Volume 36, Issue 4, Pages 642-653 (November 2009) Two-Step Recognition of DNA Damage for Mammalian Nucleotide Excision Repair: Directional Binding of the XPC Complex and DNA Strand Scanning  Kaoru Sugasawa, Jun-ichi Akagi, Ryotaro Nishi, Shigenori Iwai, Fumio Hanaoka  Molecular Cell  Volume 36, Issue 4, Pages 642-653 (November 2009) DOI: 10.1016/j.molcel.2009.09.035 Copyright © 2009 Elsevier Inc. Terms and Conditions

Figure 1 Cell-Free NER of CPDs Is Enhanced by the Presence of a Distal Bubble Structure (A) NER incision assays with DNA substrates containing the indicated UV photolesion and a bubble structure located ∼60 bp 5′ to the lesion. An internal 32P label was present at 12 nucleotides 5′ to the damage site. The substrates were incubated with the XP3BE cell extract in the presence of the indicated amounts of purified XPC-RAD23B. Dual incision products containing the 32P label were detected by denaturing PAGE followed by autoradiography. As a size marker, a 32P-labeled 28-mer oligonucleotide was electrophoresed in parallel. (B) DNase I footprinting of XPC-RAD23B on DNA fragments containing a UV lesion and/or a 5′ bubble as indicated. The 5′ end of each bottom strand (not containing the UV lesion) was labeled with 32P. The amounts of XPC-RAD23B used were 2.5 ng (lanes 3, 8, 13, 18, and 23), 5 ng (lanes 4, 9, 14, 19, and 24), or 10 ng (lanes 5, 10, 15, 20, and 25). The regions that were strongly and weakly protected by XPC-RAD23B are shown by solid and hatched bars, respectively. Arrowheads indicate the sites that became more susceptible to DNase I attack upon binding of XPC-RAD23B. As a sequence marker, Maxam-Gilbert G ladders were electrophoresed in parallel. The positions of the bubble and UV lesion are indicated by arrows. (C) NER incision assays were carried out as in (A), except that DNA substrates containing a bubble structure located on the 3′ side of the UV lesion were used. Molecular Cell 2009 36, 642-653DOI: (10.1016/j.molcel.2009.09.035) Copyright © 2009 Elsevier Inc. Terms and Conditions

Figure 2 NER Machinery Scans a DNA Strand and Excises Only the Most Upstream Lesion (A) NER incision assays using DNA substrates that contained a CPD as well as a three-base bubble at various positions 5′ to the lesion. The internal 32P label was present near the CPD site. The number of base pairs between the CPD and bubble is indicated above for each substrate. The position of a 28-mer oligonucleotide as a size marker is indicated. (B) NER incision assays using DNA substrates that contained a CPD as well as a three-base bubble with or without an AAF adduct at ∼60 bp 5′ to the CPD. The internal 32P label was present near either the CPD or bubble site, as indicated. A 32P-labeled 28-mer oligonucleotide was used as a size marker. Molecular Cell 2009 36, 642-653DOI: (10.1016/j.molcel.2009.09.035) Copyright © 2009 Elsevier Inc. Terms and Conditions

Figure 3 NER Incision Is Greatly Affected by the Orientation of a Loop Structure Present on the 5′ Side of a Lesion (A) Schematic illustration of the DNA substrates used and binding modes of XPC. (B) DNase I footprinting of XPC-RAD23B on DNA substrates that contained a CPD as well as a three-base loop or bubble structure at ∼60 bp 5′ to the CPD. The 5′ end of each bottom strand (not containing the CPD) was 32P labeled. The amounts of XPC-RAD23B used were 2.5 ng (lanes 3, 8, 13, and 18), 5 ng (lanes 4, 9, 14, and 19), or 10 ng (lanes 5, 10, 15, and 20). Strongly and weakly protected regions are indicated by solid and hatched bars, respectively. Arrowheads indicate the sites that became hypersensitive to DNase I attack upon the binding of XPC-RAD23B. As a sequence marker, Maxam-Gilbert G ladders were electrophoresed in parallel, and positions of the CPD and the loop/bubble are indicated by arrows. (C) Effect of the loop structures on dual incision around the distal CPD. NER incision assays were carried out using DNA substrates similar to those in (B), which contained an internal 32P label near the CPD site. As a size marker, a 32P-labeled 25-bp ladder was electrophoresed in parallel. Molecular Cell 2009 36, 642-653DOI: (10.1016/j.molcel.2009.09.035) Copyright © 2009 Elsevier Inc. Terms and Conditions

Figure 4 NER Is Regulated by the Binding Orientation of XPC-RAD23B (A) Schematic illustration of the DNA substrates used and binding modes of XPC. (B and C) DNase I footprinting of XPC-RAD23B on the substrates shown in (A). The 5′ end of each top strand (B) or bottom strand (C) was labeled with 32P. The amounts of XPC-RAD23B used were 2.5 ng (lanes 3, 8, and 13), 5 ng (lanes 4, 9, and 14), or 10 ng (lanes 5, 10, and 15). Strongly and weakly protected regions are indicated by solid and hatched bars, respectively. Arrowheads indicate the sites that became hypersensitive to DNase I attack upon the binding of XPC-RAD23B. These protection patterns are also superimposed in (A). As a sequence marker, Maxam-Gilbert G ladders were electrophoresed in parallel, and positions of the AAF adduct are indicated by arrows. (D) NER incision assays were carried out using the DNA substrates shown in (A), which contained an internal 32P label near the AAF adduct. The position of a 28-mer oligonucleotide as a size marker is indicated. Molecular Cell 2009 36, 642-653DOI: (10.1016/j.molcel.2009.09.035) Copyright © 2009 Elsevier Inc. Terms and Conditions

Figure 5 Assembly of NER Factors around a CPD Site Depends on the Presence of a Distal Loop Structure and ATP Hydrolysis (A) Schematic representation of the DNA beads used for the binding assays. (B) The DNA beads shown in (A) (with or without the 5′ loop) were first incubated in the presence or absence of the XPC-RAD23B complex premixed with centrin 2. After unbound proteins were washed out, the beads were further incubated with various combinations of NER factors (TFIIH, XPA, XPG, and RPA) as indicated in the presence of ATP. Unbound proteins were washed again, and DNA was digested successively with restriction endonucleases BstXI and XhoI to release the loop and CPD sites, respectively. The XPC, XPB, and XPA proteins present in each fraction were detected by immunoblotting. (C) DNA-binding assays as shown in (B), except that the second incubations, including TFIIH, XPA, and XPG, were carried out in the presence or absence of ATP or ATPγS, as indicated. (D) DNA-binding assays were carried out by using the TFIIH complex containing all wild-type subunits or TFIIH containing either the XPDK48R or XPBK346R mutant subunit. ATP was included in the second incubation of all reactions. Molecular Cell 2009 36, 642-653DOI: (10.1016/j.molcel.2009.09.035) Copyright © 2009 Elsevier Inc. Terms and Conditions

Figure 6 A Model for Two-Step Damage Recognition in Global Genome NER (A) A proposed molecular mechanism for the stimulation of CPD repair by a distal loop structure. XPC is targeted to the looped out sequence in the bottom strand, which allows loading of TFIIH from the 5′ side of the top strand and subsequent translocation of the XPD helicase in the 5′-to-3′ direction. Blockage of XPD translocation by an aberrant structure verifies the presence of damage, thereby leading to the assembly of a preincision complex. The XPB ATPase may be required for the prior opening of the DNA duplex, which may enable XPD to bind a DNA strand and start translocation. XPA may play roles in stimulating the XPD helicase activity and/or in the verification of the presence of chemical modifications, in addition to guiding other NER factors into a proper configuration of the preincision complex (through interactions with TFIIH, ERCC1-XPF, and RPA). (B) An extrapolated model for the ordinary GG-NER process. To induce productive NER incision, XPC must interact with an undamaged strand opposite a lesion. After TFIIH loading, the XPD helicase immediately encounters the lesion after beginning translocation along a DNA strand in the 5′-to-3′ direction. Molecular Cell 2009 36, 642-653DOI: (10.1016/j.molcel.2009.09.035) Copyright © 2009 Elsevier Inc. Terms and Conditions