for DNA replication in Thermococcus kodakarensis. Mariko Nagata, Sonoko Ishino, Takeshi Yamagami, Hiromi Ogino, Jan-Robert Simons,. Tamotsu Kanai ...
Supplementary Data
The Cdc45/RecJ-like protein forms a complex with GINS and MCM, and is important for DNA replication in Thermococcus kodakarensis Mariko Nagata, Sonoko Ishino, Takeshi Yamagami, Hiromi Ogino, Jan-Robert Simons, Tamotsu Kanai, Haruyuki Atomi, Yoshizumi Ishino
Supplementary Methods Cloning of the genes encoding three Rpa proteins from T. kodakarensis and preparation of the expression plasmid Similar to P. furiosus RPA (S1), the genes encoding RPA41, RPA14, and RPA32 (TK_RS09820, TK_RS09815, and TK_RS09810, respectively) are tandemly arranged in the T. kodakarensis genome and seem to consist an operon. The DNA fragment containing the three genes was amplified by PCR from the genomic DNA using the p r i m e r s e t s , r p a - F ( C G C G C ATAT G G A A G T T C T- G A C A A A G G A C G A G ) a n d r p a - R (GGGGCGGCCGCTCAGAGCGGCTCGTAGAAGCCTATC). The PCR reaction was performed by PfuDNA polymerase (Agilent Technologies), and the amplified DNA was digested by NdeI and NotI and into the corresponding sites of the pET-21a(+) expression vector (Novagen). Restriction and modification enzymes were purchased from New England Biolabs. The resultant plasmid was designated as pET-RPA. The nucleotide sequence was confirmed by the sequencing using CEQ2000XL (Beckman Coulter). Preparation of TkoRPA protein The recombinant proteins, RPA41, RPA14, and RPA32, were co-produced in E. coli BL21 codonPlusTM (DE3)RIL cells. The E. coli cells bearing the pET-RPA plasmid were cultured at 37°C in LB medium, containing 50 µg/ ml ampicillin and 34 µg/ml chloramphenicol. When the cell density reached an OD600 of 0.40, the gene expression was induced by adding IPTG to a final concentration of 1 mM, followed by further cultivation for 16 h at 25°C. The cells were harvested and were disrupted by sonication in buffer A (50 mM Tris-HCl, pH 8.0, 0.1 mM EDTA, 0.5 mM DTT, 10％ glycerol) containing 0.5 M NaCl. The soluble extracts were heated at 80°C for 20 min. The heat-resistant fraction was treated with 0.15% polyethyleneimine. The soluble fraction was subjected to an hydrophobic chromatography on HiTrap Phenyl HP column (GE Healthcare), which was developed with a linear gradient of 1–0 M (NH4)2SO4 in buffer A. The eluted protein fractions were dialyzed against buffer A, and loaded on HiTrap Heparin HP affinity chromatography column (GE Healthcare), which was developed with a linear gradient of 0–1 M NaCl in buffer A. The fractions containing the RPA41, RPA14, and RPA32 proteins, were diluted with twice the volume of buffer A, and loaded on HiTrap Q HP anion exchange chromatography column (GE Healthcare), which was developed with a linear gradient of 0.1–1 M NaCl in buffer A. The protein concentrations were determined using the absorbance at 280 nm and the extinction coefficient of 117,580 M-1cm-1 for RPA complex protein. The ssDNA-specific binding activity of the purified TkoRPA was confirmed by Electrophoresis mobility shift assay. Supplementary References S1. Komori, K, and Ishino, Y. (2001) Replication protein A in Pyrococcus furiosus is involved in homologous DNA recombination. J. Biol. Chem., 276, 25654-25660.
Supplementary Table S1. Sequences of primers used for site-specific mutagenesis Sequence (5′–3′)
Name GAN_D36A-F
CCACAGGGACGCTGcCGGCATCACGGCAG
GAN_D36A-R
CTGCCGTGATGCCGgCAGCGTCCCTGTGG
GAN_D34A/D36A-F
CATCTCCCACAGGGcCGCTGcCGGCATCACGGC
GAN_D34A/D36A-R
GCCGTGATGCCGgCAGCGgCCCTGTGGGAGATG
The substitutions are indicated in lowercase letters.
Supplementary Table S2. Sequences of oligonucleotides used in the nuclease assay and the helicase assay Number
Name
Label
Sequence (5′–3′)
Length (nt)
1
dA30FITC
3′-FITC CGAACTGCCTGGAATCCTGACGAACTGTAG
30
2
dA30ssssFITC
3′-FITC CGAACTGCCTGGAATCCTGACGAACTgtag
30
3
HJ3-54mer-FITC
3′-FITC TCACTCCGCATCTGCCGATTCTGGCTGTGGCGTGTTTCTGGTGGTTCCTAGGTC
54
4
HJ3-54merR&C
5
GACCTAGGAACCACCAGAAACACGCCACAGCCAGAATCGGCAGATGCGGAGTGA
54
HJ3-54RC34-trap
− −
GACCTAGGAACCACCAGAAACACGCCACAGCCAG
34
6
HJ3-54RC34
−
CACGCCACAGCCAGAATCGGCAGATGCGGAGTGA
34
7
cy5-hel-1-50
8
hel-2-60
9
trap
5′-Cy5 TTTCTTTCTTTCTTTGCTGGCTGTGGCGTGTTTCTGGTGGTTGGTAGGTC
50
−
GACCTACCAACCACCAGAAACACGCCACAGCCAGACACACACACACACACACACACACAC
60
−
GACCTACCAACCACCAGAAACACGCCACAGCCAG
34
Small characters represent modification to phosphorothioate bond at 5′ side to prevent the 5′ digestion of each phosphodiester backbone.
Supplementary Table S3. Combinations of the oligonucleotides to prepare the substrates Structure
Combination
blunt end
3+4
5′-overhang
3+5
3′-overhang
3+6
splayed
7+8
The numbers on the combination column correspond to those in Supplementary Table S2.
Supplementary Table S4. Primers used for amplification of the gan loci Name
Sequence (5′–3′)
gan-F
CGCGCATATGGATAAGGAGGCTTTTTTGGAGCGCG
gan-R
GGGGCGGCCGCTCAACCCTCGCCTTCACTTCCACCG
fgan-F
AAAGGATCCGAGGTAGCAGAGTTGTATCCTGCGGAG
fgan-R
AAAGAATTCCGGCTTGAATGTGCTTTGTGGCAGATCC
dgan-F
GTGGAAGTGAAGGCGAGGGTTG
dgan-R
GCGGCATCACCGGAAGAAG
Supplementary Table S5. Primers used for qPCR Name TK1251rps15-F TK1251rps15-R TK1252gan-F TK1252gan-R TK1253pcc1-F TK1253pcc1-R TK1254rps3-F TK1254rps3-R TK0765gapdh-F TK0765gapdh-R TK16S-F TK16S-R TK1620mcm3-F TK1620mcm3-R TK0536gins51-F TK0536gins51-R TK1619gins23-F TK1619gins23-R
Sequence (5′–3′) TCCAGAGGACCTCATGTTCC TTGACGAGCCTCCTGATCTT GAACCTTCCAGCTCAGCATC GAGTTCAATTGAGCCGCTTC ATCCAGGTGGACAACGAGAG TATCTTCGGCGATTTTGACC TTCAACATCACCACCAAGGA GGAGAACGAAGTCAGCGAAG AAGGGGCATTCCTGTCTATG GTGTGGCATCAACGATTACG CGGGTAGTCCTGGCTGTAAA CCCGCCAATTCCTTTAAGTT GGAGTGGGTCTACGATGTTACG TATGCTGGCGTCTACTTTCTCG ATCTCGCAGTCGAGGGTAAA CGGCCCATACTCTTTCATGT CATTCGGAGACTGGAAGAGC TAGAACTCGGCCCTCTCGTA
B
(RU) 800 600
600
Response
Response
A (RU) 800 400 200
400 200 0
0 0
120 Time
(s)
0
120
(s)
-20020406080 100 120 140 160 180 200 220 240 Time
-200
Supplementary Figure S1. Physical interactions of MCM with the GAN·GINS complex. Different concentrations of MCM (50, 100, 200, 400, and 800 nM, as the hexamer) were loaded for 120 s on a GINS-saturated GAN-immobilized chip (A) and a GAN-saturated GINS-immobilized chip (B).
ATPase (mol mol-1 min-1)
12 10 8
No DNA ssDNA dsDNA
6 4 2 0
MCM MCM MCM MCM 1 + GAN 2 + GINS 3 4 + GINS + GAN
Supplementary Figure S2. Effects of GINS and GAN on ATPase activities of MCM at 60˚C. The reaction mixtures, containing 0.5 µM MCM (as the hexamer), 2 µM GINS (as the tetramer), and 4 µM GAN, were incubated for 20 and 60 min at 60°C in the presence or absence of 7.5 µM (in nucleotides) DNA (M13mp18 ssDNA or M13mp18 RF DNA). The ATPase activity is expressed as the amount of Pi released by a constant amount of the MCM protein (as the hexamer). The SEM was calculated from three independent experiments.
B
A (Mr, ×103)
M RPA
dsDNA (45 bp)
ssDNA (45 nt)
200 116 97.2 66.4
0
5 10 25 50 100 200
0
5 10 25 50 100 200
RPA (nM)
44.3 29.0
RPA-DNA complex
20.1
free DNA
14.3
Supplementary Figure S3. Preparation and DNA-binding properties of RPA. (A) Purified the recombinant TkoRPA complex (2.5 µg) was analyzed by SDS-12.5% PAGE. The gel was stained with CBB. The Protein size markers were run in lane M, and their sizes are indicated on the left side of the gel. (B) Electrophoresis mobility shift assay of RPA with ssDNA (left) and dsDNA (right). Various concentrations (indicated on the top of the panel) of RPA were incubated with 10 nM 5′Cy5-labeled ssDNA (45 nt, CGAACTGCCTGGAATCCTGACGACATGTAGCGAACGATCACCTCA) and dsDNA (5′-Cy5-labeled ssDNA annealed with its complementary strand) at 50˚C for 15 min in a reaction solution (20 mM Tris-HCl, pH 8.0, 10 mM KCl, 6 mM (NH4)2SO4, and 2 mM MgCl2). The protein–DNA complexes were fractionated by 1.2% agarose in 0.1× TAE buffer and visualized with a Typhoon Trio+ image analyzer. The band assignments are indicated on the side of the panels. RPA specifically bound to ssDNA.
C − + − − + + + + + + + + + − − − + − − − − + + + + + − − 4 − 0.5 1 2 4 − 0.5 1 2 4
(boil)
10 0.2 1 10 −
ssDNA trapped
B Time (min) GAN 30
(GA)
M Mr, (× 103)
WT D36A
A
MCM GINS GAN-D36A (µM) 5′＊
22 19
5′＊ 5′＊
75 50
% unwound 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16
37
25
4
1 (nt)
lane 1
2
3
4
5
6
7
Supplementary Figure S4. The properties of the GAN-D36A mutant. (A) Purified GAN-WT (2 µg, same protein as in Figure 1A) and GAN-D36A (2 µg) were subjected to SDS -10% PAGE followed by CBB staining. Protein size markers were run in lane M, and their sizes are indicated on the left of the gels. (B) The exonuclease activities of 5 nM GAN-WT and GAN-D36A, using 20 nM 3′-FITC-labeled phosphorothioated DNA (dA30ssssFITC) at 70°C. Reaction products were analyzed by 8 M urea-15% PAGE in TBE. Lanes 1, 2, and 7 are the markers (19 nt and 22 nt, 1 nt, and GA ladder, respectively). (C) The 5′-Cy5-labeled splayed-arm DNA substrate (50 nM) was incubated with MCM, GINS, and GAND36A. The helicase activity is expressed at the bottom of the panel, as the relative amount of unwound DNA (%). Single-stranded and trapped DNAs were loaded in parallel as controls for the unwinding reaction (lanes 1 and 2, respectively). The same results were obtained thrice.
A
B
C
30
3′ (GA)
time (min) 20 0 0.2 1 10 20 GAN − + + + + + 30
(GA)
time (min) 20 0 0.2 1 10 20 GAN − + + + + +
(GA)
substrate 5′ time (min) GAN − + + − + − + + − + 54
22 19
22 19
35 34 33
1 (nt)
1 (nt) 1
2
3
4 5
6
7
8 9
1
2
3
4
5
6
7
8
9 1 (nt) 1 2 3 4 5 6 7 8 9 10 11 12
Supplementary Figure S5. Characterization of the exonuclease activity of GAN. (A and B) GAN (5 nM) was incubated with 20 nM 3′-FITC-labeled ssDNA at 70°C for 0, 0.2, 1, 10, and 20 min. Reaction products were analyzed by 8 M urea-15% PAGE. The substrate DNA used in panel A is a normal 30-nt DNA, and the DNA used in panel B has four successive phosphorothioate modifications at the 3′-terminal region. Lanes 1, 2, and 9 show the size marker DNAs (19 nt and 22 nt, 1 nt, and the GA ladder made of the substrate DNA, respectively). (C) GAN (20 nM) was incubated with 3′-FITC-labeled DNA (100 nM) at 70°C for 10 and 30 min. Reaction products were analyzed by 8 M urea-15% PAGE. The structures of the DNA substrates, with the position of the FITC-label (black circle), are illustrated on the top. Substrates were ssDNA (lanes 2–4), dsDNA (lanes 5 and 6), 5′-overhang (lanes 7–9), and 3′-overhang (lanes 10 and 11), respectively. Lanes 1 and 12 show the size marker (1 nt) and the GA ladder made of the substrate DNA, respectively.
A
B
3.4 kb
flanking region
fgan-F
fgan-R
1.4 kb gan-F
KUW1
coding region
gan-R
(kbp) 5.0 4.0 3.0 2.0
TK1251_rps15
1.5
(TK_RS06175)
TK1252_gan
1.0
(TK_RS06180)
0.5 TK1253_pcc1 (TK_RS06185)
TK1254_rps3 (TK_RS06190)
1 2
3 4
5′ and 3′ flanking region
coding region
Supplementary Figure S6. The gene disruption of the gan loci. (A) Genomic context of the flanking regions of the gan loci in T. kodakarensis KUW1. The expected amplified fragments from KUW1 are indicated by double-headed arrows. (B) PCR analysis of the gan loci in KUW1 and Δgan. The amplified fragments of the 5′ and 3′ flanking regions (lanes 1 and 2) and the coding regions of the gan loci (lanes 3 and 4), using the fganF/R primer set and the gan-F/R primer set, respectively. DNA size markers were run in the middle of the lanes, and their sizes (kbp) are indicated on the left of the gel.
GAN
PCNA1
1 2 3
Supplementary Figure S7. The reverted mutant of gan (Δgan::gan-D34A/D36A) produced the same amount of GAN as KUW1. Samples were analyzed by SDS-12% PAGE and western blotting. PCNA1 (T. kodakarensis has two pcna homologs, but only pcna1 is essential for its viability) was visualized as a loading control.
