Recruitment of FEN-1-XPG hybrid to local UV sites of damage in XP-G/CS cells

<p><b>Copyright information:</b></p><p>Taken from "Domain swapping between FEN-1 and XPG defines regions in XPG that mediate nucleotide excision repair activity and substrate specificity"</p><p></p><p>Nucleic Acids Research 2007;35(9):3053-3063.</p><p>Published online 22 Apr 2007</p><p>PMCID:PMC1888826.</p><p>© 2007 The Author(s)</p> Cells were irradiated at 150 J/m through a filter with 8 µm pores and fixed 30 min or 24 h after irradiation. Cells were then immunolabelled with antibodies against XPG (green), against the XPB subunit of TFIIH (red) and against 6-4PPs (green). Merged images indicate the overlay of 6-4PPs and XPB staining. Scale bars, 10 µm

Binding efficiencies of XPG, FEN1-XPG and FEN1 towards a single- and a double-flap substrate

<p><b>Copyright information:</b></p><p>Taken from "Domain swapping between FEN-1 and XPG defines regions in XPG that mediate nucleotide excision repair activity and substrate specificity"</p><p></p><p>Nucleic Acids Research 2007;35(9):3053-3063.</p><p>Published online 22 Apr 2007</p><p>PMCID:PMC1888826.</p><p>© 2007 The Author(s)</p> DNA binding was analyzed using electrophoretic mobility shift assays on the same substrates as used in . Either 2.5 nM single 5′ flap substrate (lanes 1–5) or double-flap substrate with a 1-nt 3′ flap (lanes 6–10) was incubated with increasing protein concentrations of XPG (), FEN-1-XPG () or FEN-1 () in the absence of divalent metal ions in nuclease buffer A. The protein concentrations used are indicated (in nM) on the top of each gel

FEN1-XPG does not restore NER activity in an XPG-deficient cell extract

<p><b>Copyright information:</b></p><p>Taken from "Domain swapping between FEN-1 and XPG defines regions in XPG that mediate nucleotide excision repair activity and substrate specificity"</p><p></p><p>Nucleic Acids Research 2007;35(9):3053-3063.</p><p>Published online 22 Apr 2007</p><p>PMCID:PMC1888826.</p><p>© 2007 The Author(s)</p> Cell extracts prepared from XPCS1R0 fibroblasts were incubated with a plasmid containing a single 1, 3 cisplatin adduct in absence of protein (lane 2) or in presence of 15 nM purified recombinant XPG (lane 3), FEN1-XPG (lane 4) or FEN1 (lane 5). The excision products containing the cisplatin adduct were labeled by the annealing of an oligonucleotide complementary to the excised oligonucleotides with a 5′ GGGG overhang and filling in with α-PdCTP and Sequenase enzyme. The products were separated on a 14% denaturing polyacrylamide gel and visualized by autoradiography. A MspI digest of pBR322 (lane 1) served as size marker

Endonuclease activity of FEN-1-XPG hybrid protein on flap and bubble substrates

<p><b>Copyright information:</b></p><p>Taken from "Domain swapping between FEN-1 and XPG defines regions in XPG that mediate nucleotide excision repair activity and substrate specificity"</p><p></p><p>Nucleic Acids Research 2007;35(9):3053-3063.</p><p>Published online 22 Apr 2007</p><p>PMCID:PMC1888826.</p><p>© 2007 The Author(s)</p> () Purified proteins were incubated for 45 min at 30°C with 2.5 nM of flap substrate (lanes 1–8) in the presence of 0.5 mM MnCl for XPG and 1 mM MnCl for FEN-1-XPG and FEN-1. The following protein concentrations were used: 0 nM (lane 1), 0.5 nM (lane 2), 1 nM (lane 3), 2 nM (lane 4), 4 nM (lane 5), 8 nM (lane 6), 20 nM (lane 7) and 40 nM (lane 8). The position of the 5′P label on the substrate is indicated by the asterisk. The length of the products was determined by comparison with an authentic standard and is indicated. () Incision activity of purified proteins on the bubble substrate using the conditions described in . () Cleavage efficiency of XPG, FEN-1 and FEN-1-XPG on the flap substrate (left) and bubble substrate (right) with increasing protein concentration. Graphs represent the quantification of two independent experiments. Standard deviations are represented by error bars

Endonuclease activity of XPG, FEN-1 and FEN-1-XPG on a single- and double-flap substrate

<p><b>Copyright information:</b></p><p>Taken from "Domain swapping between FEN-1 and XPG defines regions in XPG that mediate nucleotide excision repair activity and substrate specificity"</p><p></p><p>Nucleic Acids Research 2007;35(9):3053-3063.</p><p>Published online 22 Apr 2007</p><p>PMCID:PMC1888826.</p><p>© 2007 The Author(s)</p> The endonuclease activity of XPG (), FEN-1-XPG () and FEN-1 () was assessed on a single 5′ flap substrate (lanes 1–7) or on a double-flap substrate with an additional 1-nt 3′ flap (lanes 8–14). 2.5 nM of substrate was incubated for 40 min at 30°C in the presence of 0.5 mM MnCl for XPG or 1 mM MnCl for FEN-1-XPG and FEN-1. The different protein concentrations for XPG, FEN1-XPG and FEN-1 are indicated (in nM) on the top of each gel. The position of the 5′P label on the substrate is indicated by the asterisk. The length of the products was determined by comparison with authentic standards and is indicated. The percentage of substrate cleavage versus protein concentration is presented graphically (right panels) for each protein. Graphs show the quantification of two independent experiments. Standard deviations are indicated by error bars