SUPPLEMENTARY INFORMATION
PCNA directs Type 2 RNase H activity on DNA replication and repair substrates
Doryen Bubeck1,4, Martin A.M. Reijns2,4, Stephen C. Graham1,3, Katy R. Astell2, E. Yvonne Jones1, and Andrew P. Jackson2,*
1
Division of Structural Biology, Wellcome Trust Centre for Human Genetics, University of
Oxford, Oxford OX3 7BN, UK 2
Medical Research Council Human Genetics Unit, Institute of Genetics and
Molecular Medicine, Western General Hospital, Edinburgh EH4 2XU, UK 3
Current address: Cambridge Institute for Medical Research and Department of Clinical
Biochemistry, University of Cambridge, Addenbrooke's Hospital, Hills Road, Cambridge CB2 0XY, UK 4
*
These authors contributed equally to this work To whom correspondence should be addressed: Tel. +44 131 332 2471; Fax +44 131 343
2620; Email
[email protected]
1
A
Afu
co
mp
lex
eH as
A
RN
Afu
PC N
Afu
hu
ma
nP CN
A
II
Supplementary Figure S1
Afu PCNA:RNase HII complex
Afu RNase HII
B
27 kDa 23 kDa
90 ml
70 ml Fractions
C
2
Supplementary Figure S1. Purification of proteins and stereo view of the Afu PCNA:RNase HII complex. (A) Size-exclusion chromatography was used as the final step during protein purification. Aliquots from peak fractions were analyzed using SDS-PAGE to assess the purity of each sample. (B) Purified Afu RNase HII and Afu PCNA were mixed in a 1.2:1 molar ratio and injected onto a Superdex 200 16/60 column. Fractions along the elution profile (70-80 mls post-injection) were collected and analyzed using SDS-PAGE. The larger Afu PCNA:RNase HII complexes were separated from unbound RNase HII. (C) Stereo view of the Afu PCNA:RNase HII complex. Three RNase HII molecules (cyan, tan and red) bind the PCNA homotrimeric ring (grey) in unique orientations. Two binding modes (cyan and tan) extend away from the center of the ring, while the third (red) obstructs the opening. The bottom panel shows the complex rotated 90° about the plane of the PCNA ring.
3
Supplementary Figure S2
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-
-
-
-
-
20 40 80 160 320 640 640
-
2.5 10 40 160 640 2560 160 160 160 160 160 160
-
RNase HII + PCNA
RNase HII PCNA RNase HII
-
-
-
-
-
-
-
PC NA
-
B
PC NA
A
RNase HII + PCNA
RNase HII
20 40 80 160 320 640 640
0.04 0.16 0.6 2.5 10 40 2.5 2.5 2.5 2.5 2.5 2.5
-
PCNA RNase HII
18 nt 18 nt
5 nt
3 nt
RNase HII
-
-
-
-
-
-
-
20 40 80 160 320 640 640
0.16 0.6 2.5 10 40 160 5
5
5
5
5
5
-
-
PCNA RNase HII
RNase HII + PCNA
RNase HII
-
-
-
-
-
-
NA
D
NA
RNase HII + PCNA
PC
C
*D5R1D12 : D18
PC
*D3R1D14 : D18
20 40 80 160 320 640 640
0.16 0.6 2.5 10 40 160 5
5
5
5
5
5
-
PCNA RNase HII 40 nt
18 nt
7 nt 7 nt *D7R1D10 : D18 *D7R1D32 : D40
-
-
-
-
-
-
5
5
5
5
5
-
PCNA RNase HII
-
-
-
-
-
-
-
NA
NA
20 40 80 160 320 640 640
0.16 0.6 2.5 10 40 160 5
RNase HII + PCNA
RNase HII
PC
-
F
PC
E
RNase HII + PCNA
RNase HII
20 40 80 160 320 640 640
0.16 0.6 2.5 10 40 160 5
5
18 nt
5
5
5
5
-
PCNA RNase HII 18 nt
11 nt
9 nt
*D9R1D8 : D18
*D11R1D6 : D18
4
Supplementary Figure S2
10 40 160 5
-
-
160 320 640 640 5
5
-
-
-
-
160 320 640 640
- 10 40 160 5
5
5
-
PCNA RNase HII
-
-
-
10 40 160 5
-
-
RNase HII + RNase HII PCNA
160 320 640 640 5
5
-
-
-
-
PC NA
-
-
RNase HII + RNase HII PCNA
PC NA
-
H
PC NA
G
RNase HII + RNase HII PCNA
PC NA
RNase HII + RNase HII PCNA
160 320 640 640
- 10 40 160 5
5
5
-
PCNA RNase HII
18 nt 18 nt 13 nt 12 nt
-
-
-
-
-
-
PCNA RNase HII
20 40 80 160 320 640 640
0.16 0.6 2.5 10 40 160 5
5
5
5
5
5
-
RNase HII + PCNA
RNase HII
-
-
-
-
-
-
-
NA
-
-
*D16R1D1 : D18
J
NA
RNase HII + PCNA
RNase HII
*D15R1D2 : D18
PC
I
*D13R1D4 : D18
PC
*D12R1D5 : D18
15 nt
PCNA RNase HII
20 40 80 160 320 640 640
0.16 0.6 2.5 10 40 160 5
5
5
5
5
5
-
40 nt 40 nt
17 nt
21 nt
-
-
-
-
-
-
-
PCNA RNase HII
20 40 80 160 320 640 640
0.16 0.6 2.5 10 40 160 5
5
5
5
5
5
-
RNase HII + PCNA
RNase HII
-
-
-
-
-
-
-
NA
L
NA
RNase HII + PCNA
RNase HII
PC
K
*D21R1D18 : D40
PC
*D17R1D22 : D40
PCNA RNase HII
20 40 80 160 320 640 640
0.16 0.6 2.5 10 40 160 5
5
5
5
5
5
-
40 nt
40 nt 31 nt
27 nt *D31R1D8 : D40
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-
-
-
-
-
-
5
5
5
5
5
-
PCNA RNase HII
RNase HII + PCNA
RNase HII
-
-
-
-
-
-
-
A
NA
20 40 80 160 320 640 640
0.16 0.6 2.5 10 40 160 5
N
PC
M
RNase HII + PCNA
RNase HII
PC N
*D27R1D12 : D40
20 40 80 160 320 640 640
0.16 0.6 2.5 10 40 160 5
5
40 nt
5
5
5
5
-
PCNA RNase HII 40 nt 36 nt
34 nt
*D34R1D5 : D40
*D36R1D3 : D40
5
Supplementary Figure S2. PCNA enhances RNase HII cleavage of ribonucleotides in a DNA duplex, unless they are positioned within the first ten bases. Substrates (250 nM; 5’ labeled, *) were incubated for 20 min at 30°C with Afu RNase HII and/or Afu PCNA. Nucleic acids were separated by denaturing PAGE and visualized by autoradiography. All protein concentrations are given in nM. (A-E) PCNA inhibits RNase HII cleavage if the ribonucleotide is placed within the first ten bases. (F-N) PCNA stimulates RNase HII cleavage if the ribonucleotide is placed 12 or more bases away from the 5’ end. (H) RNase HII cannot cleave when the ribonucleotide is in position 2 from the 3’ end, irrespective of the presence of PCNA.
6
Supplementary Figure S3 A
RNase HII + PCNA
RNase HII -
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-
-
-
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-
15 31 63 125 250 500 500
0.2 0.8 3.1 12.5 50 200 12.5 12.5 12.5 12.5 12.5 12.5 -
B PCNA RNase HII
RNase HII -
M
RNase HII + PCNA
-
-
-
-
-
-
20 40 80 160 320 640
- 0.04 0.16 0.6 2.5 10 40 0.01 0.01 0.01 0.01 0.01 0.01
60 nt
PCNA RNase HII 60 nt
12 nt 12 nt
2.5 nM substrate
250 nM substrate
*5’-gugugugugugugugugugugugugugugu(gu) 15 -3’ CACACACACACACACACACACACACACACA(CA) 15 PCNA inhibits
PCNA stimulates
Supplementary Figure S3. PCNA enhances RNase HII cleavage of an RNA/DNA hybrid. (A) PCNA stimulates RNase HII cleavage of a 60 bp RNA/DNA substrate. Endlabeled (*) 60 nt RNA (GU)30 was annealed to complementary DNA (CA)30. This hybrid (2.5 nM) was incubated with Afu RNase HII and/or PCNA and separated on a 20% polyacrylamide gel. (B) As in A but at 250 nM substrate concentration. M = partially hydrolysed (GU)30; protein concentrations are given in nM
7
Supplemental Figure 4
1
4
- 3.1 6.3 12.5 25 50 100 100 nM PCNA pM RNase H2 16 63 250 1000 63 63 63 63 63 63 63 -
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-
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- 0.01 0.03 0.13 0.5
31
NA
RNase H2 + PCNA
RNase H2
PC
-
B
PC
eH RN
as
RNase H2
NA
RNase H2 + PCNA
2
A
63 125 250 500 10001000 nM PCNA
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-
2
8 0.13 0.13 0.13 0.13 0.13 0.13
-
nM RNase H2
40 nt 40 nt
29 nt
14 nt
*D29R1D10 : D40
*D14R1D35 : D40
D -
-
-
-
-
-
3.1 6.3 12.5 25
nM Human RNaseH2
50 100 100
- 0.005 0.02 0.08 0.31 1.25 5 0.04 0.04 0.04 0.04 0.04 0.04
-
R10D13
nM PCNA nM RNase H2
60 nt R9 8 7 6 5 4 R3
*R10D13
E
*R10D13 : D40
RNase H2 + PCNA
RNase H2
-
-
-
-
-
D17 + *R10D13 : D40
-
-
3.1 6.3 12.5 25
PC NA
-
- 0.04 0.16 0.6 2.5
NA
RNase H2
- 0.04 0.16 0.6 2.5
PC
C
- 0.04 0.16 0.6 2.5
RNase H2 + PCNA
50 100 100
- 0.002 0.01 0.04 0.16 0.63 2.5 0.04 0.04 0.04 0.04 0.04 0.04
*5’-gugugugugugugugugugugugugugugu(gu) 15 -3’ CACACACACACACACACACACACACACACA(CA) 15
D17 + *R10D13 : D40
8
-
nM PCNA nM RNase H2
Supplementary Figure S4. Recombinant human PCNA does not affect activity of human RNase H2. Substrates were incubated with recombinant human RNase H2 and/or PCNA at 30°C. Nucleic acids were separated by denaturing PAGE and visualized by autoradiography. (A, B) RNase H2 cleaves 5’ of ribonucleotides embedded in a DNA duplex (250 nM of substrate), but is not affected by the addition of recombinant human PCNA. (C) RNase H2 shows widespread cleavage of a 60 bp RNA/DNA hybrid (2.5 nM), but is not affected by PCNA. (D) Human RNase H2 cleaves Okazaki-like fragments in a similar fashion to Afu RNase HII: it has no junction ribonuclease activity on single stranded R10D13, and it shows more complete cleavage of substrate with a double stranded region upstream of the RNA primer (substrates at 2.5 nM). (E) The cleavage pattern of Okazaki-like fragments is not affected by the addition of PCNA.
9
Supplementary Figure S5 A
B
Supplementary Figure S5. Superposition of PCNA-PIP box peptide structures and their sequence alignment. The interaction of PCNA with FEN1 (1-3) as well as complexes of PCNA with RNASEH2B (this work), p21 (4) or Cdc9 peptides (5) each contain the β-zipper within the PIP box motif. (A) The PCNA component (light blue ribbon) of several human PCNA:PIP box complexes were superposed and the peptides were compared. The structure of the PCNA:p21 peptide (PDB ID: 1AXC) is pink; PCNA:PolΔ66 (PDB ID: 1U76) is cyan; PCNA:FEN1 (PDB ID: 1U7B) is yellow; PCNA:CDK (PDB ID: 1VYJ) is brown; PCNA:RNASEH2B is green. (B) Sequence alignment of the PIP box peptides compared in A
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Supplementary Figure S6 A
EGFP
DAPI
Non-extracted
EGFP
EGFP RNASEH2C
B
C
merge
EGFP
PCNA
merge
EGFP RNASEHC
merge
EdU
T7-RNASEH2B
merge
Early
Mid
D
E
EGFPRNASEH2B (FF>AA)
EGFPRNASEH2B
EGFP-NLS
PCNA
Late Detergent extracted
EGFP
DAPI
Merge
Percentage of GFP+ cells (%)
EGFP-NLS 50%
EGFP-RNASEH2B
45%
EGFP-RNASEH2B(FF>AA)
40% 35% 30% 25% 20% 15% 10% 5% 0%
G1
S-phase
11
G2
Supplementary Figure S6. RNase H2, but not RNase H1 localizes to replication foci. (A) EGFP-RNASEH2C localizes to the nucleus, with some cells showing punctate patterns of higher intensity fluorescence, reminiscent of replication foci whereas EGFP alone localizes throughout. (B) Most detergent extracted cells expressing EGFP have no remaining EGFP signal. Those that do, show no colocalizaton with PCNA at replication foci. In contrast, EGFP-RNASEH2C co-expressed with RNASEH2A-Myc/His and T7-RNASEH2B shows colocalization with PCNA at replication foci. (C) Non-transfected, detergent extracted COS7 cells show localization of PCNA at replication foci in early, mid and late S-phase. EGFP (green), PCNA (red), DAPI (blue). (D) T7-RNASEH2B colocalizes with EdU-containing DNA. pCGT-RNASEH2B transfected COS7 cells were labeled for 15 min with 10 mM EdU (Invitrogen), detergent extracted and stained for EdU (green) and T7-RNASEH2B (red). Scale bars are 10 µm. (E) Expression of EGFP-RNASEH2B (green) or EGFP-RNASEH2B (FF>AA) (red) does not affect cell cycle progression compared to EGFP-NLS (black; EGFP with SV40 nuclear localization signal). Transfected cells were fixed and propidium iodide stained for FACS analysis, 24 h after transfection. DNA content of EGFP-positive cells was measured to generate a cell cycle profile and measure the percentage of cells in G1, G2 or Sphase. Representative cell cycle profiles of a single experiment are shown, as well as the average values from three independent experiments. Error bars indicate standard deviations.
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Supplementary Figure S7 A
B
Supplementary Figure S7. Comparison of PCNA:RNase HII complex with PCNA:FEN1. (A) Similar to the PCNA:RNase HII complex, the crystal structure of human FEN1 (PDB ID:1ULI) (green ribbon) bound to PCNA (grey ribbon) captures three distinct binding modes. The PIP-box of FEN1 binds proximal to the C-terminus of PCNA, and a flexible hinge enables the rotation of FEN1 about the ring. (B) To assess the model for Okazaki fragment maturation that involves primer processing by both type II RNase H and FEN1, the structural footprint of the two enzymes on a single PCNA ring was determined. The PCNA component of each PCNA:RNase HII complex observed in the crystal structure were superposed (cyan, tan and red) on a single PCNA monomer. Similarly, the PCNA component of each PCNA:FEN1 complex (dark grey and green ribbons, respectively) were overlaid on the remaining two PCNA monomers.
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Supplementary Movie S1. Afu PCNA:RNase HII complex. The RNase HII (red ribbons) is anchored to PCNA (grey ribbons) through its PIP box and rotates about a flexible hinge. In the crystal structure of the complex, three distinct conformations are observed. The movie shows a potential transition between these binding modes. Active site residues (D6, E7, D101 and D129) are shown as yellow sticks. Coordinates for the intermediate states were generated using LSQMAN based on the three conformations observed in the structure. The animation was created with PyMOL (DeLano Scientific LLC) and VideoMach (Gromada.com).
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Supplementary Table S1. Plasmids used in this work Plasmid pGEX6P1 pDONR221 pEGFP‐N1 pEGFP‐C2 pEGFP‐C2‐Dest pEGFP‐NLS
Alias ‐ ‐ ‐ ‐ ‐ ‐
pcDNA3.1mychis‐Dest pCGT‐Dest pGEX6P1‐Afu‐rnhB
‐ ‐ pMAR177
pGEX6P1‐Afu‐rnhB D101N
pMAR182
pGEX6P1‐Afu‐rnhBΔPIP
pMAR235
pGEX6P1‐Afu‐rnhBΔSNLR
pMAR237
pENTRY‐RNASEH2B
‐
pENTRY‐RNASEH2C
‐
pEGFP‐RNASEH2B
pKA32
pEGFP‐RNASEH2B FF>AA pEGFP‐RNASEH2C
pKA31 pKA4
pEGFP‐RNASEH1
pKA33
pcDNA‐RNASEH2A pCGT‐RNASEH2B
‐ ‐
Source/Description GE Healthcare Invitrogen Clontech Clontech Gateway (Invitrogen) converted pEGFP‐C2 Expresses SV40 nuclear localization signal (NLS) fused to EGFP C‐terminal Myc/His tagging (6) N‐terminal T7 tagging (6) Archaeoglobus fulgidus rnhB (RNase HII) coding sequence cloned into pGEX6P1; SalI/KpnI D101N catalytic site mutation introduced into pMAR177 by Quikchange mutagenesis A.fulgidus RNase HII with C‐terminal deletion (PIP box) cloned into pGEX6P1; SalI/NotI Hinge (4aa; SNLR) deleted just before PIP box by Quikchange mutagenesis on pMAR177 RNASEH2B coding sequence including stop in pDONR221 RNASEH2C coding sequence including stop in pDONR221 Gateway cloned RNASEH2B from pENTRY‐RNASEH2B into pEGFP‐C2‐DEST Quikchange mutagenesis (F300A,F301A) of pKA32 Gateway cloned RNASEH2C from pENTRY‐RNASEH2C into pEGFP‐C2‐DEST Human RNASEH1 coding sequence (starting at M27) cloned into pEGFP‐N1 RNASEH2A in pcDNA3.1mychis‐Dest(6) RNASEH2B in pCGT‐Dest (6)
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Supplementary Table S2. Oligonucleotides for RNase H substrates Name
Sequence
Description & comments
DNA18‐Rev1
AGCTCCCAGGCTCAGATC
DNA3‐RNA1‐DNA14
GATcTGAGCCTGGGAGCT
DNA5‐RNA1‐DNA12
GATCTgAGCCTGGGAGCT
DNA7‐RNA1‐DNA10
GATCTGAgCCTGGGAGCT
DNA9‐RNA1‐DNA8
GATCTGAGCcTGGGAGCT
DNA11‐RNA1‐DNA6
GATCTGAGCCTgGGAGCT
DNA12‐RNA1‐DNA5
GATCTGAGCCTGgGAGCT
DNA13‐RNA1‐DNA4
GATCTGAGCCTGGgAGCT
DNA14‐RNA1‐DNA3‐F2
GATCTGAGCCTGGGaGCT
DNA15‐RNA1‐DNA2
GATCTGAGCCTGGGAgCT
DNA16‐RNA1‐DNA1
GATCTGAGCCTGGGAGcT
DNA40‐R
CCTCTAGAGTCGACCTGCAGGCATGCAAGC TTTTGTTCCC GGGAACAaAAGCTTGCATGCCTGCAGGTCG ACTCTAGAGG GGGAACAAAAGCTTgCATGCCTGCAGGTCG ACTCTAGAGG GGGAACAAAAGCTTGCAuGCCTGCAGGTCG ACTCTAGAGG GGGAACAAAAGCTTGCATGCcTGCAGGTCG ACTCTAGAGG GGGAACAAAAGCTTGCATGCCuGCAGGTCG ACTCTAGAGG GGGAACAAAAGCTTGCATGCCTGCaGGTCG ACTCTAGAGG GGGAACAAAAGCTTGCATGCCTGCAGgTCG ACTCTAGAGG GGGAACAAAAGCTTGCATGCCTGCAGGTcG ACTCTAGAGG GGGAACAAAAGCTTGCATGCCTGCAGGTCG AcTCTAGAGG GGGAACAAAAGCTTGCATGCCTGCAGGTCG ACTCuAGAGG GGGAACAAAAGCTTGCATGCCTGCAGGTCG ACTCTAgAGG GGGAACAAAAGCTTGCA ugccugcaggTCGACTCTAGAGG
18 nt DNA oligo used as reverse strand for RNase H assay substrates DRD18 with ribonucleotide in position 4, anneals to DNA18‐Rev1 DRD18 with ribonucleotide in position 6, anneals to DNA18‐Rev1 DRD18 with ribonucleotide in position 8, anneals to DNA18‐Rev1 DRD18 with ribonucleotide in position 10, anneals to DNA18‐Rev1 DRD18 with ribonucleotide in position 12, anneals to DNA18‐Rev1 DRD18 with ribonucleotide in position 13, anneals to DNA18‐Rev1 DRD18 with ribonucleotide in position 14, anneals to DNA18‐Rev1 DRD18 with ribonucleotide in position 15, anneals to DNA18‐Rev1 DRD18 with ribonucleotide in position 16, anneals to DNA18‐Rev1 DRD18 with ribonucleotide in position 17, anneals to DNA18‐Rev1 40 nt DNA oligo anneals to 40nt DRD oligos for RNase H assay DRD40 with ribonucleotide in position 8, anneals to DNA40‐R DRD40 with ribonucleotide in position 15, anneals to DNA40‐R DRD40 with ribonucleotide in position 18, anneals to DNA40‐R DRD40 with ribonucleotide in position 21, anneals to DNA40‐R DRD40 with ribonucleotide in position 22, anneals to DNA40‐R DRD40 with ribonucleotide in position 25, anneals to DNA40‐R DRD40 with ribonucleotide in position 28, anneals to DNA40‐R DRD40 with ribonucleotide in position 30, anneals to DNA40‐R DRD40 with ribonucleotide in position 32, anneals to DNA40‐R DRD40 with ribonucleotide in position 35, anneals to DNA40‐R DRD40 with ribonucleotide in position 37, anneals to DNA40‐R anneals to first 17 nt of DNA40‐R Okazaki‐like fragment containing 10 ribo and 13 deoxyribonucleotides; anneals to nt 18‐40 of DNA40‐R
DNA7‐RNA1‐DNA32‐F DNA14‐RNA1‐DNA25‐F DNA17‐RNA1‐DNA22‐F DNA20‐RNA1‐DNA19‐F DNA21‐RNA1‐DNA18‐F DNA24‐RNA1‐DNA15‐F DNA27‐RNA1‐DNA12‐F DNA29‐RNA1‐DNA10‐F DNA31‐RNA1‐DNA8‐F DNA34‐RNA1‐DNA5‐F DNA36‐RNA1‐DNA3‐F DNA17‐F RNA10‐DNA13
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