Post on 12-Nov-2018
111 122 3 4 5 6 7 8 9 10 13
EME1MUS81
Sephacryl S-300
Heparin
Q-SepharoseFT
Superdex 75 pg
HeLa nuclear extract150 mM KCl
(NH4)2SO425 - 55 %
Butyl-Sepharose
Heparin
SP-Sepharose
MonoQ
Phosphocellulose
0.1 1 M NaCl
1
0.5 0.35
0.1 1 M NaCl
0.1 1 M NaCl
670 158 44 17 kDa
0.1 1 M KCl
75 43Void 29 kDa
0.1 1 M KCl
ssDNA-cellulose
0.1 1 M NaCl
MonoS
0.1 0.8 M KCl
25 3526 27 28 29 30 31 32 33 34*
*
ResA
*
**
Suppl. Fig. S1. Extracts were prepared from 50 litres (left) or 200 litres (right) of HeLa cells and fractionated by column chromatography according to the schemes. Full details can be found in Methods.The scheme shown on the right was designed to allow the purification of ResA away from MUS81/EME1 complex. Aliquots of each fraction were assayed for their ability to resolve 5’-32P-labelled Holliday juntion X26, lead-ing to the formation of nicked duplex DNA products, respectively. The junction X26 allows branch migration through its 26 bp homologous core, thereby minimising any potential problems with sequence preferences which are often shown by HJ resolvases. FT; column flow through. 32P-labelled DNA products were detected by neutral PAGE and autoradiography. MUS81/EME1 was detected by Western blotting. The branch migration activity shown in the top panels, which gives rise to splayed arm duplexes, was shown to be due to RECQ1 helicase.
0 M (NH4)2SO4
*
*
SUPPLEMENTARY INFORMATION
doi: 10.1038/nature07470
1
MGVNDLWQILEPVKQHIPLRNLGGKTIAVDLSLWVCEAQTVKKMMGSVMKPHLRNLFFRISYLTQMDVKLVFVMEGEPPKLKADVISKRNQTRYGSSGKSWSQKTGRSHFKSVLRECLHMLECLGIPWVQAAGEAEAMCAYLNAGGHVDGCLTNDGDTFLYGAQTVYRNFTMNTKDPHVDCYTMSSIKSKLGLDRDALVGLAILLGCDYLPKGVPGVGKEQALKLIQILKGQSLLQRFNRWNETSCNSSPQLLVTKKLAHCSVCSHPGSPKDHERNGCRLCKSDKYCEPHDYEYCCPCEWHRTEHDRQLNEVENNIKKKACCCEGFPFHEVIQEFLLNKDKLVKVIRYQRPDLLLFQRFTLEKMEWPNHYACEKLLVLLTHYDMIERKLGSRNSNQLQPIRIVKTRIRNGVHCFEIEWEKPEHYAMEDKQHGEFALLTIEEESLFEAAYPEIVAVYQKQKLEIKGKKQKRIKPKENNLPEPDEVMSFQSHMTLKPTCEIFHKQNSKLNSGISPDPTLPQESISASLNSLLLPKNTPCLNAQEQFMSSLRPLAIQQIKAVSKSLISESSQPNTSSHNISVIADLHLSTIDWEGTSFSNSPAIQRNTFSHDLKSEVESELSAIPDGFENIPEQLSCESERYTANIKKVLDEDSDGISPEEHLLSGITDLCLQDLPLKERIFIKLSYPQDNLQPDVNLKTLSILSVKESCIANSGSDCTSHLSKDLPGIPLQNESRDSKILKGDQLLQEDYKVNTSVPYSVSNTVVKTCNVRPPNTALDHSRKVDMQTTRKILMKKSVCLDRHSSDEQSAPVFGKAKYTTQRMKHSSQKHNSSHFKESGHNKLSSPKIHIKETEQCVRSYETAENEESCFPDSTKSSLSSLQCHKKENNSGTCLDSPLPLRQRLKLRFQST
161121181241301361421481541601661721781841901
Suppl. Fig. S2. Mass spectrometric analysis of gel slice e (as indicated in Figure 1c) revealed five peptides (coloured) corresponding to the N-terminal region of GEN1. Green, red and blue underlines indicate the XPG-N, XPG-I and helix-hairpin-helix domains, respectively.
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Yen1
Cef
1M
gs1
Mus
81A
pn2
Rad
16S
lx4
Slx
8M
ms4
Cce
1
*
*
-
Suppl. Fig. S3. TAP-fusion proteins, selected as potential HJ resolvases in the screen of Figure 2, were further analysed for HJ resolution using 32P-labelled HJ X26. Like the Mus81-Mms4 TAP pull-downs, Yen1-TAP consistently gave rise to nicked duplex products.
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***.::*::*:* * :**** * . * **:* *: *.GEN1 MGVNDLWQILEPVKQ--HIPLR------NLGGK---TIAVDLSLWVCEA--- 38YEN1 MGVSQIWEFLKPYLQDSRIPLRKFVIDFNKSQKRAPRIAIDAYGWLFECGFI 52
*.:. :.*: * * GEN1 QTVKKMMGSVMKPHLR-----------------------------------N 55YEN1 QNID------ISPRSRSRSRSPTRSPRDSDIDSSQEYYGSRSYTTTGKAVIN 98
:: *:. * .::*: *** **: *.:* .. ::* :..* *GEN1 LFFRISYLTQMDVK--LVF--VMEGEPPKLKADVISKRN------QSRYGSS 97YEN1 FISRLKELLSLNVEFLLVFDGVMK---PSFKRKFNHEQNATTCDDEKEYYS- 146
**.*:. :.* * *::*: :*: :.*.:* *.* GEN1 GKSWSQKTGRSH----------------FKSVLRECLHMLECLGIPWVQAAG 133YEN1 --SWEQHV-KNHEVYGNCKGLLAPSDPEFISLVRK---LLDLMNISYVIACG 192
*.** *.:*:..* ** *:**.**:::*.:.: :*:: .* : * GEN1 EAEAMCAYLNAGGHVDGCLTNDGDTFLYGAQTVYRNFTMNTKDPHVDCYTMS 185YEN1 EGEAQCVWLQVSGAVDFILSNDSDTLVFGGEKILKNYSKFYDD-----FGPS 239
**.* *:. :** :*: :::***.**GEN1 SIKS-----------------------KLG---LDRDALVGLAILLGCDYLP 211YEN1 SITSHSPSRHHDSKESFVTVIDLPKINKVAGKKFDRLSLLFFSVLLGADY-N 290
:** *:**:::*:* * *..:** * .*:.GEN1 KGVPGVGKEQALKLIQI--------------------LKGQSLLQRFNRWNE 243YEN1 RGVKGLGKNKSLQLAQCEDPNFSMEFYDIFKDFNLEDLTSESL--RKSRYR- 339
*. *:* :* ***. ** * :...:GEN1 TSCNSSPQLLVTKKL-AHCSVCSHPGSPKDHE-----RNGCRLCKSDKYCEP 289YEN1 ---------LFQKRLYLYC---------KDHSVELFGRNYPVLLNQGSF--- 370
**:* ** ::* GEN1 HDYEYCCPCEWHRTEHDRQLSEVENNIKKKACCCEGFP--------FHEVIQ 333YEN1 ----------------------------------EGWPSTVAIMHYFHPIVQ 388
*.* .** ::: :*:. * *: :: *:.:: *: : :*KLVKVI---RYQRPDLLLFQ--RFTLEKMEWPNHYACEKL 375
YEN1 PYFDEEVL--SDKYINMAGNGHYRN---LNFNELKYFLQSLNLPQISSFDKW 435
:.** .: *:. ::*. * :**.: :* * :GEN1 -------LVLLTHYDMIER--KLGSRNSNQLQPIRIVKTRIRN------GVH 412YEN1 FHDSMHEMFLLREFLSIDESDNIGKGN------MRITEEKIMNIDGGKFQIP 481
**:*.: :.* : *: :*GEN1 CFEIEW------------------------EKPEHYAMEDKQHGEFALLTIE 440YEN1 CFKIRYTTFLPNIPISSQSPLKRSNSPSRSKSPTRRQMDIMEH--------- 524
:**: . * * :: *:.*:* * ****.:: : *::*** * GEN1 EESLFEAAY------PEIVAVYQKQKL-----EIKGKKQKRIK-PKENNLPE 480YEN1 PNSLWLPKYLIPQSHPLVIQYYETQQLIQKEKEKKGKKSNKSRLPQKNNLDE 576
-----EFLLNKDGEN1
GEN1 D...EAEAMCAYLNAGGHVDGCLTNDGD Yen1 EGE134 136193 195
Suppl. Fig. S4. Homology between Homo sapiens GEN1 and Saccharomyces cerevisiae Yen1. a, Alignment of the N-terminal GEN1 and Yen1 protein sequences. A significant homology was found between the first 480 amino acids of GEN1 and the first 576 amino acids of Yen1, while little homology was found in their C-terminal regions (not shown). The green, red and blue lines indicate the positions of the XPG-N, XPG-I and helix-hairpin-helix domains, respectively. The black boxes show the regions containing the acidic residues modified in the mutagenesis experiments. Asterisks, colons and dots indicate identity (red), strong similarity (orange) or weak similarity. b, Conserved acidic residues (red) that were mutated to generate catalytically inactive Yen1 and GEN1 mutants.
a
b 15730
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Rad2 1031Yen1 759
Rad27 382Din7 430Exo1 702
XPG 1186GEN1 908
FEN1 380EXO1 846
S. cerevisiae
H. sapiens
Suppl. Fig. S5. The Rad2/XPG family of proteins in S. cerevisiae and H. sapiens. Three characteristic sequence motifs are indicated: the N-terminal (green) and internal (red) nuclease domains, and a helix-hairpin-helix domain (blue). Total amino acid residues are indicated.
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Suppl. Fig. S6. a, Immunoprecipitates of TAP-fusion versions of Cce1 and representative members of the Rad2/XPG family in S. cerevisiae were analysed for HJ cleavage activity using 32P-labelled X26. b, The presence of each TAP fusion protein was analysed by Western blotting using peroxidase anti-peroxidase soluble complex antibody (Sigma). Predicted molecular weights for Cce1, Yen1, Exo1, Rad2 and Rad27 are 41, 87, 80, 117 and 43 kDa, respectively. The TAP-tag has a molecular weight of ~20 kDa. The strong signal obtained for Rad27 reflects the relatively high expression level of this protein.
97
191
Cce1
Yen1
Exo1
Rad2
Rad2
7
64
51
39
2819
14
kDa
Cce1
*
*
Yen1
Exo1
Rad2
Rad2
7
-
a b
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GCAA
G
ATGT
C
G
G
C
A
AT
C
T
GT
CC
GG
C
AA
A
AG
T
TG
C
G
G
ACA
CT
C
TTTGCC
GC
TA
TA
AT
CG
CG
TA
AT
AT
CG
GC
AT
TA
AT
TA
CG
GC
TA
TA
AT
GC
GC
TA
AT
AT
CG5’
5’
AGC
T1 2
4 3
40
36
3126
40
36
3126
1*3* 1*3* 1*3* 1*3*
40
36
26
31
2 3 4 5 6 7 81
Yen1
Yen1
mut
RuvC
-
a b
Suppl. Fig. S7. Resolution of HJ X26 by Yen1.a, The junction was 5’-32P-end labelled in either strand 1 or strand 3, and incubated with extracts preparedfrom yeast cells over-expressing either wild-type or mutant Yen1. E. coli RuvC was used as a control. Theproducts were detected by denaturing PAGE. Preferential sites of cleavage, leading to the formation offragments 31 and 36 nucleotides in length for Yen1, and 26 and 40 nucleotides in length for RuvC, weremapped using oligos of defined length (not shown). The slight differences in migration, observed witholigos of the same length, are due to sequence effects.b, The major Yen1 (black) and RuvC (red) induced sites of cleavage, which occur with perfect symmetryare shown. Strands 1 to 4 are indicated. The central 26 base pairs region of homology through which thejunction can migrate is indicated by dotted lines. Resolution in the other orientation of the junction, causedby cleavage of strands 2 and 4, is not shown.
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2* 4* 2*4* 2*4* 2*4*
GEN
1
ResA
RuvC
b-
GCAA
G
ATGT
C
G
G
C
A
AT
C
T
GT
CC
GG
C
AA
A
AG
T
TG
C
G
G
ACA
CT
C
TTTGCC
GC
TA
TA
AT
CG
CG
TA
AT
AT
CG
GC
AT
TA
AT
TA
CG
GC
TA
TA
AT
GC
GC
TA
AT
AT
CG
5’
5’
AGC
T1 2
4 3
4238
29
28
42 38
29
28
a
Suppl. Fig. S8. Resolution of HJ X26 by GEN1.a, The junction was 5’-32P-end labelled in either strand 2 or strand 4, and incubated with affinity purifiedGEN1-FLAG. E. coli RuvC, and ResA from HeLa cells, were used as controls. The products were detectedby denaturing PAGE. Preferential sites of cleavage, leading to the formation of fragments 29 and 38 nucle-otides in length for GEN1, and 28 and 42 nucleotides in length for RuvC, were mapped using oligos ofdefined length (not shown). ResA gave an identical cleavage pattern to that produced by GEN1. Note thatthe slight differences in migration, observed with oligos of the same length, are due to sequence effects.b, The major GEN1 (black) and RuvC (red) induced sites of cleavage, which occur with perfect symmetryare shown. The central 26 base pairs region of homology through which the junction can migrate is indicated by dotted lines. Resolution in the other orientation of the junction, caused by cleavage of strands 1 and 3, is not shown.
42
38
28 29
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0.1
ScExo1
ScDin7
Ce28728
Dmtosca
HsEXO1AtEXO1
AtFEN1ScRad27
DmFEN1CeCRN1
HsFEN1
CeXPG
AtUVH3
ScRad2
DmXPG
HsXPGCe01401 ScYen1
AtGEN
HsGEN1
DmGEN
Class I:Nucleotide excisionrepair endonucleases
Class IV:Holliday junctionresolvases
Class II:replication flapendonucleases
Class III:recombination/repairexonucleases
Suppl. Fig. S9. Yen1 and GEN1 belong to the Rad2/XPG family of structure-specific nucleases. Phylo-genic relationships of representative Rad2/XPG nucleases from S. cerevisiae (Sc), C. elegans (Ce), D. melanogaster (Dm), H. sapiens (Hs), and A. thaliana (At). The scale bar indicates distance.
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RAD51C
XRCC3
GEN1
Cont
rol
GEN1-FLAG
FLAG-IP
Suppl. Fig. S10. Analysis for the presence of RAD51C-XRCC3 in the affinity purified preparationof GEN1-FLAG. Immunoprecipitation was performed using anti-FLAG agarose using cell linestransfected (GEN1) or not (control) with the GEN1-FLAG construct. M: Active resolvase fractionsfrom the sephacryl S-300 column (Supp. Fig. S1) were probed for RAD51C and XRCC3 using thesame antibodies.
M
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MUS81-EME1BLM-TopIIIα-RMI1
dissolution symmetricresolution
GEN1
asymmetriccleavage
Suppl. Fig. S11. Pathways for Holliday junction processing in mammalian cells. Dou-ble Holliday junctions produced by strand exchanges between homologous molecules(blue and red), may be processed in a variety of ways. These involve junction dissolu-tion by BLM complex, in which junctions are branch migrated and decatenated bycombined helicase/topoisomerase actions, symmetric HJ resolution by GEN1, or byasymmetric cleavage mediated by MUS81/EME1 complex. Green arrows indicatebranch migration. Black arrows indicate cleavage sites. Broken lines indicate newlysynthesised DNA.
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X26-1 GCGCTACCAGTGATCACCAATGGATTGCTAGGACATCTTTGCCCACCTGCAGGTTCACCC
X26-2 GGGTGAACCTGCAGGTGGGCAAAGATGTCCTAGCAATCCATTGTCTATGACGTCAAGCTC
X26-3 GAGCTTGACGTCATAGACAATGGATTGCTAGGACATCTTTGCCGTCTTGTCAATATCGGC
X26-4 GCCGATATTGACAAGACGGCAAAGATGTCCTAGCAATCCATTGGTGATCACTGGTAGCGC
X0-1 ACGCTGCCGAATTCTACCAGTGCCTTGCTAGGACATCTTTGCCCACCTGCAGGTTCACCC
X0-2 GGGTGAACCTGCAGGTGGGCAAAGATGTCCATCTGTTGTAATCGTCAAGCTTTATGCCGT
X0-3 ACGGCATAAAGCTTGACGATTACAACAGATCATGGAGCTGTCTAGAGGATCCGACTATCG
X0-4 CGATAGTCGGATCCTCTAGACAGCTCCATGTAGCAAGGCACTGGTAGAATTCGGCAGCGT
X0-2.1 GGGTGAACCTGCAGGTGGGCAAAGATGTCC
X0-3.1 CATGGAGCTGTCTAGAGGATCCGACTATCG
X26-2S GGGTGAACCTGCAGGTGGGCAAAGATGTCCTAGCAATCCATTGTCTATGACGT
X26-3S ACGTCATAGACAATGGATTGCTAGGACATCTTTGCCGTCTTGTCAATATCGGC
Suppl. Table 1. Oligonucleotides employed for the preparation of the various DNA substrates. All the sequences are given as 5’-3’.
Holliday junction X26, containing a 26 bp region of homology through which the junction can move, was prepared by annealing oligos X26-1, X26-2, X26-3, and X26-4. The immobile Holliday junction X0 was generated by annealing oligos X0-1, X0-2, X0-3, and X0-4. The splayed arm substrate contained X0-1 and X0-4. The 5’-flap structure contained X0-1, X0-2.1 and X0-4. The 3’-flap structure contained X0-1, X0-3.1 and X0-4. The replication fork substrate contained X0-1, X0-2.1, X0-3.1 and X0-4. The asymmetric HJ (X26-S) employed for ligation assays contained oligos X26-1, X26-4, X26-2S and X26-3S.
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