SUPPLEMENTAL FIGURES EVOLUTIONARY AND FUNCTIONAL ... · SUPPLEMENTAL FIGURES EVOLUTIONARY AND FUNCTIONAL CONSERVATION OF THE DNA NONHOMOLOGOUS END-JOINING PROTEIN, XLF/CERNUNNOS Pierre

SUPPLEMENTAL FIGURES

EVOLUTIONARY AND FUNCTIONAL CONSERVATION OF THE DNA NONHOMOLOGOUS END-JOINING PROTEIN, XLF/CERNUNNOS

Pierre Hentges, Peter Ahnesorg, Robert S. Pitcher, Chris K. Bruce, Boris Kysela, Andrew J. Green, Julie Bianchi, Thomas E. Wilson, Stephen P. Jackson and Aidan J. Doherty

Supplementary Figure 1XLF proteins are predicted to share the same structural fold as XRCC4. The sequences of human, S. pombe and N. crassa XLF homologues were submitted to Phyre (Protein Homology/analogY Recognition Engine; 1). The best hits were based on the crystal structure of human XRCC4.

Supplementary Figure 2A – sequence alignment of metazoan XLF homologues

Hsap/1-299Agam/1-261Dmel/1-459Drer/1-309Ggal/1-298Mmus/1-295Tnig/1-354Xlae/1-311

jnetpred

Conservation

Consensus


jnetpred

Conservation

Consensus


jnetpred

Conservation

Consensus

11111111

5850525561586169

M - - - E E L EQ G L L MQ P WA W- - - L Q L A E N S - - - - - - L L A K V F I T K Q G Y A L L V S D L QQ V W- - H EQ V D T S V V SQ R AM - - T D F C E L T L N R K G F Y G - - - V D F S K D F - - - - - - - - - - - - - - - D R I K C V V F DM E G L W- - M E E L P V A T L I K RQM - - - L L K V V N I N G L Q H L F - - - A C T E GQ H - - - - - - - - - - - - - - D E T L T I H V F R P R E K WT Y S E T L M K R D Y QQ R LM - - - - - - E A V L S A L P WV P - - - V N I S G S D - - - - - - L L A K A W F GD SQ Y R V L L T D L S T V W- - E E EM S T D D I Q S R AM E H S E E L E S R L L T Q P WA S - - - V C F D E S T - - - - - - F I A K A C F GD T G Y M L L I S D L S GV W- - Y E S A D A E A V GQ R SM - - - E E L EQ D L L L Q P WA W- - - L Q L A E N S - - - - - - L L A K V S I T K H G Y A L L I S D L QQ V W- - H EQ V D T S V V SQ R AM E A C D A A P D V L L Q R P W L P - - - V N I G GC R - - - - - - L L A K S C L GA S S Y H L L L T D T H C V W- - E E R A GP S D I Q R R AI - A V S R H R T C L C R K K WM P D S C S C P G GR S A SQ T V F FM GK V C F T E S S Y A L L L S D L S SMW- - C E E A K A D I I Q D R A

9 - - - 0 0 1 2 3 2 + 4 3 5 3 7 5 4 - - - 5 5 4 4 3 3 2 - - - - - - - - - - - - - - 0 3 4 7 3 8 4 9 5 7 5 3 4 5 * - - 2 * 5 7 2 2 3 3 6 5 4 * 5

M E A - E E L EQ V L L RQ P WA P - - - V C+ + G+ S - - - - - - L L A K V C F+ D S G Y A L L + S D L S+ V W- - + E E A D T S D+ QQ R A

5951535662596270

125107110124127125131135

K E L N K R L T A P P A A F L C H L D N L L R P L L K D A A H P - - S E A T F S C D C V A D A - L I L R V R S E L S G L P F Y - - WN F H CM LK E R N P T M D C T E A L I DQ T L L E A G- - - - - - - - - - - - C S GN Y S A T I A E A G - V H F T A K Y Y L E N Y P I T - - FQ F F L V PK T L N A R V K F P E E L V RQ V L L N K - - - - - - - - - - - - - D P L H V EQ D Y Q H P L - N V L K L K Y A M A N F K V F L K W EWH L R PQ D L N K R L R A P A Q A F F S H L C S V A R P C F S G L D E DQ I S A A Q A A L EQ H G E S - L T V K L K S E L A G L P F Y - - W E F R C T TK E L N K R L T A H V S S V L Q R L H G L L S P A L A GQ P H - - - A A T S F S CQ R P A GG - L S L R V R S E L S G L P F Y - - WD F R C R PK E L N K R L T A P P A A L L C H L D E A L R P L F K D S A H P - - S K A T F S C D R G E E G - L I L R V Q S E L S G L P F S - - WH F H C I PQ E L N K R L R A P V E A F F T H L CQ V V R P C L S G S A GP P D GG P Q I S L I R E D D G D L R I K V K S E L A G L P F R - - W E F H C R PR E L N K R L K A P V S S F L S Y L SQ I V F P V L N S K D N - - - GQ N I F S C H R S E A E - L L L Q V K SQ L S G L P F Y - - W S F H C K E

7 7 5 * 3 7 8 5 7 3 2 4 5 7 2 6 3 * 1 4 4 1 - - - - - - - - - - - - 5 2 3 2 5 4 5 2 3 0 2 3 3 - 5 2 7 6 7 7 6 3 9 4 6 7 4 7 3 - - 9 3 9 5 8 4 3

K E L N K R L T A P V A A F L Q H L + + + L R P + L + G S A H P - - S A A + F S C D R - E+ G - L I L + V K S E L S G L P F Y - - W E F H C R P

126108111125128126132136

192174179191194192188202

A S P S L V SQ H L I R P L M GM S L A L Q CQ V R E L A T L L HM K D L E I Q D Y Q E S GA T L I R D R L K T E P F E E N S F L EQ - -A R A Q G I S C H Y I K P L WR T V L L Q Y A Q I R A L A E E L K R K D A E I A Q Y Q A E GA R L N R T S V Q T V P F D EQ T F R H E - -M D A A Q L Y SQM F L N T M S T A L G L R A Q I P L L L G I I H A K D K E L NQ Y R T E GCQ L R R V T V E T Q P F D L E A FM N E H KT P V A V V C RQ L V R P L L A M T L V L Q RQ A E D L A A L L A R K D A E I Q D Y Q E N GA V L S R A R LQ T E P F E V HQ Y K E N - -A P V A T V C R H L V R P L V RM S L A L Q HQ V Q E L T S L L L R K D A E I E D Y R E S GA T L S R D R L R T E P FQ E E T F L Q N - -A S S S L V SQ H L I H P L M G V S L A L Q S H V R E L A A L L RM K D L E I Q A Y Q E S GA V L S R S R L K T E P F E E N S F L EQ - -A S L A V V C A Q L V R P L L A M S H L L Q RQ - - G L C G L A A P P L F S A P A WK GQ GG V P Q R H H - - - - - - - - R P V M E T - -A T V S T V C R H L V R P L K SM T E A L E SQ NQ E L C L L L K K K D A E I Q E Y Q D S GA V L T R D R L K T E V F D E L K FQ K S - -

6 3 4 6 5 9 5 4 6 7 7 4 7 6 4 4 6 6 3 7 7 5 3 6 0 0 3 * 5 3 5 7 2 3 4 4 3 6 7 3 4 + 7 4 6 * 9 4 6 3 * 1 4 2 1 2 0 1 2 1 0 2 3 6 5 4 6 - -

A S V A + V C R H L V R P L M+ M S L A L Q+ Q V R E L A + L L + R K D A E I Q D Y Q E S GA V L S R D R L K T E P F+ E+ + F L E+ - -

metazoan XLF

Supplementary Figure 2B – sequence alignment of yeast homologues of human XLF

Spom/1-189Afum/1-255Dhan/1-256Gzea/1-263Ncra/1-253Ylip/1-283

jnetpred

Conservation

Consensus


jnetpred

Conservation

Consensus


jnetpred

Conservation

Consensus


jnetpred

Conservation

Consensus

111111

454952504454

M S- - - - - - - - W- - - - VQ L R GH R F H F I Q A S F D A E SN EA VQ V I H I T D L V - - R VW SC T C SR NMM S SK - - - - - WR R L H L P KQ D G L P P L L - L R Y SR T L D SY N - - V YM T D L S- - N VWT EHM SRQM F D L P L - - - SWN S I F I P F V N P R K V I Y GYQ C D F N N K A Y S- - I Y I T D F K - - N VW L ER L SKQM S L N K S- - - - WR P L P L Q P SP D L P V L L - V S F H T ET SA Y T - - I H I T DM A - - NMWT E S L D R K- - - - - P - - - - WR P L P V T - A P G I P N L L - V A T N F T AQ SY K - - V H L T D L A - - N VWV ENM D R RM V EA N P K A GAW- - V P I T T D D G S FQ F F - A R L T I SN GGY T - - L H L A D V A S FQ FW I ED L R K D

1 0 - - - - - - - - * - - 2 0 9 7 0 2 4 1 3 3 2 8 7 - 5 4 5 3 2 6 4 3 4 6 3 - - 9 8 9 7 * 7 3 - - 7 7 * 5 5 3 8 5 9 5

M S- - K P - - - - WR P L P+ + - - + G+ P V L L - + + F D F+ N - + Y T - - I H I T D L A - - N VWT E- L SRQ

465053514555

9410010810998112

Q I V NQ A E S ER C P I D P FQ T F ET L V D V L A EV L V N V S S E S- - - - - - - - - - R I Q L R G SN D EG SD I L N R A D ED A T T I D P S ED P EQ F EV L L R K I G EA L SG E SGC T A S L EN G L R - - - - - - - - ED SD I I D K A - - N SY G I ED T ED - EK L H Y L L EV L R N GV EC GD V D K T L L F S I T K I N R T M SD N EN TQ I SM R GWN EN T S I D P S ET Q D NM A K F L A S I ST A L D S SQ P GH Y ET S L C L D H D D R S EA G ED DP I M GR A SA ED T S I D P T D GN EN L Q R L L DW I R A A F D I D D P GH R N T S L T L - - - - - GQ GK D D SR V T I R A ED D D C L I D - A N D P T Q H A L L L D K L GQ A L R EGQ I D I R K T SR G L R I SV SM KM GGGT

4 9 5 3 7 + 1 0 8 3 6 4 * 9 1 5 7 5 0 7 6 5 1 2 7 * 3 1 9 3 3 6 7 5 3 4 6 0 1 0 0 0 0 0 0 0 0 2 - - - - 0 1 0 0 4 5 7

+ I - N R A E- ED T S I D P+ ED P E+ L A - L L + K I + - A L + S+ + P+ H R - T S L G L R I - - R+ S- G ED S

95101109110

99113

137155159163153156

L K I F C T SK I A K D I F - L EWNWT L W L T P P E- - - - T I A KM VW F P L I N H H - - - - - - - - - - - L FM K L T V ST K L P A P L R P L I WT I Y L SK EPQ S- - - - ST SQ H L I L P L L R A EA GWE SRQ R T L L D HI Q A K F T G E I - - - - - - - DWD F SV V KQ SP SN A - I D V I SQ I N FQQ F EN H N Y L N Y K V EQ L ER LL V L R I T C E I P E- L Q P L QWPM Y L K K L P A I - - - - N I A T N L V L P L I Q T H H A K D L E I A S L I Q SI EVQ I T C I L P EP F K P L KWP L Y L K K C SA S- - - - T L A T E L V L P L I Q SH EA R AQ E I N H L I A TF E- - - - - - - - - - - - - - - WT F R V SQ V T ET R E L V D L Q R T F F SG L I T V SH S L L SQ V E F L E SQ

8 3 1 0 1 2 1 0 2 - - - - - - - 0 * 6 7 4 9 2 5 4 7 3 3 - - - - 7 6 5 5 3 7 4 4 5 7 8 6 2 3 0 0 0 0 0 1 0 0 0 2 0 2 2

+ + L - I T C+ I P EP L - P L - W+ F Y L + K - P+ S- - - - + + A T - L V L P L I Q - H H A L - S E+ E- L + - -

138156160164154157

189212208216206213

E S ET D T SP G S L M S I I R DQ K S L L N D K T W F K EA L N ER S S S SN P ST P R K R SA F A D - - - - -L SK K DWV L GK L F D K I E SM G I D L ST I F P GV A G L R K A H GD T L L SH A A N Y V K GV A P F D EQI N I K D H Y I V Y L S EN Y K A I N GD E L I K K Y R K N N K A D - - - - - - - - A K Y L H K Y D R K L W SR KL GQ K D A V L T R L L D K L EAM GT GM EH I F N A L SGK K K V SR A V - - - - A A EK V P G L A P F D R RI R D K D A V I N R L V D K L EA N G I G L EH I F N K L P GK R K V GR K E- - - - A EGK I K G L A T F V EAL S L K D Y H I GAMQ K L L A D T EP GR ST EY R P R R F T EA A Y K T D P V K L T E SWK E ER P A V T EK

5 4 3 6 * 3 2 6 5 3 9 4 4 4 7 3 5 5 3 4 3 4 3 2 4 4 3 2 4 2 4 4 3 3 0 0 0 0 0 - - - - 3 2 2 6 3 1 3 3 2 0 1 1 2 0

L S- K D A V I GR L - D K L EAM G I G L + + I F N - + - GK R K+ SR - - - - S- A+ - K+ K G+ A P F D EK

yeast XLF

Supplementary Figure 2C – alignment of yeast homologues of S. cerevisiae NEJ1

Supplementary Figure 2 Sequences belonging to metazon XLF (A), yeast XLF (B) and yeast NEJ1 (C) were aligned using Muscle (2 ) in Jalview (3). Secondary structure prediction was carried out by JNet (4 ). Amino acids are coloured according to hydrophobicity/hydophilicity, and the degree of conservation within the alignment. Abbreviations and sequences used: 2A: Hsap, Homo sapiens (gi: 48146699); Agam, Anopheles gambiae (19611762:470082-478082); Dmel, Drosophila melanogaster (16769408); Drer , Danio rerio (46250370) ; Ggal, Gallus gallus (50750592); Mmus, Mus musculus (47564022) ; Tnig, Tetraodon nigroviridis (47214397); Xlae, Xenopus laevis (50415440). 2B: Spom, Schizosaccharomyces pombe (7160259); Afum, Aspergillus fumigatus (70989185) ; Dhan Debaryomyces hansenii (50425493); Gzea, Gibberella zeae (46107826); Ncra, Neurospora crassa (85115253); Ylip, Yarrowia lipolytica (50549523). 2C: Scer, Saccharomyces cerevisiae (6323295); Scas, Saccharomyces castellii (574.5d); Kwal, Kluyveromyces waltii (56.22583); Dhan, Debaryomyces hansenii (50425493); Cgla, Candida glabrata (50292109); Calb, Candida albicans (77022494); Agos, Ashbya gossypii (44986293).

Scer/1-342Scas/1-346Kwal/1-351Klac/1-300Dhan/1-335Cgla/1-314Calb/1-324Agos/1-270

jnetpred

Conservation

Consensus


jnetpred

Conservation

Consensus


jnetpred

Conservation

Consensus

11111111

6765665959666060

M D - S E L K GQQ L SD - A EWC V K K I N G EGN C L L L F L PM S SP T T I VM I V L V S L ER L V P Y - - - - - - - - - - - V F K L SQ T Q L SQQ CQM E- R E S I GRQ T S E L L EWH S C T I N - - GQ P F L F S L N L SD N E E S T L F F I N A DQ H P T F - - - - - - - - - - - - K A C I S SN D I K AQ C TM SA S SP L K V D F S E L N EW L P Y T I D - - SR T I F G S F SH V K EAQ K I T L C F ST L K GC P V - - - - - - - - - - - - V V I F D A K D I K KQ A AM SNQ EA T G- - - - - - - SWQ F L H I N - - - N D I S L A K I T K E S S S S I I GV L K L C ED EK F L - - - - - - - - - - - R F R V S S EQ I P K E L GM F - D L P - - - - - - - - L SWN S I F I P F - V N P R K V I Y GYQ C D F N N K A Y S I Y I T D F K N VW- - - - - - - - - - - L E R L SKQ D I I D K A NM V - N A - - - - - - - - - - - WK V G E I D - - GR Y N L L KQ D I D R D GT K L L K I T I HM T EA L L V PQ S ST L K K S L K T Y A V K L D SA R KQ L KM S- L F P - - - - - - - - T QW I A K S I P F L GQ EK P C I Y G F T Y N S EC Y Y Y G L Y L C N F EN VW- - - - - - - - - - - KQ EV D KQ K L V H I A KM S- R KM D S ER L ET H I KWR L F D I K - - - - N E L Y F V G L H C D D G L V L Y I Y SP FQ D L V - - - - - - - - - - - - - Q T K I T D E E L HQ A - -

* 2 - 2 1 0 - - - - - - - - - 1 * 3 3 3 2 * 3 - - - 1 3 2 1 5 2 3 2 3 1 2 4 3 4 3 3 4 3 4 5 2 1 3 5 2 1 3 0 - - - - - - - - - - - - 4 2 2 7 4 2 5 4 7 1 2 2 1 0

M S- + EP - G- Q L S E L L EW- + K T I N F - GN P I L L+ + G L SC D S+ + + L Y+ L + L+ + + + + VW- - - - - - - - - - - + F+ V S+ + D I K KQ A K

6866676060676161

132128128118133139120118

- SQ G F T D S I S L N L I K L K LM D I LQ A PQ E I NQ I G L V D SN L V F S - - - - - - - - - - - - - - F D V SA D I T V S I N S V P SH V T K DM F YM- I Q G F P SD SV T K I T K - ET Y KQ I HQ L R S F K K I - ED G SR LQ L T - - - - - - - - - - - - - - I V L SGD I T L N L T T I I L G L SMQ EQ L T- GQ G I V V E SD I T E- - - Q V A H L L R EP SK A LQ I H I D E NQ V V L E - - - - - - - - - - - - - - I T I SR D I T L S L T F SA C R L E E SQ I SAC SN GV D S I S- - - - - - - E L Y N I L T K GV EG S S I V AM D K K I I I S - - - - - - - - - - - - - - I Q R K K A S SV E I ET A I I A I T G EH L T Q- SY G I ED T ED E- - - - - K L H Y L L EV L R N GV EC GD V D K T L L F S I T K I N R T M SD N EN T I Q A K F T G E I DWD F SV V KQ SP SN A I D- SQ G F D LQ S SD N L I K - I V I E F L N N L N C Y T K N N Y K D SK C EM S L H K - - - - - SDM K I T I K L D A T I K A T I N L G F V R L T D EQ G L L- T I G I EDM SD D - - - - - D L V E L I N D I SR Y I P EKM V F EQ D D V N - - - - - - - - - - - - - - V R A R T I GD I EWR F N L SRQ NQ EQ T I E- K YQ F ST K E- - - - - - - AM E I L L T Q GK D V E F T F EQ D N V LQ L G- - - - - - - - - - - - - - L L L H SGT R A T L E L P Y N K VQ N V ER Y D

- 3 4 6 7 2 3 2 3 - - - - - - - 2 6 1 2 6 + 4 2 5 3 3 5 2 1 4 0 2 3 3 4 4 4 4 7 4 - - - - - - - - - - - - - - 7 3 3 3 3 3 5 4 7 6 7 6 4 3 3 4 3 6 6 2 3 3 2 4 3

- SQ G F+ D - SD D N L I K - + L Y E L L+ Q L + E+ - + I G EV D S+ L+ L S - - K - - - - - SD - - - T I Q L SA D I T + + + + F S I + R L T - EQ - + D

133129129119134140121119

207202202192207206194192

I LQ S L CM L L L K L V N L ST Q Y H Y VQ R D I L N EKQ K C L D F L L I S L R D L D GG SK V I SQWA P EN SK N Y E S LQQ C T D D D I I KL S EN L I RQ T L R I I H I Q G L Y SN H L L N V I T EK D SV I Q F L SD S L N D L - GA SK I I K N F A P K H S I N Y EVM N K Y N DQ F N L SV F R A L SC S L F N N L I V F K Y A I D D L ER T I SH K D N A L S F L A D SV ED A - G F K SV I K KWA P I G S F N D E L L R K F N L A EWL HL L L EC N T Y L M R N V L V L N Y VQ N Y T SH T A L EK D R A I E F L GN T A K D Y - GA N N A I L RWA P EN S L N Y R A LM K Y K P E L V LQV I SQ I N FQQ F EN H N Y L N Y K V EQ L ER L I N I K D H Y I V Y L S EN Y K A I - N GD E L I K K Y R K N N K A D A K Y L H K Y D R K LWSRI L L E L SH F L S ET LWYWSR V C S- - - - - L I D S E SN T K N L N N C L I E L - - - N R I A EA K E SNQ I Y GR D L A K L F I I R EK L LF L Y K L N YQQ F EN I C FM K FQ V D S L K E I I SV K DQ Y SR F L A T N F KQ S- H GM DM I N N Y K K N NQ SD I E F I ER F SP K KWD KVM S S L S FWQM R S I L A L T SM N E E LQ NM L V EK D SA I E F L GY T A K EY - GG SR L I R RWA P I G SQ N Y E S L T K F K P GVWK K

7 4 1 3 8 4 4 4 6 4 6 4 4 3 5 4 4 2 2 2 4 0 0 0 2 0 7 4 2 5 7 4 3 5 2 5 * 4 3 6 5 3 4 4 - 0 1 3 4 7 7 2 4 6 3 3 3 3 3 2 5 1 6 4 7 3 5 7 2 2 3 2 3 2 2

V L+ + L+ FQ L F+ N I + + L+ Y+ V+ - L E+ + I + EK D SA I E F L + + S L K D L - GG S+ + I K+ WA P N N S- N Y E+ L - K F+ P K+ WL K

yeast NEJ1

Supplementary Figure 3Combined sequence alignment of metazoan XLF, yeast XLF and yeast NEJ1. The three alignments shown in supplementary Fig.2 were combined as profiles using T-Coffee (5) and then manually edited. Amino acids are coloured according to hydrophobicity/hydophilicity, and the degree of conservation within the alignment.

Agos/1-270Calb/1-324Cgla/1-314Dhan/1-335Klac/1-300Kwal/1-351Scas/1-346Scer/1-342Spom/1-189Ylip/1-283Afum/1-255Gzea/1-263Ncra/1-253Agam/1-261Drer/1-309Dmel/1-459Ggal/1-298Hsap/1-299Mmus/1-295Tnig/1-354Xlae/1-311

Quality


Quality


Quality


Quality

111111111111111111111

484641454552515338474243373742374845454856

- m s r k md s e r l e t h i k w r l f d i k - - - - n e l y - - - - f v g l h c d d g l v l y i y s p f q d l v - -- - - - - - - - - m s l f p t q w i a k s i p f l g q e k p c - - - - i y g f t y n s e c y y y g l y l c n f e n v w- - - - - - - - - - - - m v n aw k v g e i d - - g r y n l l - - - - k q d i d r d g t k l l k i t i h m t e a l l v- - - - - - - - - m f d l p l s w n s i f i p f - v n p r k v - - - - i y g y q c d f n n k a y s i y i t d f k n v w- - - - - - - m s n q e a t g s w q f l h i n - - - n d i s l - - - - a k i t k e s s s s i i g v l k l c e d e k f lm s a s s p l k v d f s e l n ew l p y t i d - - s r t i f g - - - - s f s h v k e a q k i t l c f s t l k g c p v -- m e r e s i g r q t s e l l ew h s c t i n - - g q p f l f - - - - s l n l s d n e e s t l f f i n a d q h p t f -- - md s e l k g q q l s d a ew c v k k i n g e g n c l l l - - - - f l p m s s p t t i v m i v l v s l e r l v p y- - - - - - - - - - - - - - m s w - - - - v q l r - g h r f h - - f i q a s f d a e s n e a v q v i h i t d l v r v w- - - - - - m v e a n p k a g aw - - v p i t t d - d g s f q - - f f - a r l t i s n g g y t l h l a d v a s f q f w- - - - - - - - - - - mm s s k w r r l h l p k q - d g l p p - - - - l l - l r y s r t l d s y n v y m t d l s n v w- - - - - - - - - - m s l n k s w r p l p l q p s - p d l p v - - - - l l - v s f h t e t s a y t i h i t d m a n mw- - - - - - - - - - - - - - - p w r p l p v t - a - p g i p n - - - - l l - v a t n f t a q s y k v h l t d l a n v w- - - m t d f c e l t l n r k g f y g v d f s - - - k d f - - - - - - - - - - - - - - - - d r i k c v v f d m e g l w- - - - - - - m e a v l s a l p w v p v n i s - - - g s d - - - - - - l l a - k a w f g d s q y r v l l t d l s t v w- - - - m l l k v v n i n g l q h l f a c t e - - - g q h - - - - - - - - - - - - - - - d e t l t i h v f r p r e k w- m e h s e e l e s r l l t q p w a s v c f d - - - e s t - - - - - - f i a - k a c f g d t g y m l l i s d l s g v w- - - - m e e l e q g l l mq p w a w l q l a - - - e n s - - - - - - l l a - k v f i t k q g y a l l v s d l q q v w- - - - m e e l e q d l l l q p w a w l q l a - - - e n s - - - - - - l l a - k v s i t k h g y a l l i s d l q q v w- m e a c d a a p d v l l q r p w l p v n i g - - - g c r - - - - - - l l a - k s c l g a s s y h l l l t d t h c v w- - i a v s r h r t c l c r k k w mp d s c s c p g g r s a s q t v f f mg - k v c f t e s s y a l l l s d l s s mw

494742464653525439484344383843384946464957

8486968584949498818985868072877392909093100

- - - - - - - - - - - q t k i t d e e l h q a - - - - k y q f s t k e - - - - - - - - a m e i l l t q g k d v e f t f- - - - - - - - - - - k q e v d k q k l v h i a k - - t i g i e d m s d - d - - - - - d l v e l i n d i s r y i p e kp q s s t l k k s l k t y a v k l d s a r k q l k - - s q g f d l q s s - d n l i k - i v i e f l n n l n c y t k n n- - - - - - - - - - - l e r l s k q d i i d k a n - - s y g i e d t e d - e - - - - - k l h y l l e v l r n g v e c g- - - - - - - - - - - r f r v s s e q i p k e l g - c s n g v d s i s - - - - - - - - e l y n i l t k g v e g s s i v- - - - - - - - - - - v v i f d a k d i k k q a a - - g q g i v v e s d - i t e - - - q v a h l l r e p s k a l q i h- - - - - - - - - - - k a c i s s n d i k a q c t - - i q g f p s d s v - t k i t k - e t y k q i h q l r s f k k i -- - - - - - - - - - - v f k l s q t q l s q q c q - - s q g f t d s i s - l n l i k l k l md i l q a p q e i n q i g- - - - - - - - - - - s c t c s r n q i v n q a e s e r c p i d p f q t - - f e - - - t l v d v l a e v l v n v s s e- - - - - - - - - - - i e d l r k d r v t i r a e d d d c l i d - a n d - - p t - - - q h a l l l d k l g q a l r e g- - - - - - - - - - - t e h m s r q d i l n r a d e d a t t i d p s e d - - p e - - - q f e v l l r k i g e a l s g e- - - - - - - - - - - t e s l d r k q i s m r g w n e n t s i d p s e t - - q d - - - n m a k f l a s i s t a l d s s- - - - - - - - - - - v e n md r r p i mg r a s a e d t s i d p t d g - - n e - - - n l q r l l d w i r a a f d i d- - - - - - - - - - - m e e l p v a t l i k r q k e r n p t md c t e a l i d q - - - t l l e a g - - - - - - - - - -- - - - - - - - - - - e e e m s t d d i q s r a q d l n k r l r a p a q a f f s - - - h l c s v a r p c f s g l d e d- - - - - - - - - t y s e t l m k r d y q q r l k t l n a r v k f p e e l v r q - - - v l l n k - - - - - - - - - - -- - - - - - - - - - - y e s a d a e a v g q r s k e l n k r l t a h v s s v l q - - - r l h g l l s p a l a g q p h -- - - - - - - - - - - h e q v d t s v v s q r a k e l n k r l t a p p a a f l c - - - h l d n l l r p l l k d a a h p- - - - - - - - - - - h e q v d t s v v s q r a k e l n k r l t a p p a a l l c - - - h l d e a l r p l f k d s a h p- - - - - - - - - - - e e r a g p s d i q r r a q e l n k r l r a p v e a f f t - - - h l c q v v r p c l s g s a g p- - - - - - - - - - - c e e a k a d i i q d r a r e l n k r l k a p v s s f l s - - - y l s q i v f p v l n s k d n -

8587978685959599829086878173887493919194

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e q d n v l q l g - - - - - - - - - - - - - - - l l l h s g t r a - - - - - - - t - - l e l p y n k v q n v e r - y dm v f e q d d v n - - - - - - - - - - - - - - - v r a r t i g d i - - - - - - - e - - w r f n l s r q n q e q t - i ey k d s k c e m s l h k - - - - - s d m k i t - i k l d a t i k a - - - - - - - t - - i n l g f v r l t d e q g - l ld v d k t l l f s i t k i n r t m s d n e n t - i q a k f t g e i - - - - - - - d - - w d f s v v k q s p s n a - i da md k k i i i s - - - - - - - - - - - - - - - i q r k k a s s v - - - - - - - e - - i e t a i i a i t g e h l - t qi d e n q v v l e - - - - - - - - - - - - - - - i t i s r d i t l - - - - - - - s - - l t f s a c r l e e s q i - s ae d g s r l q l t - - - - - - - - - - - - - - - i v l s g d i t l - - - - - - - n - - l t t i i l g l s mq e q - l tl v d s n l v f s - - - - - - - - - - - - - - - f d v s a d i t v - - - - - - - s - - i n s v p s h v t k d m f - y ms - - - - - - - - - - r i q l r g s n d e g s - l k i f c t s k i a k d i f - l e - - w n w t l w l t p p e - - - - tq i d i r k t s r g l r i s v s m k mg g g t - f e - - - - - - - - - - - - - - - - - w t f r v s q v t e t r e l v ds g c t a s l e n g l r - - - - - - - - e d s - m k l t v s t k l p a p l r p l i - - w t i y l s k e p q s - - - - sq p g h y e t s l c l d h d d r s e a g e d d - l v l r i t c e i p e - l q p l q - - w p m y l k k l p a i - - - - nd p g h r n t s l t l - - - - - g q g k d d s - i e v q i t c i l p e p f k p l k - - w p l y l k k c s a s - - - - t- - - c s g n y s a - - - - - - - t i a e a g - v h f t a k y y l e n y - - p i t - - f q f f l v - - - p a r a - q gq - i s a a q a a l - - - - - - - e q h g e s - l t v k l k s e l a g l - - p f y - - w e f r c t - - - t t p v - a v- - - d p l h v e q - - - - - - - d y q h p l - n v l k l k y a m a n f - - k v f l k w e w h l r - - - p md a - a q- - - a a t s f s c - - - - - - - q r p a g g - l s l r v r s e l s g l - - p f y - - w d f r c r - - - p a p v - a t- - - s e a t f s c - - - - - - - d c v a d a - l i l r v r s e l s g l - - p f y - - w n f h c m - - - l a s p - s l- - - s k a t f s c - - - - - - - d r g e e g - l i l r v q s e l s g l - - p f s - - w h f h c i - - - p a s s - s lp - d g g p q i s l - - - - - - - i r e d d g d l r i k v k s e l a g l - - p f r - - w e f h c r - - - p a s l - a v- - - g q n i f s c - - - - - - - h r s e a e - l l l q v k s q l s g l - - p f y - - w s f h c k - - - e a t v - s t

119121140134119129129133123131130138128113130116133131131137141

174176188189174184184189167186185193183166183169186184184182194

v m s s l s f w q m r s i l a l t s mn e e l q n m l v e k d s a i e f l g y t a k e y - g g s r l i r r w a p if l y k l n y q q f e n i c f m k f q v d s l k e i i s v k d q y s r f l a t n f k q s - h g md m i n n y k k ni l l e l s h f l s e t l w y w s r v c s - - - - - l i d s e s n t k n l n n c l i e l - - - n r i a e a k e s nv i s q i n f q q f e n h n y l n y k v e q l e r l i n i k d h y i v y l s e n y k a i - n g d e l i k k y r k nl l l e c n t y l m r n v l v l n y v q n y t s h t a l e k d r a i e f l g n t a k d y - g a n n a i l r w a p ev f r a l s c s l f n n l i v f k y a i d d l e r t i s h k d n a l s f l a d s v e d a - g f k s v i k k w a p il s e n l i r q t l r i i h i q g l y s n h l l n v i t e k d s v i q f l s d s l n d l - g a s k i i k n f a p ki l q s l c m l l l k l v n l s t q y h y v q r d i l n e k q k c l d f l l i s l r d l d g g s k v i s q w a p ei a k m v w f p l i n h h - - - - - - - - - - - l f e s e t d t s p g s l m s i i r d q - k s l l n d k t w f k el q r t f f s g l i t v s h s l l s q v e f l e s q l s l k d y h i g a mq k l l a d t - e p g r s t e y r p r rt s q h l i l p l l r a e a g w e s r q r t l l d h l s k k d w v l g k l f d k i e s m - g i d l s t i f p g v ai a t n l v l p l i q t h h a k d l e i a s l i q s l g q k d a v l t r l l d k l e a m - g t g m e h i f n a l sl a t e l v l p l i q s h e a r a q e i n h l i a t i r d k d a v i n r l v d k l e a n - g i g l e h i f n k l pi s c h y i k p l w r t v l l q y a q i r a l a e e l k r k d a e i a q y q a e g a r l - n r t s v - - q t v p fv c r q l v r p l l a m t l v l q r q a e d l a a l l a r k d a e i q d y q e n g a v l - s r a r l - - q t e p fl y s q m f l n t m s t a l g l r a q i p l l l g i i h a k d k e l n q y r t e g c q l - r r v t v - - e t q p fv c r h l v r p l v r m s l a l q h q v q e l t s l l l r k d a e i e d y r e s g a t l - s r d r l - - r t e p fv s q h l i r p l mg m s l a l q c q v r e l a t l l h m k d l e i q d y q e s g a t l - i r d r l - - k t e p fv s q h l i h p l mg v s l a l q s h v r e l a a l l r m k d l e i q a y q e s g a v l - s r s r l - - k t e p fv c a q l v r p l l a m s h l l q r q - - g l c g l a a p p l f s a p a w k g q g g v p - q r h h - - - - - - - -v c r h l v r p l k s m t e a l e s q n q e l c l l l k k k d a e i q e y q d s g a v l - t r d r l - - k t e v f

Supplementary Figure 4Sequence alignment of the XLF/NEJ1 and Lif1/XRCC4 superfamilies. The alignment is based on sequence-structure threading onto the XRCC4 crystal structure by Fugue (6). Amino-acids are coloured according to hydrophobicity/hydophilicity, and the degree of conservation within the alignment. Sequences used in addition to those already shown in Fig.2. : HsapXRCC4 60115830 H. sapiens XRCC4 ; AthaXRCC4 85701292 Arabidopsis thaliana XRCC4 ; ScerLIF1 51830317 S. cerevisiae LIF1.

01HsapXLF/1-18502GgalXLF/1-18703DrerXLF/1-18403EDmelXLF/1-16504NcraXLF/1-18405SpomXLF/1-16606YlipXLF/1-18407DhanNEJ1/1-19008CalbNEJ1/1-17709KlacNEJ1/1-17510ScerNEJ1/1-18612HsapXRCC4i1/1-33613AthaXRCC4/1-24814ScerLIF1/1-421

Quality


Quality


Quality

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5962565151505959605967575869

11711911691106100112116103101112115115131

k e l n k r l t a - - - p p a a f l c h l d n l l - - - - r p l l k d a a h p - - s e a t f s c d c v a d a l i l r v r s e l s g l p fk e l n k r l t a - - - h v s s v l q r l h g l l - - - - s p a l a g q p h - - - a a t s f s c q r p a g g l s l r v r s e l s g l p fq d l n k r l r a - - - p a q a f f s h l c s v a - - - - r p c f s g l d e d q i s a a q a a l e q h g e s l t v k l k s e l a g l p fk t l n a r v k f p e e v r q v l l n k - - - - - - - - - - - - - - - - - - d p l h v e q - - - - - - - d y q h p l n v l k l - - k y as a e d t s i d p t d g - n e n l q r l l d w i r - - - - a a f d i d d p g h r n t s l t l - - - - - g q g k d d s i e v q i - - t c ie s e r c p i d p f q t - f e t l v d v l a e v l - - - - v n v s s e s - - - - - - - - - - r i q l r g s n d e g s l k i f c - - t s ke d d d c l i d - a n d - p t q h a l l l d k l g - - - - q a l r e g q i d i r k t s r g l r i s v s m k mg g g t f e - - - - - - - -n - - s y g i e d t e d e - - k l h y l l e v l r - - - - n g v e c g d v d k t l l f s i t k i n r t m s d n e n t i q a k f - - t g ek - - t i g i e d m s d d - - d l v e l i n d i s - - - - r y i p e k m v f e q d d v n - - - - - - - - - - - - - - v r a r t - - i g dg - c s n g v d s i s - - - - e l y n i l t k g v - - - - e g s s i v a md k k i i i s - - - - - - - - - - - - - - i q r k k - - a s sq - - s q g f t d s i s l n l k l md i l q a p q - - - - e i n q i g l v d s n l v f s - - - - - - - - - - - - - - f d v s a - - d i td d - - - - - - - m a m e k g k y v g e l r k a l l s g a g p a d v y t f n f s k e s c y f f f e k n l k d v s f r l g s f n - - l e ka q - - - - w d q p v s e y l k l a e q y l g f q q p n s - v y s f s d a l e g s k r l s w t f e k e g t k l e w r - - - w k - - c k pi f i d - - - e e s k k y v w y e l l k m l y i a s l d e k v v f t k w t c r mq d d e v w k v v m e l e s s a i i r k i a e l t - - l

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y w n f h c m l a s p s l v s q h l i r p l mg m s l a l q c q v r e l a t l l h m k d l e i q d y q e s g a t l i r d r l k t e p f -y w d f r c r p a p v a t v c r h l v r p l v r m s l a l q h q v q e l t s l l l r k d a e i e d y r e s g a t l s r d r l r t e p f -y w e f r c t t t p v a v v c r q l v r p l l a m t l v l q r q a e d l a a l l a r k d a e i q d y q e n g a v l s r a r l q t e p f -m a n f - - k v f w e w h l r - - - p md a a q l y s q m f l n t m s t a l g l r a q i p l l l g i i h a k d k e l n q y r t e g c q ll p e p f k p l k w p l y l k k c s a s - - - t l a t e l v l p l i q s h e a r a q e i n h l i a t i r d k d a v i n r l v d k l e a ni a k d i f - l e w n w t l w l t p p e - - - t i a k m v w f p l i n h h - - - - - - - - - - - l f e s e t d t s p g s l m s i i r d q- - - - - - - - - w t f r v s q v t e t r e v d l q r t f f s g l i t v s h s l l s q v e f l e s q l s l k d y h i g a mq k l l a d ti - - - - - - - d w d f s v v k q s p s n a i d v i s q i n f q q f e n h n y l n y k v e q l e r l i n i k d h y i v y l s e n y k a ii - - - - - - - e w r f n l s r q n q e q t i e f l y k l n y q q f e n i c f m k f q v d s l k e i i s v k d q y s r f l a t n f k q sv - - - - - - - e i e t a i i a i t g e h l t q l l l e c n t y l m r n v l v l n y v q n y t s h t a l e k d r a i e f l g n t a k d yv - - - - - - - s i n s v p s h v t k d m f y m i l q s l c m l l l k l v n l s t q y h y v q r d i l n e k q k c l d f l l i s l r d lv e n p a e v i r e l i c y c l d t i a e n q a k n e h l q k e n e r l l r d w n d v q g r f e k c v s a k e a l e t d l y k r f i l vs d d s k k i t v g i l d f l m e a n i r l s e e v v n k t r s f e k m r s e a e r c l a q g e k l c d e k t e f e s a t y a k f l s vh p v k k g e i d l f e m a d k l y k d i c c v n d s y r n i k e s d s s n r n r v e q l a r e r e l l d k l l e t r d e r t r a mm v

Supplementary Figure 5Vegetative xlf1d cells are not sensitive to DNA damaging treatments. Ten-fold serial dilutions were made of log-phase cultures of wild-type, lig4d and xlf1d cells. Cells were then spotted on rich agar containing DNA damaging drugs as indicated and incubated at 30°C for 3 days.

Supplementary Figure 6Synteny of the Lif1 locus, as annotated at the Yeast Gene Order Browser (YGOB; 7). A single homologue of S. cerevisiae (dark blue) Lif1 is present at the syntenic locus in the post-duplication yeasts C. glabrata (olive green) and S. castelii (pink). In the pre-duplication yeasts, a likely Lif1 homologue is present in K. waltii (brown), but appears to be absent in K. lactis (orange), A. gossypii (green) and S. kluyveri (yellow).

>ScerLIF1 212 MVTLLNEKKKKIRELHEILR 23.512 >SparLIF1 212 VVALLNEKKKKIRQLHEILR 22.850 >SsclXlf 156 LVELLGEKDAVIQKLVDTLE 22.145 >SbayLIF1 214 VVTLLNEKKKKIRELNETLR 21.932 >SmikLIF1 213 MVTLLNEKKKRIRELHGILQ 21.731 >SparNEJ1 155 QRDILNEKQKCLDFLLRSLG 21.643 >SkudLIF1 209 VVILLNEKKKKIKELHEILR 21.549 >BfucXlf 157 LIELLEEKDAVIQKLVDTLE 21.104 >SkudNEJ1 155 QRDVLNEKQKCLDFLLRSVN 21.083 >AnidXlf 153 LIEELNKKDWVLSKLFDKFE 20.905 >UreeXlf 151 LIEKLKEKDWALGKLFDKLE 20.845 >ScerNEJ1 155 QRDILNEKQKCLDFLLISLR 20.658 >SmikNEJ1 156 QQDILNEKQQCLDYLLRSLN 20.625 >AfumXlf 152 LLDHLSKKDWVLGKLFDKIE 20.450 >MgrisLIF1 200 FRLLLNEKKAKIREQHDVIK 20.365 >SbayNEJ1 155 QRDVLGEKQKCVDFLLRSVK 20.322 >AoryXlf 150 LIDQLHKKDWILAKLFDKIE 20.252 >AfumLIF1 191 FVQLLNEKKLKIRNQQRLLA 19.637 >AnidLIF1 186 IVQLLNEKKLKIRNQQRLLA 19.410 >ScasNEJ1 151 LLNVITEKDSVIQFLSDSLN 19.302 >GzeaLIF1 180 FRDLLNEKKIKIREQQQALA 19.164 >NcraLIF1 197 FRALLNEKKAKIRQQQQALS 18.839 >CglaLIF1 195 MIGLLNEKKARIRILTSLME 18.605 >ScasLIF1 204 VVGLLNEKKKKIAELRKRLL 18.213 >PguiNEJ1 140 LHQLLAGRDYYIMFLSQNLK 17.868 >DhanNEJ1 156 LERLINIKDHYIVYLSENYK 17.780 >NcraXlf 216 LIATIRDKDAVINRLVDKLE 17.760 >GzeaXlf 160 LIQSLGQKDAVLTRLLDKLE 17.751 >AoryLIF1 194 FVQLLNEKKLKIRNQQRLPA 17.364 >CtroNEJ1 143 LKDIISVKDQYSRFLATNFK 17.028 >MgriXlf 165 LLAVIKEKDGVISKVLDKLD 16.801 >CalbNEJ1 143 LKEIISVKDQYSRFLATNFK 16.523 >AgosNEJ1 141 LQNMLVEKDSAIEFLGYTAK 15.920 >KwalNEJ1 151 LERTISHKDNALSFLADSVE 15.674 >YlipLIF1 205 LQSLIAEKRRYEKELVNKFS 14.435 >ClusNEJ1 143 LESMIRTRDNYMLYLEENYK 13.575 >YlipXlf 153 LESQLSLKDYHIGAMQKLLA 12.978 >CglaNEJ1 144 LSHFLSETLWYWSRVCSLID 12.333 >CalbLIF1 70 LNILISEKEKTEQELEKLTK 11.653 >SpomXlf 155 QKSLLNDKTWFKEALNERSS 11.625 >KlacNEJ1 141 TSHTALEKDRAIEFLGNTAK 11.547

conservation

Supplementary Figure 7AA. A conserved block can be identified in yeast Lif1/Nej1/Xlf sequences by NOMAD (Neighborhood Optimization for Multiple Alignment Discovery; 8) as an ungapped multiple alignment.

Supplementary Figure 7B

Sequence signature of the conserved block in the stalk region of the yeast Lif1 and yeast Nej1/Xlf families. Each motif was determined from an ungapped alignment of the sequences of twenty of either LIF1 or XLF/NEJ1 homologues. The combined motif results from an alignment of forty LIF1 and XLF/NEJ1 sequences. This figure was prepared using the sequence logo generator, WebLogo (9).

Supplementary References

1. Kelley, L. A., MacCallum, R. M., and Sternberg, M. J. (2000) J. Mol. Biol. 299, 499-520

2. Edgar, R. C. (2004) Nucleic Acids Res. 32, 1792-1797

3. Clamp, M., Cuff, J., Searle, S. M. and Barton, G. J. (2004) Bioinformatics 20, 426-427

4. Cuff J. A., and Barton, G.J (1999) Proteins 40, 502-511

5. Notredame, C., Higgins, D., and Heringa, J. (2000) J. Mol. Biol. 302, 205-217

6. Shi, J., Blundell, T. L., and Mizuguchi, K. (2001) J. Mol. Biol. 310, 243-257

7. Byrne, K.P., and Wolfe, K.H. (2005) Genome Res. 15, 1456-146

8. Hernandez, D., Gras, R., and Appel, R. (2006) Eur. J. Oper. Res. (in press)

9. Crooks, G.E., Hon, G., Chandonia, J.M., and Brenner, S.E. (2004) Genome Res. 14, 1188-1190

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SUPPLEMENTAL FIGURES EVOLUTIONARY AND FUNCTIONAL ... · SUPPLEMENTAL FIGURES EVOLUTIONARY AND FUNCTIONAL CONSERVATION OF THE DNA NONHOMOLOGOUS END-JOINING PROTEIN, XLF/CERNUNNOS Pierre