Lesion recognition by XPC, TFIIH and XPA in DNA excision repair

57

58

59

60

61

62

63

64

65

66

67

Yasuda, G. et al. In vivo destabilization and functional defects of the xeroderma

pigmentosum C protein caused by a pathogenic missense mutation. Mol Cell Biol

27, 6606-6614, doi:10.1128/MCB.02166-06 (2007).

Henricksen, L. A., Umbricht, C. B. & Wold, M. S. Recombinant replication protein

A: expression, complex formation, and functional characterization. The Journal of

biological chemistry 269, 11121-11132 (1994).

Kim, M.-S., Lapkouski, M., Yang, W. & Gellert, M. Crystal structure of the V(D)J

recombinase RAG1-RAG2. Nature 518, 507-511, doi:10.1038/nature14174

(2015).

Schorb, M., Haberbosch, I., Hagen, W. J. H., Schwab, Y. & Mastronarde, D. N.

Software tools for automated transmission electron microscopy. Nat Methods 16,

471-477, doi:10.1038/s41592-019-0396-9 (2019).

Zheng, S. Q., Palovcak, E., Armache, J.P., Verba, K.A., Cheng, Y., and Agard,

D.A. MotionCor2: anisotropic correction of beam-induced motion for improved

cryo-electron microscopy. Nat Methods 14, 331-332 (2017).

Zhang, K. Gctf: Real-time CTF determination and correction. J Struct Biol 193, 112, doi:10.1016/j.jsb.2015.11.003 (2016).

Fernandez-Leiro, R., and Scheres, S.H.W. A pipeline approach to single-particle

processing in RELION. Acta Crystallogr D Struct Biol 73, 496-502 (2017).

Pettersen, E. F. et al. UCSF Chimera--a visualization system for exploratory

research and analysis. J Comput Chem 25, 1605-1612, doi:10.1002/jcc.20084

(2004).

Sanchez-Garcia, R. et al. DeepEMhancer: a deep learning solution for cryo-EM

volume post-processing. Commun Biol 4, 874, doi:10.1038/s42003-021-02399-1

(2021).

Varadi, M. et al. AlphaFold Protein Structure Database: massively expanding the

structural coverage of protein-sequence space with high-accuracy models. Nucleic

acids research 50, D439-D444, doi:10.1093/nar/gkab1061 (2022).

Emsley, P., Lohkamp, B., Scott, W.G., and Cowtan, K. Features and development

of Coot. Acta Crystallogr D Biol Crystallogr 66, 486-501 (2010).

Adams, P. D. et al. PHENIX: a comprehensive Python-based system for

macromolecular structure solution. Acta Crystallogr D Biol Crystallogr 66, 213-221,

doi:S0907444909052925 [pii] 10.1107/S0907444909052925 (2010).

24

Kim et al., 2022

733

734

735

736

737

738

739

740

741

68

69

70

Chen, V. B. et al. MolProbity: all-atom structure validation for macromolecular

crystallography.

Acta

Crystallogr

Biol

Crystallogr

66,

12-21,

doi:10.1107/S0907444909042073 (2010).

Swint-Kruse, L., and Brown, C.S. Resmap: automated representation of

macromolecular interfaces as two-dimensional networks. Bioinformatics 21, 33273328 (2005).

Kucukelbir, A., Sigworth, F. J. & Tagare, H. D. Quantifying the local resolution of

cryo-EM density maps. Nat Methods 11, 63-65, doi:10.1038/nmeth.2727 (2014).

742

25

Kim et al., 2022

743

744

745

746

747

748

749

750

751

752

753

754

755

756

757

758

759

760

761

762

763

764

765

766

767

768

769

770

771

Acknowledgements

The authors thank Drs. R. Craigie, M. Gellert, H. Lans, D. Leahy and W. Vermeulen

for critical reacting of the manuscript and J. Li for preparing carbon-filmed cryoEM grids.

This work utilized the Cryo-Electron Microscopy Core facility, NIDDK, and the NIH MultiInstitute Cryo-EM Facility (MICEF). This research was supported by National Institute of

Diabetes, Digestive and Kidney Disease (DK075037) to W.Y., and Grants-in-Aid

(KAKENHI) (Grant Number JP16H06307 and JP21H03598) to K.S.

Author contribution

J.K. carried out biochemical and structural studies; C.L.L. carried out dual incision

assays; F.M.G. developed the GraFix protocol; H.W. and Y.C. helped with cryoEM grid

preparation and data acquisition, Y.C. and X.C. helped with cryoEM data processing and

map improvement; K.S. and W.Y. conceived the research project; W.Y. supervised

experimental design and data interpretation; K.S. and F.H. helped with data interpretation;

all authors were involved in writing the paper and adhere to the “Inclusion & Ethics”

regulation.

Data and Code Availability

The structures and cryoEM maps have been deposited with PDB and EMDB with

accession codes of 8EBS, 8EBT and 8EBU, EMD-27996, 27997 and 27998 for C7CD,

C7CAD and C7AD of Cy5; 8EBV, 8EBW, 8EBX and 8EBY and EMD-27999, 28000,

28001 and 28002 for C7CD1, C7CD2, C7CAD and C7AD of AP. The focused refinement

maps of XPC-lesion DNA in Cy5_C7CD and the C-terminal domain of XPC in C7CAD

and C7AD have been deposited with EMDB with accession codes EMD-29674 and 29673,

respectively. These data will be released immediately upon publication. Other research

materials reported here are available upon request.

Competing interests

The authors declare no competing interest.

26

Kim et al., 2022

772

Extended Data

773

774

775

776

777

778

779

780

781

782

783

784

785

786

787

788

789

790

791

792

793

794

795

796

797

798

799

800

801

802

803

804

805

806

807

808

809

810

811

812

813

814

815

816

Extended Data Fig. 1 Structures of NER complexes. (a) Yeast Rad4 (XPC) complexed

with Core7 and damaged DNA (orange and yellow) (PDB: 7K04 at 9.25 Å). (b) Human

XPA and Core7 are complexed with undamaged but branched DNA (PDB: 6RO4). These

structures are superimposed at XPB. The DNA damage site is far away and upstream of

the lesion sensor Fe4S4 (marked by the grey arrowheads). (c) For comparison, the

structures reported here, human XPC and Core7 complex with Cy5-DNA (C7CD), is

shown after superposition with 7K04. (d) In human XPC, XPA and Core7 complexed with

Cy5-DNA (C7CAD), the DNA lesion (Cy5) is downstream of the XPD motor (5′ to 3′) and

close to the lesion sensor Fe4S4 of XPD. when XPD translocates along the lesion strand

(orange), Cy5 would be “seen” and stall the XPD motor.

Extended Data Fig. 2 Structure determination of three Cy5 structures. (a) Diagram of

Cy5-DNA and Cy5. (b) The workflow of cryoEM data processing and model generation.

(c) FSC analysis of the quality and map resolution and model fit of each complex structure.

(d) For each complex, angular distributions of particles used for the final threedimensional reconstruction, and a surface presentation of its map colored according to

the local resolution estimated by ResMap with the scale bar on the side, are shown. (e)

Representative regions of the three cryoEM maps are superimposed with the final

structural models.

Extended Data Fig. 3 Structure determination of four AP structures. (a) Diagram of

the AP-DNA, and EMSA results of 5 nM 32P-labeled AP-DNA binding by 5 nM each of

XPA, Core7, Core7 and XPA (C7A), XPC, XPC and XPA (CA), Core7 and XPC (C7C)

and Core7 with XPC and XPA (C7CA). The EMSA results were replicated at least six

times. (b) The workflow of cryoEM data processing and model generation. (c) FSC

analysis of the quality and map resolution and model fit of each complex structure. (d)

For each complex, angular distributions of particles used for the final three-dimensional

reconstruction, and a surface presentation of its map, colored according to the local

resolution estimated by ResMap with the scale bar on the side, are shown. (e)

Representative regions of the three cryoEM maps (DNA) are superimposed with the final

structural models. For gel source data of 3a, see Supplementary Figures 3.

Extended Data Fig. 4 Structure-based sequence alignment of human XPC and yeast

Rad4. Conserved residues are highlighted in yellow (hydrophobic core), grey (structural

stability), green (subunit interface), cyan (DNA binding, and underscore indicating base

interactions), and red (disease mutation). Protein secondary structures are indicated by

box (for helix) and arrow (strand). They are labeled alphabetically for helices and

numerically for strands. In BHD domains 1-3, secondary structures are preceded by

domain name “1”, “2” and “3”. Disordered regions are indicated by dashed lines.

Extended Data Fig. 5 cryoEM maps of DNA bound by XPC and XPA. (a) The flipped out

T26 in Cy5_C7CD. (b) The LHN has close contacts with Cy5 and the non-lesion strand

across the minor groove. The cryoEM map in the above two panels are shown as semitransparency grey surface. (c) cryoEM map corresponding to XPA and DNA in

27

Kim et al., 2022

817

818

819

820

821

822

823

824

825

826

827

828

829

830

831

832

833

834

835

836

837

838

839

840

841

842

843

844

845

846

847

848

849

850

851

852

853

854

855

856

857

858

859

860

861

862

Cy5_C7CAD. Map volume is color coded and labeled. (d) A close-up of the C-terminal

52 residues of XPC (aa 889-940). XPB, p52, and p8 of Core7 and XPC are represented

by the cryoEM map of C7CAD and C7AD of Cy5-DNA and color coded. Helices L, M and

N of XPC are show as ribbon cartoons and labeled. The penultimate K939 of XPC, which

is shown in a stick model, caps the carboxyl end of helix N. Potential interactions between

the sidechain amine of K939 (shown as a sphere) and carbonyl oxygens are indicated by

dashed yellow lines. Residues F935, P936 and F937 of XPC are anchored in a

hydrophobic pocket in XPB (green).

Extended Data Fig. 6 Domain comparison of XPC, Rad4, RAD23 and CETN2. (a)

Superposition of TGD of XPC (slate blue) and Rad4 (semi-transparent grey). (b)

Superposition of BHD1 of XPC and Rad4. (c) Superposition of BHD2 of XPC and Rad4.

(d) Superposition of BHD3 of XPC and Rad4. (e) Superposition of Rad23 and RAD23

(pale green cartoon with molecular surface) reveals that TGD domains of XPC (blue) and

Rad4 (grey) differ by a 16° rotation. (f) Superposition of TGD domains of XPC and Rad4

reveals that BHD1, BHD2 and BHD3 diverge increasingly. (g) Crystal structures of

CETN2 (2GGM in pink and 2OBH in light blue) complexed with XPC peptide (LHC, blue)

are superimposed. Symmetry mate of XPC is shown in pale green. (h) CETN2 (light

green) and XPB (dark green) in C7CD are included in superposition. The LHC (XPC, dark

blue) is shifted and interacts with the C-terminal helix of XPB when complexed with Core7.

Extended Data Fig. 7 Structure comparison of C7CD with PIC and XPB with SF2

helicase. (a) Superposition of XPB (green) in C7CD and in human PIC (PDB: 7NVW,

light grey) shows the bent DNA associated with XPB and different position of XPD (cyan

in C7CD and light grey in PIC) in the U-shaped Core7. (b) Superposition of HD2 of four

SF helicases, XPB, Rad26 (CSB homolog), Snf2 and NS3 reveals that the tracking

strands superimpose well in all cases.

Extended Data Fig. 8 Repetitive and flexible structure of TFIIH (Core7). (a) The Ushaped Core7 in C7CD. The N-terminal helices of p44 that contact XPB are outlined in a

rounded rectangle. The XPD (left) and XPB (right) arm are well separated. (b) The σshaped Core7 in C7CAD with p34 superimposed to C7CD and viewed in the same

orientation as in panel a. The interface at p34-p44 and p34-p52 (inside the dashed oval)

remain unchanged. (c) The stable interfaces of p34 with p44-RING finger (RF) and p52.

The C-terminal p34-DZF (double Zinc finger) and p44-ZR (Zing Ribbon) domain are

labeled. (d) A β hairpin of p34-DZF in C7CD is changed to a short α helix in C7CAD. A

part of p62 becomes disordered in C7CAD. (e) The third domain of p52 (DRD fold)

contacts the N-terminal DRD domain (blueish) of XPB, which is followed by the second

DRD domain (greenish) of XPB. The N-terminal helices of p44 (pink) contact the back

side of XPB. (f) The fourth domain of p52 (grey) and p8 (light purple) form a heterodimer.

Extended Data Fig. 9 Comparison of DRD (Damage Recognition Domain) domains.

Two MutS DRDs (domains I and VI from 1EWQ) are shown on the left side for comparison.

Five DRD domains in TFIIH are shown after superposition with MutS DRDs. Each DRD

is colored in rainbow fashion from the blue N- to red C-terminus. Four β strands are

labeled 1 to 4, and strands 2 and 4 are each followed by an α helix (A and B). In the P5228

Kim et al., 2022

863

864

865

866

867

868

869

870

871

872

873

874

875

876

877

p8 heterodimer, the two subunits complement each other by supply the partner DRD with

the first β strand (shown in semi-transparent blue and labeled 1’).

Extended Data Fig. 10 Length of Cy5_DNA substrate required for efficient dual

incision. (a) Sequence of three Cy5 DNA substrates, each of which contains a total 94

bp but different upstream (left) and downstream length from Cy5 (right). (b) Diagrams of

the three DNA substrates. (c) Dual incision results of each DNA substrate (sub) after

incubation with Core7, XPC, XPA, RPA, XPF and XPG at 37°C for 60 min. DNA cleavage

intermediate (int) and final product (prod) are marked. (d) Means and standard deviations

(error bars) of triplicated dual incision reactions as well as individual data points are shown

in the bar graph. For gel source data, see Supplementary Figures 5.

Extended Data Table 1. CryoEM data collection and processing, and structural

model refinement

878

879

29

782

(kDa)

FeS HD1 Arch

HD2

760

200

150

120

100

85

70

60

50

125 164 281 346

PH

17

p52

Bndl 548

127 132

305

401

D1 D2 DRD P8L 462

54 240

328 387

395

vWA ZR RF

250

292

308

vWA

DZF

69

p8 71

167

635

684 741 866 913

p44

p34

p8

XPC

TGD

RAD23

CETN2

24

102 135

XPA

CB

409

25

20

15

CETN2

10

P8

(TTDA)

C 172

197 234

ZF

C7CD

331

XPC

XPB

XPD

p62

RAD23

p52

p44

XPA

p34

40

30

940

BH1 BH2 BH3

271

XPA

723

HD2

Core7

500

HD1

483

p62

292

273

non-lesion

lesion

strand

XPC

C7CAD

XPC

C7AD

CETN2

XPD

XPB

Cy5

XPB

XPD

Cy5

XPC

CETN2

p44

p62

XPB

p62

p44

p52

XPD

XPA

RAD23

p8

DN

+X

+C A

ore

+C 7

7A

+X

PC

+C

+C

7C

+C

7C

XPD

163

DRD DRD

XPC

52

XPB

MW

p34

p8

p44

p62

XPA

p52

p34

p52

p34

Fig. 1

CETN2

XPC

RAD23

BHD3

CETN2

EF-N

34

EF-C

Ca2+

Ca

BHD2

24

31

33

23 14

22

2B

21

32

3B

2+

Cy5

BHD1 non-lesion

1A 12

strand

2C

upstream

LHC

LHN

51

C B

D TGD

downstream

lesion

strand

LH

F186

CETN2

RAD23

R192

Cy5

Y 189

R196

2B

P806

LH

T26

2C

1A

1B

Y656

A28 T27

H685 23 22 11

13 12

F756

2A

14

F733

1C

3A

31

34

21

P703

24

BHD1

BHD2

BHD3

33

3B

F797

T27

BHD3 A28 T26 T26

T27

Cy5-DNA (C7CD)

BHD2

A28

T27

T26

AP-DNA

Cy5

Cy5-DNA

Cy5

DNA (Rad4, 2QSH)

Fig. 2

XPC

RAD23

ZnF

XPA

BHD3

CETN2

C7CAD (XPA)

BHD3

XPC

BHD2

BHD1

TGD

LHC

CETN2

BHD1

Q197

Cy5

non-lesion

strand

LHN

A718

TTDA

(p8)

Cy5

XPD

LHC

XPD

p52

domain

35Å

p34

LHA

XPA

domain

W175

R211 T142

K217

T239

H242

XPB

p8

p44

H244

H266

L268

Y270

XPA

K259

p62

domain

M273

p52

p52

R207

Q146

S233

N p34

p8

p62

lesion

strand

LHA

XPB

p44

W235

XPB

E238

K706

XPC

LHA

C7CD

LHC

K218

-8 -7

XPA

domain

XPC

XPB

Cy5_C7CAD

Q893

AP_C7CAD

F546 F550

Cy5_C7CD

F658

L L900

M273

R89

Y660

P936

XPC

F937

K939

F935

S462

p8

A931

Q906

(O)

R908 W904 L934

V241 E911

p52

p52(p8-like) p8

C7CD

XPA

XPC

50°

bend

Cy5

C7CAD

Fig. 3

XPA

HD1

domain

non-lesion

strand

K218

XPA

R207

W175

K221

Q629

Fe4S4

W175

R228

lesion

strand

Y627

F508

R511

HD2

94

75

54

35

20

sub

int

prod

-F

- GGR

-F

AP

NER

Cy5

NER

min

-F

- GGR

-F

XPD

min

Dual incision prod (%)

XPB

lesion

strand

80

60

Cy5

40

20

AP

0 10 20 30 40 50 60

(min)

Fig. 4

GGR

XPC+TFIIH (C7CD)

C7CAD

XPA

XPB

XPC

XPC

XPB

XPA

XPB

lesion

XPD

XPD

stalled

XPD

XPC

XPA

XPA

Stalled RNAP+TFIIH (PIC-like)

RNA pol, CAK

XPB

RNA pol

XPB

ATP

dual incision

XPD

bypass

ATP

XPA

XPA

C7AD + 6RO4

XPB

XPD

XPD

TCR

Fig. 5

...