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Results: 7

1.
Scheme 1

Scheme 1. From: Adenine Removal Activity and Bacterial Complementation with the human MutY homologue (MUTYH) and Y165C, G382D, P391L and Q324R variants associated with colorectal cancer.

Minimal kinetic scheme used in the analysis of the adenine glycosylase activity of MUTYH [16].

Sucharita Kundu, et al. DNA Repair (Amst). ;8(12):1400-1410.
2.
Figure 1

Figure 1. From: Adenine Removal Activity and Bacterial Complementation with the human MutY homologue (MUTYH) and Y165C, G382D, P391L and Q324R variants associated with colorectal cancer.

MUTYH variations associated with MAP. (A) Bacillus stearothermophilus MutY-DNA cocrystal structure (1RRQ) with residues corresponding to variations in MUTYH involved in MAP highlighted: Y88, black; G260, pink; P269, dark blue; FeS cluster; brown and orange; adenine, violet; OG, green; DNA, grey; N-terminal domain, light blue; C-terminal domain, yellow, linker region between domains, black dotted line. The sequence present only in MUTYH shown in green dotted line harbors position of Q324. This region is not based on the structure, and was added into a structure generated from coordinates (1RRQ) from the reported structures. (B) Summary of conserved residues across different species. X denotes no matching residue found on sequence alignment using Clustal W.

Sucharita Kundu, et al. DNA Repair (Amst). ;8(12):1400-1410.
3.
Figure 6

Figure 6. From: Adenine Removal Activity and Bacterial Complementation with the human MutY homologue (MUTYH) and Y165C, G382D, P391L and Q324R variants associated with colorectal cancer.

Representative binding data of WT MUTYH. Rate of adenine removal (kobs) was determined at 37°C and 150 mM buffer NaCl concentration. Reaction conditions included and OG:A mismatch-containing duplex DNA substrate (0.01 nM) and enzyme concentrations between 0.02 and 1.5 nM. The kobs value at each concentration was determined at least three times to provide an average value and the error bars represent the standard deviation from the average. The line represents the fit of the data to a single binding site isotherm and provided a Kd of 0.3 ± 0.1 nM.

Sucharita Kundu, et al. DNA Repair (Amst). ;8(12):1400-1410.
4.
Figure 4

Figure 4. From: Adenine Removal Activity and Bacterial Complementation with the human MutY homologue (MUTYH) and Y165C, G382D, P391L and Q324R variants associated with colorectal cancer.

Adenine glycosylase assays of MUTYH under single-turnover conditions with OG:A and G:A substrates. (A) Representative storage phosphor autoradiogram of denaturing PAGE experiment. Bands derived from substrate and product are shown. The minus (−) lane represents the G:A-containing DNA with no enzyme added as a control. (B) Plot of adenine removal activity by WT MUTYH with OG:A- (close circles) and G:A- (open squares) containing substrates at 37 °C. Lines represent fits of the data to a single exponential (equation 2). For this particular experiment, k2 = 1.5 min−1 and <0.002 min−1, for OG:A and G:A, respectively. Reaction conditions: 0.1 nM DNA, 1.5 nM active enzyme in 150 mM NaCl-containing assay buffer.

Sucharita Kundu, et al. DNA Repair (Amst). ;8(12):1400-1410.
5.
Figure 5

Figure 5. From: Adenine Removal Activity and Bacterial Complementation with the human MutY homologue (MUTYH) and Y165C, G382D, P391L and Q324R variants associated with colorectal cancer.

Representative plot of adenine removal activity by WT MUTYH and variants under single-turnover conditions with an OG:A-containing duplex DNA substrate at 37 °C in 150 mM buffer salt concentration. Reaction conditions include 0.1 nM DNA and 1.5 nM active enzyme. WT MUTYH (closed circle), Y165C (open circle), Q324R (open triangle), G382D (open square), P391L (open rhombus). The lines represent fits to a single exponential curve (equation 2) to obtain kobs (= k2). The values for the rate constants determined from at least three measurements for each enzyme (from different enzyme preparations) were averaged and are listed in Table 1.

Sucharita Kundu, et al. DNA Repair (Amst). ;8(12):1400-1410.
6.
Figure 2

Figure 2. From: Adenine Removal Activity and Bacterial Complementation with the human MutY homologue (MUTYH) and Y165C, G382D, P391L and Q324R variants associated with colorectal cancer.

Adenine glycosylase assays of WT MUTYH under multiple-turnover conditions (A) Representative plot of adenine removal activity by WT MUTYH under multiple-turnover conditions at 37 °C and 150 mM buffer salt concentration with an OG:A-containing duplex DNA substrate (10 nM). Line represents fit to equation 1 to determine A0 and k3, which are = 0.92 and 0.008 min−1, respectively, for this particular data set. (B) Stability assays of WT MUTYH based on A0 values determined after incubation of WT MUTYH in assay buffer show no significant reduction in enzyme activity as a function of time. The activity of MUTYH without prior incubation was normalized to 1. Aliquots were removed at intervals of 2, 5 and 10 minutes for adenine glycosylase assays under multiple-turnover conditions, and active enzyme concentrations were determined from the burst amplitudes of the progress curves. The adenine glycosylase activity at each time point was measured in at least three separate experiments. The grey bars represent the average on the basis of the initial amplitude and the error bars represent the standard deviation from the average (as a percentage of the normalized value).

Sucharita Kundu, et al. DNA Repair (Amst). ;8(12):1400-1410.
7.
Figure 3

Figure 3. From: Adenine Removal Activity and Bacterial Complementation with the human MutY homologue (MUTYH) and Y165C, G382D, P391L and Q324R variants associated with colorectal cancer.

Comparison of burst amplitudes of MUTYH variants to the WT enzyme.
(A) Representative plot of adenine removal activity by WT MUTYH and variants under multiple-turnover conditions at 37 °C and 150 mM buffer salt concentration with an OG:A containing duplex DNA substrate (10 nM). WT MUTYH (closed circles), Y165C (closed squares), Q324R (closed stars), G382D (open triangles), P391L (open rhombus) were added to the reaction mix to provide equal total protein concentrations (30 ug as measured by Bradford assay). Lines represent fit to equation 1, with values of A0 obtained for the various enzymes of WT = 0.93; Y165C = 0.19; Q324R= 0.38; G382D = 0.19; P391L = 0.24.
(B) Stability assays of WT MUTYH and variants in assay buffer as measured by glycosylase assays show no significant reduction in enzyme activity as a function of time. Aliquots were removed at intervals of 0, 5 and 10 minutes for adenine glycosylase assays under multiple-turnover conditions and fit to equation 1 to determine the burst amplitudes, A0. The activity of MUTYH without prior incubation was normalized to 1. The bars at 5 and 10 min represent the average loss of activity on incubation. The adenine glycosylase activity at each time point was measured in at least three separate experiments and the error bars represent one standard deviation from the average (as a percentage of the normalized value).

Sucharita Kundu, et al. DNA Repair (Amst). ;8(12):1400-1410.

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