PMC

G132R and G156C do not change the overall structure of MGMT. Multiple circular dichroism spectra for WT, G132R, and G156C purified human MGMT were measured, averaged, and normalized. The overall shapes of the spectra, with peaks of intensity at 208 and 217 nm, were the same for all three proteins.

G156C does not react with O⁶BG. 2 × 10⁶ EMT6 mouse mammary carcinoma cells were incubated with tritated benzylguanine. Free benzylguanine was removed by washing with methanol and the amount of tritium transferred to cells was measured by scintillation counting. Averages and standard deviations for triplicate assays are shown. **P < 0.001; P < 0.0001.

MGMT G132R and G156C are near the active site of MGMT. MGMT bound to the minor groove of DNA (black, bottom center), with one DNA base flipped into the MGMT active site (acceptor residue at CYS145) []. Dotted lines represent measured distances, numbers are distances measured in Ångströms (Å). Mutations to cysteine and arginine could result in residues larger than glycine and may distort the structure. Generated in MacPyMol using the 1T38 crystal structure [].

MGMT G132R and G156C do not fully rescue methyl-transferase deficient E. coli. (A) Western blot for MGMT expression in GWR111 E. coli shows the ratio of WT:G132R:G156C expression to be 1:11.0:11.2. There is no discernable MGMT expression in empty vector cells. (B) Survival curves for E. coli. Log-phase GWR111 cultures expressing wild type (■) or variant (● G132R, ▽G156C) human MGMT and the empty pBAD expression vector (✖) were incubated with graded doses of MNNG for 30 min then plated in serial dilutions. Surviving colonies were counted the next day. Representative survival curves are shown.

MGMT G132R and G156C cannot fully rescue MGMT-deficient mammalian cells. (A) Expression of the variants in EMT6 cells. The ratio of WT:G132R:G156C expressed in these pools is 1:1.3:0.7. (B) Survival of EMT6 cells after treatment with MNNG. EMT6 mouse mammary carcinoma cells transfected with empty pRVY expression vector (×), pRVY containing WT MGMT (■), G132R MGMT (●), or G156C MGMT (▽) were treated with graded doses of MNNG and plated over a range of dilutions. Colonies were stained 10 d later and counted. Points are geometric means from three separate experiments on pools within a range of three passages and error bars represent the standard error of the mean. * P = 0.03 and P = 0.04 for WT versus G132R and G156C, respectively, paired t-test.

MGMT variants have decreased affinity for substrate. (A) Schematic of substrates and the cutting ability PvuII on those substrates. (B) Gel showing separation of repaired and unrepaired products of time course for 33 nM of G132R with 5.3 nM substrate. Time is given in seconds. U is undamaged control substrate. (C) Determination of pre-steady state k_obs for G132R. Fraction of substrate repaired is plotted against time. The scale of the X-axis changes at 100 s to allow visualization of points in the early timepoints of the curve, before saturation. 5.3 nM substrate was reacted with varying concentrations (○ 500 nM, ▲ 333 nM, * 33 nM, ◆25 nM, + 5 nM, ■ 2 nM) of G132R MGMT in separate time courses. Repair was evaluated via gel electrophoresis followed by quantification on a phosphorimager and normalized to a no damage control. A single exponential equation was fit to the plot to yield a k_obs for each concentration. (D) Single hyperbolic plot to determine K_d and k_r for MGMT G132R. The k_obs values as calculated in (A) were plotted against nM concentration of MGMT and fit to a single hyperbolic equation to determine the reaction rate (k_r) and the binding efficiency (K_d).