Supplementary Table 1 Crystals of hPolη ternary complexes

Structure name / Protein / DNA sequence / Incoming dNTP
Nrm
(Type 3) / WT / 3’ TCGCAGTATTACT 5’
5’ TAGCGTCAT 3’ / dAMPNPP
TT1
(Type 3) / WT / 3’ TCGCAGTATTAC 5’
5’ TAGCGTCAT 3’ / dAMPNPP
TT2
(Type 2) / WT / 3’ CGCAGTATTCAAT 5’
5' TGCGTCATA 3’ / dAMPNPP
TT3
(Type 3) / WT / 3’ CGCAGTATTCAAT 5’
5’ ACGTCATAA 3’ / dGMPNPP
TT4
(Type 3) / C406M / 3’ CGAGCATTACTAC 5’
5’ TCTCGTAAT 3’ / dGMPNPP
Native (Type 2) / C406M / 3’ ATCGCAGTATTA 5’
5’ TAGCGTCAT 3’ / dAMPNPP
SeMet (Type 2) / C406M / 3’ ATCGCAGTATTA 5’
5’ TAGCGTCAT 3’ / dAMPNPP
Type 1 / WT / 3’ CCCCCTTCCTAAGTTTCT 5’
5’ GGGGGAAGGATTC 3’ / dATP
WT / 3’ GCACGGATCGCATGTATG 5’
5’ GTGCCTAGCGTA 3’ / dCTP
WT / 3’ CACGCACGGATCGCATGTATG 5’
5’ GTGCGTGCCTAGCGTA 3’ / dCTP

TT denotes cis-syn thymine dimer

Supplementary Table 2 Data collection, phasing and refinement statistics

Nrm / TT1 / TT2 / TT3 / TT4 / SeMet / Native / Type 1
Data collection
Space group / P 61 / P 61 / P212121 / P 61 / P 61 / P212121 / P212121 / P 61
Cell dimensions
a, b, c (Å) / 98.46
98.46
82.50 / 98.24
98.24 82.31 / 61.24
80.26
139.76 / 99.13
99.13
81.70 / 98.04
98.04
82.05 / 63.26
81.45
139.39 / 63.51
80.16
139.26 / 137.06
137.06
65.59
Peak / Inflection / Remote
Wavelength (Å) / 1.0000 / 1.0000 / 1.0000 / 1.0000 / 1.0000 / 0.9794 / 0.9796 / 0.9494 / 1.0000 / 1.0000
Resolution (Å) / 45.0-1.83 / 40.0-1.75 / 30.0-2.15 / 30.0-1.80 / 30.0-1.90 / 60-3.50 / 60-3.50 / 60-3.50 / 40-2.64 / 30-2.90
Rsym (%) / 6.3 (51.7) / 8.0 (59.6) / 8.6 (59.3) / 6.6 (57.4) / 7.8 (53.4) / 12.1 (23.8) / 8.2 (31.1) / 9.1 (29.8) / 4.5 (62.7) / 9.6 (67.4)
I/σI / 17.9 (3.2) / 13.9 (3.5) / 12.43(2.9) / 15.1 (2.6) / 14.5 (3.3) / 10.5 (5.1) / 11.8 (4.1) / 10.9 (4.5) / 18.3 (2.0) / 23.6 (2.0)
Completeness (%) / 98.9 (90.6) / 99.5 (99.6) / 99.0
(97.1) / 99.3 (99.6) / 98.9 (98.0) / 97.2 (95.1) / 93.9 (95.8) / 95.5 (95.9) / 92.1 (93.8) / 99.0 (92.0)
Redundancy / 4.4 (4.0) / 4.0 (4.0) / 8.4 (6.8) / 4.1 (4.1) / 3.3 (3.2) / 3.4 (2.0) / 2.0 (2.0) / 2.0 (2.0) / 2.4 (2.4) / 7.2 (5.7)
Refinement
Resolution (Å) / 45.0-1.83 / 40.0-1.75 / 30.0-2.15 / 30.0-1.80 / 30.0-1.90
No. reflections / 39732 / 45282 / 37705 / 42013 / 34965
Rwork/Rfree / 18.3/ 19.7 / 19.0/ 20.8 / 23.5/ 26.3 / 19.6/ 21.0 / 20.1/ 21.8
No. atoms
Protein/DNA / 3370/407 / 3333/406 / 3310/402 / 3348/395 / 3336/390
Ligand/ion / 68 / 68 / 75 / 63 / 76
Water / 404 / 436 / 116 / 318 / 295
B-factors
Protein/DNA / 22.0/25.4 / 19.0/22.5 / 68.4/81.5 / 25.3/31.2 / 23.5/29.2
Ligand/ion / 28.6 / 26.1 / 78.7 / 27.0 / 36.6
Water / 36.2 / 33.7 / 71.9 / 26.8 / 32.7
R.m.s deviations
Bond lengths (Å) / 0.010 / 0.014 / 0.014 / 0.008 / 0.018
Bond angles (º) / 1.8 / 1.9 / 2.0 / 1.9 / 2.1

*Highest resolution shell is shown in parenthesis.

Supplementary Table 3 Kinetic parameters of dNTP incorporation by WT, Q38A and R61A Pol on normal and CPD templates

Enzyme / Template : dNTP / KM(M) / kcat(min-1) / kcat/KM (M-1・min-1)
Pol (1-511) WT / ND (3’T) : dATP / 4.1 ± 1 / 200 ± 10 / 49
ND (3’T) : dGTP / 17 ± 1 / 50 ± 10 / 3.0
CPD (3’T) : dATP / 4.1± 0.7 / 90 ±5 / 20
CPD (3’T) : dGTP / 110 ± 40 / 60 ±3 / 0.55
Pol (1-511) Q38A / ND (3’T) : dATP / 8.8 ± 0 / 210 ± 20 / 24
ND (3’T) : dGTP / 80 ± 0 / 90 ±7 / 1.1
CPD (3’T) : dATP / 15 ± 1 / 160 ± 20 / 11
CPD (3’T) : dGTP / 330 ± 30 / 40 ± 2 / 0.12
Pol(1-511) R61A / ND (3’T) : dATP / 6.5 ±0.4 / 150 ±6 / 22
ND (3’T) : dGTP / 170 ± 50 / 80 ± 16 / 0.47
CPD (3’T) : dATP / 13± 2 / 100 ± 3 / 7.5
CPD (3’T) : dGTP / 450 ± 40 / 20 ± 1 / 0.044