing hALKBH1. Cells were plated at low cell density, harvested at the indicated time point by trypsinization and counted. Survival curves for Alkbh1^−/− fibroblasts expressing hALKBH1 or containing a vector control when treated with various concentrations of H₂O₂ (C) or MMS (D). Error bars indicate standard deviations derived from three (H₂O₂) or two (MMS) independent experiments.</p

Ig class switching to IgG1 is not reduced in Alkbh1^−/− splenic B cells.

<p>A. Flow cytometry results showing surface antibody expression four days after treatment to switch to IgG1. B. Analysis of flow cytometry data related to formation of IgG1 in ALKBH1 deficient splenic B cells normalized to the switching rate of Alkbh1^+/+ B cells. Four mice were used for each genotype and error bars represent standard deviations from these four independent experiments.</p

Characterization of ALKBH1 deficient mice.

<p>A. Offspring distribution of crosses between heterozygous male and female mice. Numbers of pups from nine different litters are shown, demonstrating non-Mendelian inheritance of the knockout allele. B. Offspring distribution after crosses between Alkbh1^+/− females and Alkbh1^−/− males. C. Male-to-female ratios of Alkbh1^+/+, Alkbh1^+/−, and Alkbh1^−/− mice show a distorted sex ratio in favor of males for the knockout mice. D. Different sizes of Alkbh1^−/− and wild type (larger pup) littermates at two weeks of age. E. Weight development of Alkbh1^−/− and wild type pups. Alkbh1^−/− and wild type pups were derived from breeding either two ALKBH1 deficient or two wild type parent animals. For each time point, the weights of at least twelve animals were used to calculate the average and the error bars represent standard deviations.</p

ALKBH7 Variant Related to Prostate Cancer Exhibits Altered Substrate Binding

<div><p>The search for prostate cancer biomarkers has received increased attention and several DNA repair related enzymes have been linked to this dysfunction. Here we report a targeted search for single nucleotide polymorphisms (SNPs) and functional impact characterization of human ALKBH family dioxygenases related to prostate cancer. Our results uncovered a SNP of <i>ALKBH7</i>, rs7540, which is associated with prostate cancer disease in a statistically significantly manner in two separate cohorts, and maintained in African American men. Comparisons of molecular dynamics (MD) simulations on the wild-type and variant protein structures indicate that the resulting alteration in the enzyme induces a significant structural change that reduces ALKBH7’s ability to bind its cosubstrate. Experimental spectroscopy studies with purified proteins validate our MD predictions and corroborate the conclusion that this cancer-associated mutation affects productive cosubstrate binding in ALKBH7.</p></div

Difference absorption spectra of WT ALKBH7 and its R191Q variant.

<p>The spectra of the anaerobic proteins (0.3 mM) were recorded in the presence of 2 mM α-kg and 100 μM Fe(II). The difference spectra were obtained by subtracting the spectra for proteins with α-kg, but without the metal. <b>A</b>, WT ALKBH7; <b>B</b>, R191Q ALKBH7.</p

Structural and dynamic comparison between WT and R191Q ALKBH7 with bound α-kg.

<p><b>a,</b> Overlay of representative structures for WT (gray) and R191Q mutant (yellow) forms of ALKBH7. Active site residues and α-kg as well as the site undergoing substitution are displayed (licorice). <b>b,</b> 180 degree rotation and close-up of the substituted site. <b>c,</b> 90 degree rotation and close-up of the active site, with each relevant active site residue and α-kg labeled. Dashed lines in gray represent the original bonds to the metal ion in the crystal structure, and dashed lines in orange represent the new bonds to the metal ion near the end of the trajectory for the variant protein. <b>d,</b> Correlation difference for each residue in the WT protein with respect to the R191Q variant mapped onto the protein structure using the mutation site as the reference. <b>e,</b> Distance analysis for key residues in the SNP variant and active sites (with respect to their centers of mass) throughout the simulation trajectory.</p

Average binding enthalpies for cosubstrate α-kg and Fe at the active site (in kcal/mol) for the simulation.

<p>Results for the product (succinate) are reported in the Supplementary Information.</p

Hydrogen bond analysis for the WT/R191Q variant with α-kg.

<p>Residues colored in red denote amino acids involved in H-bonds for over 30% of the WT trajectory and broken for over 90% of the R191Q variant trajectory. Residues colored in orange are involved in hydrogen bonds for both trajectories, but are present for at least 30% less of the time in the variant trajectory. The hydrogen bonds between these residues are displayed in blue. The corresponding analysis for the WT/SNP variant with succinate are given in the Supplementary Information.</p

SNPs of <i>ALKBH</i> family genes with significant association (p ≤ 0.05) with prostate cancer phenotype.

<p>SNPs of <i>ALKBH</i> family genes with significant association (p ≤ 0.05) with prostate cancer phenotype.</p