Base Excision Repair of Oxidative DNA Damage Activated by XPG Protein Arne Klungland, Matthias Höss, Daniela Gunz, Angelos Constantinou, Stuart G Clarkson, Paul W Doetsch, Philip H Bolton, Richard D Wood, Tomas Lindahl  Molecular Cell  Volume 3, Issue 1, Pages 33-42 (January 1999) DOI: 10.1016/S1097-2765(00)80172-0

Figure 7 DNA Base Excision Repair by Human Enzymes The proteins assumed to be responsible for the various steps are indicated. Left: Pathway for repair of oxidized pyrimidines. Right: Branched pathway for removal of uracil from DNA, showing the major route involving replacement of a single nucleotide and a second route for longer-patch repair. Molecular Cell 1999 3, 33-42DOI: (10.1016/S1097-2765(00)80172-0)

Figure 1 Reconstitution of Base Excision Repair of the Toxic Oxidative DNA Lesion Thymine Glycol with Human Enzymes (A) BER of a double-stranded oligonucleotide containing a thymine glycol (Tg) residue opposite adenine. Substrate DNA was incubated with the purified human enzymes hNth1 (3750 fmol), HAP1 (145 fmol), DNA polymerase β (0.5 fmol), and DNA ligase III-XRCC1 heterodimer (100 fmol) as indicated. Reaction products were analyzed by autoradiography after electrophoretic separation in a denaturing 20% polyacrylamide gel. Sizes (nucleotides, nt) and positions of reaction products are indicated. (B) Structure of Tg and substrate DNA. The sequence of the Tg containing strand is given, and the Tg residue is shown as †. (C) Predicted partial or complete reaction products. Asterisks indicate the position of the 32P label. Molecular Cell 1999 3, 33-42DOI: (10.1016/S1097-2765(00)80172-0)

Figure 3 Promotion of hNth1 Activity by XPG Protein (A) Nicking assay with a Tg containing DNA substrate (Figure 1B). A limiting amount (75 fmol) of human Nth1 was incubated under standard assay conditions with the double-stranded oligonucleotide substrate and increasing amounts of human XPG, as indicated. Reaction products were analyzed by autoradiography after electrophoretic separation on denaturing 20% polyacrylamide gels. (B) Fragmented and full-length material from (A) were measured and results with hNth1 containing fractions are shown. The amount of cleaved product in the absence of hNth1 was <2%. (C) Nicking assay with a dihydrouracil-containing oligonucleotide substrate. Details as in (A). (D) Fragmented and full-length material from (C) was quantified as in (B). Molecular Cell 1999 3, 33-42DOI: (10.1016/S1097-2765(00)80172-0)

Figure 4 Effect of Human XPG Protein on the Activities of S. pombe Nth and E. coli Nth with a Dihydrouracil-Containing DNA Substrate Reaction conditions and product analysis were as in Figure 3. (A) A limiting amount of S. pombe Nth was incubated with increasing amounts of XPG protein as indicated. (B) A limiting amount of E. coli Nth was incubated with XPG protein as indicated. Molecular Cell 1999 3, 33-42DOI: (10.1016/S1097-2765(00)80172-0)

Figure 2 Reconstitution of Base Excision Repair of the Mutagenic DNA Lesion Dihydrouracil (A) BER of a double-stranded oligonucleotide containing a dihydrouracil opposite a guanine residue. Substrate DNA was incubated with purified human enzymes as in Figure 1. (B) Structure of dihydrouracil and substrate DNA. The sequence of the lesion-containing strand is given, and the dihydrouracil residue is shown as ‡. (C) Scheme as in Figure 1C. Molecular Cell 1999 3, 33-42DOI: (10.1016/S1097-2765(00)80172-0)

Figure 5 Improved Binding of Human Nth1 at DNA Lesions in the Presence of XPG Protein (A) Electrophoretic mobility shift assay. An amount of hNth1 limiting in standard enzyme assays was incubated with a double-stranded 60-mer oligonucleotide containing a single reduced abasic site. Reaction mixtures were supplemented with XPG protein or BSA, as indicated. (B) Same electrophoretic mobility shift assay, using E. coli Nth instead of human Nth. Lower amounts of the much more active bacterial enzyme were employed. Molecular Cell 1999 3, 33-42DOI: (10.1016/S1097-2765(00)80172-0)

Figure 6 Stimulation of hNth1 Activity by Mutant and Wild-Type XPG Proteins Nicking assays used a dihydrouracil-containing substrate (see Figure 2B). Reactions were carried out as in Figure 3A except that immunoprecipitated XPG protein on antibody beads (1 to 4 μl) was used, either baculovirus-produced wild type (P, lane 4), or proteins expressed in a vaccinia system: wild-type (wt, lane 5), E791A (lane 6), A792V (lane 7). Negative controls were mock immunoprecipitations from a cell extract overexpressing Sendai virus N protein in the vaccinia system (S, lane 8) or from buffer alone (C, lane 9). Baculovirus-produced XPG without immunoprecipitation gave the stimulation in lane 3 (P). Molecular Cell 1999 3, 33-42DOI: (10.1016/S1097-2765(00)80172-0)