Ip Suppl. Fig. S1 - media.nature.com · Cce1 Yen1 Cef1 Mgs1 Mus81 Apn2 Rad16 Slx4 Slx8 Mms4 *...

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.

doi: 10.1038/nature07470 SUPPLEMENTARY INFORMATION

www.nature.com/nature 2

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.



***.::*::*:* * :**** * . * **:* *: *.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


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.



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



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.



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



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.



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



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.




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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