Stimulation of DNA Glycosylase Activities by XPC Protein Complex: Roles of Protein-Protein Interactions

Abstract

We showed that XPC complex, which is a DNA damage detector for nucleotide excision repair, stimulates activity of thymine DNA glycosylase (TDG) that initiates base excision repair. XPC appeared to facilitate the enzymatic turnover of TDG by promoting displacement from its own product abasic site, although the precise mechanism underlying this stimulation has not been clarified. Here we show that XPC has only marginal effects on the activity of E. coli TDG homolog (EcMUG), which remains bound to the abasic site like human TDG but does not significantly interacts with XPC. On the contrary, XPC significantly stimulates the activities of sumoylated TDG and SMUG1, both of which exhibit quite different enzymatic kinetics from unmodified TDG but interact with XPC. These results point to importance of physical interactions for stimulation of DNA glycosylases by XPC and have implications in the molecular mechanisms underlying mutagenesis and carcinogenesis in XP-C patients.

Figure 1

DNA substrates used in this study.

Figure 2

XPC stimulates the activity of TDG that cleaves uracil from G/U mismatches. The activity of TDG was measured by using 1.6 nM of 60-bp DNA containing a single G/U mismatch as a substrate. The reaction was done at 30°C for the indicated time with 0.42 nM His-TDG in the presence of various concentrations of XPC-RAD23B. The DNA samples were then purified and subjected to alkali-treatment to cleave the resulting AP sites and separated with denaturing PAGE. The ratio of the cleaved product was calculated and plotted as a graph. The mean values and standard errors were calculated from at least two independent experiments.

Figure 3

XPC physically interacts with SUMO-1-conjugated TDG, but not with EcMUG. Glutathione-Sepharose beads (20 μl) were incubated in 100 μl of the binding mixture containing 10 nM of GST (negative control), GST-TDG (positive control), GST-EcMUG, or SUMO-1-GST-TDG in the presence of the same concentration of XPC-RAD23B. After extensive washing, bound proteins were eluted with buffer containing 10 mM glutathione. One-fourth of each eluate was mixed with whole cell extract from XP4PASV cells which do not express XPC and subjected to 8% SDS-PAGE followed by immunoblotting with anti-XPC antibody (upper panel). The same samples were also subjected to 12% SDS-PAGE followed by immunoblotting with anti-GST antibody (lower panel).

Figure 4

The effect of XPC on the activity of EcMUG that cleaves uracil from G/U mismatches. The activity of EcMUG was measured by using 1.6 nM of 60-bp DNA containing a single G/U mismatch as a substrate. The reaction was done at 30°C for the specified time with 0.8 nM His-EcMUG and indicated concentrations of XPC-RAD23B. The DNA samples were then purified and subjected to alkali-treatment to cleave the resulting AP sites and separated with denaturing PAGE. The ratio of the cleaved product was calculated and plotted as a graph. The mean values and standard errors were calculated from at least two independent experiments.

Figure 5

The effect of XPC on the activity of SUMO-1-modified TDG that cleaves uracil from G/U mismatches. (a) Silver staining of the purified recombinant nonmodified His-TDG and SUMO-1-modified His-TDG. M represents the size marker. (b) The sumoylation of TDG was verified with western blot analyses using anti-TDG antibody (left) or anti-SUMO-1 antibody (right). (c) The activity of sumoylated His-TDG was measured by using 1.6 nM of 60-bp DNA containing a single G/U mismatch as a substrate. The reaction was done at 30°C for the time indicated with 2 nM SUMO-1-conjugated His-TDG in the presence of XPC-RAD23B. The DNA samples were then purified and subjected to alkali-treatment to cleave the resulting AP sites and separated with denaturing PAGE. The ratio of the cleaved product was calculated and plotted as a graph. The mean values and standard errors were calculated from at least two independent experiments.

Figure 6

The effect of XPC on the activity of other DNA glycosylases involved in G/T and/or G/U mismatch repair. The activity of UNG2 (a), MBD4 (b), or SMUG1 ((c) and (d)) was measured in the presence of various amounts of XPC-RAD23B. In each reaction, 1.6 nM of 60-bp DNA containing a single G/U mismatch ((a) and (c)), G/T mismatch (b), or single stranded 30-mer oligonucleotide containing a single uracil residue (d) was used as the substrate. The reaction was done at 30°C for the time indicated with specified concentration of purified recombinant proteins as shown. The DNA samples were then purified and subjected to alkali-treatment to cleave the resulting AP sites and separated with denaturing PAGE. The ratio of the cleaved product was calculated and plotted as a graph. The mean values and standard errors were calculated from at least two independent experiments.

Figure 7

XPC interacts physically with SMUG1 in vitro. Glutathione-Sepharose beads (20 μl) were incubated in 100 μl of the binding mixture with 10 nM of either GST or GST-TDG in the presence of the same concentration of XPC-RAD23B. After extensive washing, bound proteins were eluted with buffer containing 10 mM glutathione. One-fourth of each eluate was mixed with whole cell extract from XP4PASV cells which do not express XPC and subjected to 8% SDS-PAGE followed by immunoblotting with anti-XPC antibody (upper panel). The same samples were also subjected to 12% SDS-PAGE followed by immunoblotting with anti-GST antibody (lower panel).