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Figure 1

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Figure 2

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Figure 3

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Figure 4

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Figure 5

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Figure 6

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Figure 7

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Supplemental Information Tables S1-S7

Table S1 SILAC differential mass-spectrometry identified RPB1-K1268 ubiquitination in UVirradiated cells (1 h, 10 J/m2), Related to Figure 1

Annotated

Sequence

in RPB1

(P24928)

Qvality

PEP

Qvality

q-value

PSMs

IMNSDE

N(K)MQ

EEEEVV

DKMDD

DVFLR

(1xGG

[K8])

9.5698

×10-8

Positions

# Missed

Cleavages

Theoretical

MH+ (Da)

Abundances

(scaled)

(WT)

Abundances

(scaled)

(∆UVSSA)

Abundance

Ratio*

(∆UVSSA) /

(WT)

12611286

3272.43918

177.8

22.2

0.125

*SILAC ratio. Ubiquitinated lysine residue is indicated by boldface. WT, HCT116 wild-type cells;

∆UVSSA, UVSSA-deleted HCT116 cells.

Table S2 Mass-spectrometry identified RPB1-K1268 ubiquitination after UV irradiation (1 h,

20 J/m2), Related to Figure 1

Positions

in RPB1 (P24928)

GlyGly (K)

Intensity

Intensity

probabilities

in detected

(Replicate. 1) (Replicate. 2)

peptides*

Modifications

0 NICEGGEEMDNK

(1)FGVEQPEGDEDLTK 1xGG [K12]

163

4285100

177

13760000

0 FGVEQPEGDEDLTK

(0.869)EK (0.131)

1xGG [K14]

758

1949200

TGSSAQK

0 (0.996)SLSEYNNFK

(0.004)

1xGG [K7]

853

1268

20479000

1350

2449300

1245900 EGLIDTAVK

(1)TAETGYIQR

10857000 IMNSDENK

(1)MQEEEEVVDK

0 VLSEK (1)DVDPVR

1xGG [K9]

1xGG [K8],

Oxidation [M2]

1xGG [K5]

*Ubiquitinated lysine residue is indicated by boldface. Probabilities of ubiquitination for each

lysine residue were shown in parentheses. Wild-type HeLa cells were UV irradiated.

Table S3 CRISPR/Cas9-based gene editing and cell lines used in this study, Related to Figure 1

and STAR Methods

Cell line

CRISPR/Cas9

guide RNA

(gRNA)

(1) HCT116

UVSSA cell line

UVSSA

GTGTGGAGGT

CCCTGAGAAG

Homology directed repair (HDR) oligos

clone # used

in this study

#1

(2) HeLa RPB1-KR mutants

RBP1K163R

RBP1K177R

RBP1K642R/

K643R

RBP1K710R

RBP1K767R

RBP1K796R

RBP1K812R

RBP1K853R

GATGGACAAC

AAGTTCGGTG

CCTCCAGGCCTCTGACCCCTCCTTCCCA

AAAGTCTCCGCCAGCCCCAGCCACCTTT

TCTTTGGTCAGATCCTCGTCACCCTCAG

GTTGTTC[g**]ACACCGAAC[C*]TGTTGTC

CATCTCCTCCCCACCCTCGCATATGTTTT

GGATCTGACC CCAGCCCCTCCTGTTTCCTTCCCTTCCAG

AAAGAAAAGG TTTCCTCCCTCCAGGCCTCTGACCCCTCC

TTCCCAAAAGTCTCCGCCAGCCCCAGCC

ACCTT[c**]TCT[C*]TGGTCAGATCCTCGT

CACCCTCAGGTTGTTCCACACC

GTGCCCAGAG ATTTGGACGTGGGAGCCAGGACCAGAG

ACTTCTTACAC CAGGGGCCTTGAGTGGGTGCTTTGTCCT

TAGGTGGTGGTGGAGAATGGGGAGCTG

ATCATGGGCAT[t**]CTGTGTA[G*]GA[G*]

GTCTCTGGGCACGTCAGCTGGCTCCCTG

GTCCACATC

GCACTATTAA

ACTCCCTTTGCTCTTGATGATGCTAACTT

GAAGGCCAAG CGAAGTCCCTGGAAACCCCTTATTCCGT

CTCTGGTGGCCTCCCCTCTTACCTCTATT

ACGTC[t**]TGC[C*]TGGCCTTCTTAATAG

TGTTCTGAATGTCCTGGTAAGT

GTTCAAGTCTA TCCTTCCTCCTCCCAAACTTCACAGCGG

TGGTCGTGTC

CCCCGTATATGGAAAAACAAGGCTTCTC

ACCTGGGAGATGTTAATCTTGGAACCTT

TAGCTCCG[ctg**]ACGACCATAGAC[C*]T

GAAGTTATTGTATTCAGACAGGGATTTC

TGAGCAGA

GAGCAGAACG GGTAGGAGTTCTCCACAAAGCCACGGCT

TCGAGGGCAA CTCAGGCCCGTAGTCATCCTTGATGAAG

TGAGGCAGAGTCCGGTGCTTGAAGCCA

AATGGAAT[t**]C[t**]C[C*]TGCCCTCGAC

GTTCTGCTGTCCAACGACAGCAATGAC

GACTCTGCCTC CCCCCATGGCGTGGAAAAAGAACTCAG

ACTTCATCA

TGGGTGTGAGGCCGGCTAGGTAGGAGT

TCTCCACAAAGCCACGGCTCTCAGGCCC

GTAGTCATC[t**C*]TGATGAAGTGAGGCA

GAGTCCGGTGCTTGAAGCCAAA

GCTGTCAAGA GAGCATTTCCGCTCCCCACCTGTTAGGG

CTGCTGAGAC GTTTCTCAGCCTGCAGCAGTCCCTGCTA

ACAGCCCAAGGAAGACCCCTGAGGAAA

#4

#8

# 16

# 23

#2

# 14

# 32

# 29

RBP1K866R

GCGGCGGCTG

ATCAAGTCCA

RBP1K874R

GTGAAGTACG

ACGCGACTGT

RBP1K1225R

GACTGACCGG

AAGCTCACCA

RBP1K1268R

GCGATGAGAA

CAAGATGCAA

RBP1K1350R

GGTGCTGAGT

GAGAAGGACG

GCCTCACC[g**]GT[t**]TCAGCAGTC[C*]T

GACAGCCGTGTCAATGAGCCCCTCACGA

CCCCCCAT

CCAGGCCGTCTTCGCCGTAGCGCAGCTG

CACCACCTGGTTGATGGAGTTCCGCACA

GTCGCGTCGTACTTCACCATCACTGACT

CCATGGAC[C*]TGATCAGCC[t**]CCGCTG

GATGTATCCTGGAGGGAAGTAAGGGGA

TGA

GCTTAAGCGTAGCCAGGTTCTGGAACTC

AACGCTCTCGCCTGCCAGGCCGTCTTCG

CCGTAGCGCAGCTGCACCACCTGGTTGA

TGGAGTT[t**]C[t**]CACAGTCGCGTCGTA

C[C*]TCACCATCACTGACTCCATGGACTT

GATCAGCCGCCG

ACGATAGGTGGTAGCCCAGAGAGCGGG

GCTCCTGAGCCAGGCCAGCCCTCCTAGG

CTTACCAGCATTGATCTTTTCAGCAATC

TGCTCCAT[t**]GT[c**]A[a**]C[C*]TCCGGT

CAGTCATGTGCTTCCGATCCAGCTCCAC

CCG

AGGTTTTGGTGACGACTTGAACTGCATC

TTTAATGATGACAATGCAGAGAAGCTG

GTGCTCCGTATTCGCATCATGAACAGCG

ATGAGAACA[G*]GATGCAAGAGGTAAT

GGGGGTCCTAGAAGTCAGCGTG

AGCCAGAGATCCACGAAAGGCAGCTAG

GCAGCACACACGGGCTCACCGTGAAGA

TCTCCACAATGTCATTGGACGTGGTGCG

TACGGGGTC[g**]ACGTC[t**C*]TCTCACT

CAGCACCCGCATCAAGCTCACGCCGTCC

GT

#3

#4

# 21

# 7 (Fig. 1B,

1C),

# 9 (all Figs.)

#8

(3) HeLa ∆TCR mutants

CSA

CSB

UVSSA,

UVSSAΔK414

GTCCGCACGC CAAACGGGTT

GCTTCTCCACG TCAACGAGCT

GTGTGGAGGT CCCTGAGAAG

#5

#1

#5

#1

(4) HEK293 GFP-RPB1 expressing cell lines

RPB1GCCTGCCTCCG #1

WT***

CCATGCACG

RPB1#7

K1268R***

RPB1-WT, same as above

# 13

CSA***

*Lys->Arg target mutations; **silent mutations (lower cases). ***endogenous POLR2A alleles were

deleted.

Table S4 Polr2aK1268R/K1268R single mutant mice phenotype, Related to Figure 7

Parameter

Sex

Age

(days)

Polr2aWT/WT

Bodyweight

(g)

80±5

Polr2aWT/KR

Polr2aKR/KR

pvalue*

27.0±1.8 (6) 26.5±1.7 (6)

26.9±2.1

(10)

1.00

110±5

28.0±2.1 (6) 28.3±2.0 (6)

28.4±2.3

(10)

1.00

130±5

29.3±2.2 (6) 29.0±2.6 (6)

29.3±2.6

(10)

1.00

80±5

19.9±1.1 (7)

20.3±1.1

(16)

20.1±1.4 (8)

0.866

110±5

20.9±0.92

(7)

21.2±1.2

(16)

21.5±1.2 (8)

0.866

130±5

21.3±1.1 (7)

21.7±0.97

(16)

21.8±1.1 (8)

0.866

Fertility

♂+♀

60->365

Normal

Normal

Normal

Gait

abnormalities

♂+♀

90->365

Hind limb

dystonia

♂+♀

20->365

Kyphosis

♂+♀

90->365

Remarks

Results

from a tail

suspension

test.

Values are average ± S.D; number of tested animals are shown in parentheses. *P-values were

calculated to test the difference in bodyweights between Polr2aWT/WT vs Polr2aKR/KR animals with

the Mann-Whitney U-test, and were corrected by the Benjamini-Hochberg method. WT, wild type;

KR, K1268R.

Table S5 Polr2aK1268R/K1268R / Xpa+/- mice phenotype (Xpa heterozygous deletion), Related to

Figure 7

Parameter

Sex

Age

(days)

Polr2aWT/WT

/ Xpa+/-

Bodyweight

(g)

80±5

24.5±1.4 (5) 24.9±1.5 (8)

110±5

150±7

Polr2aKR/KR

/ Xpa+/-

pvalue*

26.0±1.2

(12)

0.172

27.8±1.3 (6)

0.240

27.7±2.3 (4) 29.0±1.2 (6) 29.1±1.2 (6)

0.202

26.6±0.84

(4)

Polr2aWT/KR

/ Xpa+/-

27.4±1.1

(10)

260±10

32 (1)

32.2±2.1 (4) 33.6±1.1 (2)

80±5

19.6±1.2 (6)

20.1±0.87

(12)

20.7±0.0 (2)

0.241

110±5

20.7±0.65

(4)

21.6±0.85

(12)

25.1 (1)

NA

150±7

22.1±1.57

(3)

22.4±1.0 (9)

NA

NA

Fertility

♂+♀

60->365

Normal

Normal

Normal

Gait

abnormalities

♂+♀

90->365

Hind limb

dystonia

♂+♀

20->365

Kyphosis

♂+♀

90->365

Remarks

NA

Results

from a tail

suspensio

n test.

Values are average ± S.D; number of tested animals are shown in parentheses. *P-values were

calculated to test the difference in bodyweights between Polr2aWT/WT / Xpa+/- vs Polr2aKR/KR / Xpa+/animals with the Mann-Whitney U-test, and were corrected by the Benjamini-Hochberg method.

WT, wild type; KR, K1268R.

Table S6 DM mice phenotype, Related to Figure 7

Parameter

Sex

Age

(days)

Polr2aWT/WT

/ Xpa-/-

Polr2aWT/KR

/ Xpa-/-

Polr2aKR/KR

/ Xpa-/(DM)

pvalue*

Remarks

Bodyweight

(g)

80±5

26.2±1.3 (9)

22.5±3.4

(22)

18.5±2.4

(4)

0.009

52

110±5

27.9±1.2 (6)

24.2±2.8

(10)

18.2±1.6

(4)

0.009

52

150±7

28.9±1.5 (8)

26.5±2.1

(12)

16.2±1.3

(5)

0.004

66

Differences

between

WT/WT and

WT/K1268R

are also

statistically

significant in

male mice (<

160 days).

275±5

32.5±3.3 (3)

31.2±0.75

(3)

NA

NA

80±5

19.0±1.1

(11)

19.3±1.0

(16)

14.8±2.0

(4)

0.000

754

110±5

20.7±0.8

(12)

20.7±0.9

(16)

14.9±2.0

(4)

0.004

35

150±7

21.4±0.7

(10)

21.6±1.0

(14)

14.8±0.8

(5)

0.004

01

NA

NA

275±5

25.5±3.6 (4) 23.8±1.5 (7)

Fertility

♂+♀

60>365

Normal

Normal

Not Tested

Gait

abnormalities

♂+♀

90

Moderate

120180

Prominent

Hind limb

dystonia

♂+♀

20-180

Kyphosis

♂+♀

90

Moderate

150180

Prominent

Depigmentati

on

(animals)

♂+♀

90-180

0 (21)

0 (40)

2 (12)

Cataract

(animals)

♂+♀

90-180

0 (21)

0 (40)

2 (12)

Results from a

tail

suspension

test.

Monocular

cases.

Values are average ± S.D; number of tested animals are shown in parentheses. *P-values were

calculated to test the difference in bodyweights between Polr2aWT/WT / Xpa-/- vs Polr2aKR/KR / Xpa-/(DM) animals with the Mann-Whitney U-test, and were corrected by the Benjamini-Hochberg

method. WT, wild type; KR, K1268R.

Table S7 ChIP-seq data summary, Related to STAR Methods

Analysis in which sequence data were used

Cell type

UV

Time after

Replicate

Antibody

(Hela Mutant)

(J/m2)

irradiation

ngsplot

SSI-scatter

SSI-RI

replicate-

SSI

check

difference

T-T site

SRA Run ID

PreCR

number

Fig. 4B, 4C,

Fig. 4G,

Fig. 5B, 5C, Fig. 5D, 5E,

Fig. 6A-E,

Fig. S4D

S4A, S4B

S4E

S5B-D

S5E, S5F

Fig. 4F

S6A

RPB1-K1268R

0h

3E10

SRR9722058

RPB1-K1268R

0h

3E10

SRR9722057

RPB1-K1268R

5m

3E10

SRR9722060

RPB1-K1268R

30m

3E10

SRR9722059

RPB1-K1268R

1h

3E10

SRR9722062

RPB1-K1268R

1h

3E10

SRR9722061

RPB1-K1268R

3h

3E10

SRR9722064

RPB1-K1268R

3h

3E10

SRR9722063

RPB1-K1268R

6h

3E10

SRR9722056

RPB1-K1268R

6h

3E10

SRR9722055

RPB1-K1268R

12h

3E10

SRR9722145

RPB1-K1268R

12h

3E10

SRR9722144

RPB1-K1268R

12h

3E10

SRR9722143

WT

0h

3E10

SRR9722142

WT

0h

3E10

SRR9722156

WT

5m

3E10

SRR9722155

WT

30m

3E10

SRR9722147

WT

1h

3E10

SRR9722146

WT

1h

3E10

SRR9722158

WT

3h

3E10

SRR9722157

WT

3h

3E10

SRR9722079

WT

6h

3E10

SRR9722080

WT

6h

3E10

SRR9722077

WT

12h

3E10

SRR9722078

WT

12h

3E10

SRR9722083

WT

12h

3E10

SRR9722084

WT

12h

3E10

SRR9722081

!CSA

0h

3E10

SRR9722082

!CSA

0h

3E10

SRR9722075

!CSA

5m

3E10

SRR9722076

!CSA

30m

3E10

SRR9722068

!CSA

1h

3E10

SRR9722067

!CSA

1h

3E10

SRR9722070

!CSA

3h

3E10

SRR9722069

!CSA

3h

3E10

SRR9722072

!CSA

6h

3E10

SRR9722071

!CSA

6h

3E10

SRR9722074

!CSA

12h

3E10

SRR9722073

!CSA

12h

3E10

SRR9722066

!CSB

0h

3E10

SRR9722065

!CSB

0h

3E10

SRR9722097

!CSB

5m

3E10

SRR9722098

!CSB

30m

3E10

SRR9722099

!CSB

1h

3E10

SRR9722100

!CSB

1h

3E10

SRR9722101

!CSB

3h

3E10

SRR9722102

!CSB

3h

3E10

SRR9722103

!CSB

6h

3E10

SRR9722104

!CSB

6h

3E10

SRR9722095

!CSB

12h

3E10

SRR9722096

!CSB

12h

3E10

SRR9722094

!UVSSA

0h

3E10

SRR9722093

!UVSSA

0h

3E10

SRR9722092

!UVSSA

30m

3E10

SRR9722090

!UVSSA

5m

3E10

SRR9722091

!UVSSA

1h

3E10

SRR9722089

!UVSSA

1h

3E10

SRR9722088

!UVSSA

3h

3E10

SRR9722087

!UVSSA

3h

3E10

SRR9722086

!UVSSA

6h

3E10

SRR9722085

!UVSSA

6h

3E10

SRR9722121

!UVSSA

12h

3E10

SRR9722122

!UVSSA

12h

3E10

SRR9722119

!CSA

0h

3E8

SRR9722120

!CSA

3h

3E8

SRR9722117

!CSA

6h

3E8

SRR9722118

!CSA

12h

3E8

SRR9722115

!CSB

0h

3E8

SRR9722116

!CSB

3h

3E8

SRR9722123

!CSB

6h

3E8

SRR9722124

!CSB

12h

3E8

SRR9722110

!UVSSA

0h

3E8

SRR9722109

!UVSSA

3h

3E8

SRR9722112

!UVSSA

6h

3E8

SRR9722111

!UVSSA

12h

3E8

SRR9722106

RPB1-K1268R

0h

3E8

SRR9722105

RPB1-K1268R

3h

3E8

SRR9722108

RPB1-K1268R

6h

3E8

SRR9722107

RPB1-K1268R

12h

3E8

SRR9722114

WT

0h

3E8

SRR9722113

WT

3h

3E8

SRR9722152

WT

6h

3E8

SRR9722153

WT

12h

3E8

SRR9722154

!CSA

0h

A304-405A

SRR9722052

!CSA

3h

A304-405A

SRR9722148

!CSA

6h

A304-405A

SRR9722149

!CSA

12h

A304-405A

SRR9722150

!CSB

0h

A304-405A

SRR9722151

!CSB

3h

A304-405A

SRR9722053

!CSB

6h

A304-405A

SRR9722054

!CSB

12h

A304-405A

SRR9722137

!UVSSA

0h

A304-405A

SRR9722136

!UVSSA

3h

A304-405A

SRR9722135

!UVSSA

6h

A304-405A

SRR9722134

!UVSSA

12h

A304-405A

SRR9722141

RPB1-K1268R

0h

A304-405A

SRR9722140

RPB1-K1268R

3h

A304-405A

SRR9722139

RPB1-K1268R

6h

A304-405A

SRR9722138

RPB1-K1268R

12h

A304-405A

SRR9722133

WT

0h

A304-405A

SRR9722132

WT

3h

A304-405A

SRR9722127

WT

6h

A304-405A

SRR9722128

WT

12h

A304-405A

SRR9722125

WT

0h

ab5095

SRR9722129

WT

3h

ab5095

SRR9722126

WT + PreCR-20min

3h

ab5095

SRR9722131

WT + PreCR-2h

3h

ab5095

SRR9722130

The sequence data were deposited in the NCBI Short Read Archive (SRA), with the BioProject

accession number, PRJNA548234.

Supplemental Figure S1

Click here to access/download;Supplemental

Figure;NakazawaFigS1R3.tiff

Supplemental Figure S2

Click here to access/download;Supplemental

Figure;Nak ...