Supplementary Figure 1 Amino acid sequence alignments of CDC7 and DBF4 orthologs (next 4 pages). (a) Alignment of distant CDC7 orthologs from Homo sapiens (mammalian), Gallus gallus (avian), Anolis carolinensis (reptile), Xenopus laevis (amphibian), Danio rerio (fish), Danaus plexippus (insect) and Saccharomyces cerevisiae (yeast). (b) Mammalian CDC7 orthologs. (c) Alignment of motif –M and –C regions of distal DBF4 orthologs. (d) Mammalian DBF4 orthologs. Secondary structure elements elements are indicated atop the alignment. Portions of the protein sequence not present in the crystallized construct are highlighted in gray (Δ1–36, Δ228–359 and Δ484–529); the kinase insert sequences (black boxes) and DBF4 motifs –N, –M and –C and the charged region (CR) are indicated and boxed. (e) Alignment of MC regions from human DBF4 and DRF1. Amino acid sequence identity for motifs –M and –C are indicated; the overall sequence identity in DBF4 – DRF1 alignment is ~22%. Conserved amino acid residues are shown in bold print and those invariant within the alignment are highlighted in yellow. Invariant CDC7 and DBF4 residues discussed in the text are shown in red and indicated.

Nat

ure

Str

uctu

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0.10

38/n

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2404

Hum

Hum

Hum

Hum

Hum

1 10 20 30 40 50 60 70 80 90 100 110

Mammal_Hs L P KIGEGTFSSVY A K T P RI EL L G K V DI Y AV QL NVFKI LA A L L HLIP SH I AA QC TVAG QDN...MEASLGIQMDEPMAFSPQRDRFQAEGSL KNEQNFKLAG KK EK E S ED T Q........... QVGPEEKI Bird_Gg L P KIGEGTFSSVY A K T P RI EL L G R I DI Y AV QL NVFKI LA A L L HLIP SH L AA QC TVAG QDN..........METESKSHCDEQHPHQAEDTS KHMQSSKLSG KK EK E V KE T Q........... QTGCEEKM Reptile_Ac L P KIGEGTFSSVY A K T P RI EL L G K A DI Y AV QL NLFKI LA A L HLIP SH V AA QC VAG QDN.............MEASHQIEHPPLHGEDPC KYSQNKKLSG ST EK E G KG V Q...........GRGNQEEKV I Amphibian_Xl L P KIGEGTFSSVY A K T P RI EL L G V EI Y AV QL NIF I A L L HLIP SH AA QC SVAG DN..................................MSSGDNSG AK EK A H Y KS F IGR........... RSGEDAKF T E Fish_Dr L P KIGEGTFSSVY A K T P RI EL L G R V EI Y VL QL IFRI LA A M L HLIP SH V AA QC TVAG TEN.............MEEANTVQRSSHRSVEKG ..SRHKISRD ET AY E SR ID E Q........... TDSSRRLF Insect_Dp L P KIGEGTFSSVY A K T P RI EL L G K I L L IF V L L L I HLVP H A C Q IG D MSRIRGIEAKVQKEIEENLENRRNKYKEVIK KNDICTKVDSEKLQ VE LYK K DK D HR GS KQ.......HAQ PDDQKRWF A EH R D K YFungus_Sc L P KIGEGTFSSVY A K T P RI EL L G K I EM Y L I N YKL A L KI S N IM S ............................MTS TKNIDDIPPE KE IQ HD G E E ID K KDITGKITKKFASHFWNYGSNYV YV S Q YN L Y T SR

120 130 140 150 160 170 180 190 200 210 220 230 240

Mammal_Hs V R PY H F Y L AL H G HRD KP NFL LVDFGLA MGV CF K DHVVIAM LE E LDIL LSF EVR M K KRI F IV V S YNR LK YA QGT DTKIELLK V KY N S NS Q E LN F Q R K H F QSEAQQERCSQNKSHIITGNKIPLSGPVPKELBird_Gg V R PY H F Y L AL H G HRD KP NFL LVDFGLA MGV CF K DHVVIVM LE E LDIL LSFEEVK MF K RRI F IV V S YNR LK YA QGT DTKIELLK A KY N S NS E N F H R Q P T HSEGQQGSYSQSNPNIALGNGVSVGVTAPKQIReptile_Ac V R PY H F Y L AL H G HRD KP NFL LVDFGLA MGV CF K DHVVIVM LE E LDIL LSFEEVR MF LK KRI F IV V S YN LK YA QGT ETKIELLK I KC N S NS D N H QQ Q P V QSEAPQGSCTYTKPQITLGSQVSVTSTAPRHSAmphibian_Xl V R PY H F Y L AL H G HRD KP NFL LVDFGLA MGV CF DHVVIVM LE E ADIL LSFEE K MF LK RHI F IV V S FNR LK FA QGT DTKIDLLK L KY NK C HS T E N S S K S V QP...............K..............Fish_Dr V R PY H F Y L AL H G HRD KP NFL LVDFGLA MGV CF K HVVIVM ME VDIV LSFEDVR IY LK KHI F II I T FNR R YA QGT DT IELLK L TY EH QT GL H H K QK E P Q G LS..............................Insect_Dp V R PY H F Y L AL H G HRD KP NFL LVDFGLA IGV C H D IV VM I E V M EEVR M L RHV F VI V S Y R R YL Q DL L L T F P RK S Y GD DA C RA V S D EN R RVVSDGPSPPVPPPAHAN...........................Fungus_Sc V R PY H F Y L AL H G HRD KP NFL LVDFGLA APL D VIAVL E L IK IW LR K V II I T FN L V Q D K I CDAK VR Q YP E RTFYRD PIKG K E F SK LE GRG EA M Y SM SSQNDYDN............................

250 260 270 280 290 300 310 320 330 340 350 360 370

Mammal_Hs R R A RA R E LMK K V V S L TK A S M S RK LTC CYA DKVCSICLS QV P DQQSTTKASVKRPYTN..AQIQIKQGKDGKEGSVGLSVQRSVFGE NFNIHSSISH SPAVK QS T D L K A KK I TKV N AVM TASSCPAS D T RQ Bird_Gg R R A RA R E LIK K I V S L TK I S V N RK LTC CYA DRVCSVCLS QV P AQQLASRATDKSSHSSSHSKIQIKQGRGGKEDSVHHSAQRSVFGE NFNIYCSTYQ NLNTK QS M D S K I KK I TKA N GMG AASGCPSN N T CQ Reptile_Ac R R A RA R D LVK K I S L K A S L N KK LTC CYA DRVCSICLS QV P THQSATKTANKRPCSAS..QTQIKEGHKRKEGQECFAHQRSVFGE NFNVRSPAFQ RSTVK QP VTD P K SA KK T T.. N GLA AASSCPAN D K RQ Amphibian_Xl R R A RA R D AAK K I V T L TR V T S KK LTC CYA D VCNICL QV P ...KQ.........................DGLVGSSTQRSVFGE NFNVHSAVTI NTTLK PS T D T K A KT S KS.TS AVP AASTCQTS D K Q A TR Fish_Dr R R A RA V K V A V R L Q L K LTC CYM DRVCNICLS QV P .........................................................KPKKEE IPR I S KH S PV AP N KQK PAESQ PKPAAVNPL N T KQ Insect_Dp R R A RA K D R L S S L K C C VCS C P ...........................................SL R.....PREE ENIQSEK FALD STR STS AAQSPKKVS PKVQIS QKLPKVSPGV S SNAGS P GA AAAR Fungus_Sc R R A RA M S Q M T K N K T ..................................................YAN.TNHDGGYS RNHEQFCPCIM NQY PNSHN TPP V IQNG ....VVHLN VNGVDLT GYPKNE RIKR N

380 390 400 410 420 430 440 450 460 470 480 490

Mammal_Hs GT GFRAPEVL K Q D W G S L R DD L G G L L P CP TTAI M SA VI L L S YPFYKA LTA AQIMTIR SRETI AAKTF KSILCS V R LCE RG T N F G S Q KE PA......QD K R MDSSTPKLTSDIQGHASHQPAISEKTDHKASBird_Gg GT GFRAPEVL K Q D W G S L R DD L G G L L P CP TTAI M SA IV L L S YPFYKA LTA AQIMTVR SRETI AAKTF KSVLCT V R LCE RG V T T F G S Q QV PA......QN T K TNGSCNRSHGDVPSKSGDESALP EADKQCAReptile_Ac GT GFRAPEVL K Q D W G S L R DD L G G L L P CP STAI I SA II L L S YPFYKA LTA AQIMTIR SRETI AARTF KSILCS I R LCE RG A I H F D G Q KE VA......QD K R NSTSFDNSTGDVQIKP.QESALP EISNAFEAmphibian_Xl GT GFRAPEVL K Q D W G S L R DD L G G L L P CP TTAI M SA II L L S Y FF A MNA AQIMTIR SKETI A K F KSVLCS L R LCE R M T H F G H N A Q S C KE PS......KD T G SAIVLPNGNQHDIQKQR..AALQ. RIMENQDFish_Dr GT GFRAPEVL K Q D W G S L R DD L G G L L P CP TAI M SA VI L L S YPFFKA L A QIMTIR SKETI AAKTF KSIVCS L R LCE RG A T N G L G S I T E RE PR......LD I T .............LRSWDDASLP EFQASHNInsect_Dp GT GFR PEVL K Q D W G S L R DD L G G L L P AV A VV A M T YPFFRA ASA A LA L T A S R MV S R L RG Q P L S R GP T A L A S E D L LPLQRT A L R T QPRRG......LC K AAR ..............GAP..PGPPPPALPACEFungus_Sc GT GFRAPEVL K Q D W G S L R DD L G G L L CG ST I I SV VI L L FP F A L L TI KE A S I K V R M A K L GR M QSL DS E C F W LRKC ALH LGFEA GL WDKPNGYSNG EFVYD LN...........KECTIGTFPEYS AFETFGF

500 510 520 530 540 550 560 570

Mammal_Hs P R A L P SN GW VPDEAY LLDKLLDLN AS IT EA H FFKDM CLVQTPPGQYSGNSFKKGD SCEHCFDEYNTNLE NE D E L SL................................. Bird_Gg P R A L P S GW VPDEAY LLDKLLDLN AT IT DA H FFKDM PVTLRKEIQHLKSCQEDDGA ......ENKAADMK DQ D K L RL................................. Reptile_Ac P R A L P GW VPDEAY LLDKLLDLN AT IT DA H FFR M ASLQGPQKHIHYQKQHHHGGDGRDVRITEKGADPK DN D K L N KQ................................. Amphibian_Xl P R A L P SS GW VP EAY LLDRLLDMN AT IT EA H FK M GWFLPESPDITPDSPAVVR CVSTPDNMEQSNHN DR N H E I L N R.................................. Fish_Dr P R A L P ST GW VPDEVY LLDKLLDLN AT IT A H FKDL PAEEKEE.....AESPALK GSRLCRSSAELKEI DR D ST Q L SE................................. Insect_Dp P R A L P S PDEAF LA RLLD AT DA H F D HCHLPLPHCLCRDETVKAT ITP.......N..LT.IAGF S A PD RA Q T LA .................................... Fungus_Sc P R A L P TN F VLE EM S DL FF EL LQQELHDRMSIEPQLPDPK MDAVDAYELKKYQEEIWSDHYWC Q QCF D QK S E KT N NENTYLLDGESTDEDDVVSSSEADLLDKDVLLISE

N lobe C lobe Kinase insert 1

Kinase insert 2

Kinase insert 3

ΔN (1–36)

Δ2q (228–359)

Δ3b (484–529)

DFG

P loop

APE Q391

Nα1 Nα2 β1 β2 β3 αC

β4 β6 αD αE β7 β5 KI2α1

αG αEF αF αG KI3α1 KI3α2 KI3β1

αH αI

RD

K90 E104

Mammal (H. sap.) Avian (G. gal.) Reptile (A. car.) Amphibian (X. lae.) Fish (D. rer.) Insect (D. ple.) Yeast (S. cer.)

Mammal (H. sap.) Avian (G. gal.) Reptile (A. car.) Amphibian (X. lae.) Fish (D. rer.) Insect (D. ple.) Yeast (S. cer.)

Mammal (H. sap.) Avian (G. gal.) Reptile (A. car.) Amphibian (X. lae.) Fish (D. rer.) Insect (D. ple.) Yeast (S. cer.)

Mammal (H. sap.) Avian (G. gal.) Reptile (A. car.) Amphibian (X. lae.) Fish (D. rer.) Insect (D. ple.) Yeast (S. cer.)

Mammal (H. sap.) Avian (G. gal.) Reptile (A. car.) Amphibian (X. lae.) Fish (D. rer.) Insect (D. ple.) Yeast (S. cer.)

N182

CDC7

Supplementary Figure 1a | Amino acid sequence alignment of distal CDC7 orthologs. N

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human_cdc7

human_cdc7

human_cdc7

1 10 20 30 40 50 60 70 80 90 100 110 120

human_cdc7 S KK EQ K DIE L EAVPQL FKI DKI GEGTFSSVYLATA L G EE IALKHLIPTSHP RIAAELQCLTVAGGQDNVMG KYCFR NMEASLGIQMDEPMAFSP R R QAEG L NF L GVKK K Y NV Q QV P K I V K Q ..D F N A. S E Mouse_Cdc7 S KK EQ K DIE L EAVPQL FKI DKI GEGTFSSVYLATA L G EE IALKHLIPTSHP RIAAELQCLTVAGGQDNVMG KYCFR N MEEPMAFS R R AD L S L GIKR NV K Q Q K M L K ........ SL GSD CP D Y V S. E C V E H Dog_Cdc7 S KK EQ K DIE L EAVPQL FKI DKI GEGTFSSVYLATA L G EE IALKHLIPTSHP RIAAELQCLTVAGGQDNVMG KYCFR NMEASLGI MD PMAFSP R QVDG L NF LP GVKK K Y GNL K Q V P K I V K H Q R R.DQV H . R Horse_Cdc7 S KK EQ K DIE L EAVPQL FKI DKI GEGTFSSVYLATA L G EE IALKHLIPTSHP RIAAELQCLTVAGGQDNVMG KYCFR NMEASLGIQMDDPLAFSP R R QADG L NF LP KK K Y GSV K QV P K I V K L ..D F H GMH H Cow_Cdc7 S KK EQ K DIE L EAVPQL FKI DKI GEGTFSSVYLATA L G EE IALKHLIPTSHP RIAAELQCLTVAGGQDNVMG KYCFR NME LGIQMDEPMAFSP R QAEG L NF P GVKK K Y GNL K Q QV P K I V K PA LG..G F Q P . Pig_cdc7 S KK EQ K DIE L EAVPQL FKI DKI GEGTFSSVYLATA L G EE IALKHLIPTSHP RIAAELQCLTVAGGQDNVMG KYCFR NMEASLGVQMDEPMAFSP H R QADG L NF LP GVKK K Y GNV K Q QV P V V K L ..G F H . T Panda_Cdc7 S KK EQ K DIE L EAVPQL FKI DKI GEGTFSSVYLATA L G EE IALKHLIPTSHP RIAAELQCLTVAGGQDNVMG KYCFR NMEASLGIQMDEPMAFSP R QADG L NF LP GVK K Y GNL K Q QV P K I V K Q ..DQV N . Q Elephant_Cdc7 S KK EQ K DIE L EAVPQL FKI DKI GEGTFSSVYLATA L G EE IALKHLIPTSHP RIAAELQCLTVAGGQDNVMG KYCFR NMEASLGIQMDEPVAFSP R QVDG L SF LP GVKK K Y GNV K Q QV P K I V PC..N L D . E Marsupial_cdc7 S KK EQ K DIE L EAVPQL FKI DKI GEGTFSSVYLATA L G EE IALKHLIPTSHP RIAAELQCLTVAGGQDNVMG KYCFR NMEASLGIQ D L Q D NY L GV K K Y GSV K Q M P K I V K D QQP SPMHD...QL T D F Y T. E K

130 140 150 160 170 180 190 200 210 220 230 240 250

human_cdc7 DHVVIAMPYLEHESFLDILNSLSFQEVREYM NLF AL RIHQFGIVH RDVKPSNFL NRRLKKYALVDFGLAQGT DTK ELLK VQSE QE NK K L PK Q K K K Y H I F AQ CSQ SHIITGN I SGPV ELDQ T S L .R P . S T A Mouse_Cdc7 DHVVIAMPYLEHESFLDILNSLSFQEVREYM NLF AL RIHQFGIVH RDVKPSNFL NRRLKKYALVDFGLAQGT DTK ELLK VQSE QE NK K L PK Q K Y K Y R I F AQ CS H V G S PA VDQ T TS V D. R Y G V H GL R . T C P Dog_Cdc7 DHVVIAMPYLEHESFLDILNSLSFQEVREYM NLF AL RIHQFGIVH RDVKPSNFL NRRLKKYALVDFGLAQGT DTK ELLK VQSE QE NK K L PK Q K F K K F H I F AQ SCSQ SHVITGN IS SGPA ELDQ T TS . A P M Horse_Cdc7 DHVVIAMPYLEHESFLDILNSLSFQEVREYM NLF AL RIHQFGIVH RDVKPSNFL NRRLKKYALVDFGLAQGT DTK ELLK VQSE QE NK K L PK Q K F K K Y H I F AQ SC Q SHVITGN IS SG A ELDQ T TS . L L A P T Cow_Cdc7 DHVVIAMPYLEHESFLDILNSLSFQEVREYM NLF AL RIHQFGIVH RDVKPSNFL NRRLKKYALVDFGLAQGT DTK ELLK VQSE QE NK K L PK Q K F K K Y H I F A SCSQ S VITGN IS SGPA ELDQ T TS H . Y A S P Pig_cdc7 DHVVIAMPYLEHESFLDILNSLSFQEVREYM NLF AL RIHQFGIVH RDVKPSNFL NRRLKKYALVDFGLAQGT DTK ELLK VQSE QE NK K L PK Q K F R K Y H I F AQ SCSQ S VITGS IS SGPA ELD T TS . Y A P P T Panda_Cdc7 DHVVIAMPYLEHESFLDILNSLSFQEVREYM NLF AL RIHQFGIVH RDVKPSNFL NRRLKKYALVDFGLAQGT DTK ELLK VQSE QE NK K L PK Q K F K K Y H I F AQ S SQ SHVITGN IS SGPA E DQ T TS . Y K S P V Elephant_Cdc7 DHVVIAMPYLEHESFLDILNSLSFQEVREYM NLF AL RIHQFGIVH RDVKPSNFL NRRLKKYALVDFGLAQGT DTK ELLK VQSE QE NK K L PK Q K F R K Y H I F AQ S SQ SHIITGN IS SGPA E DQ TS . S . P PIT Marsupial_cdc7 DHVVIAMPYLEHESFLDILNSLSFQEVREYM NLF AL RIHQFGIVH RDVKPSNFL NRRLKKYALVDFGLAQGT DTK ELLK VQSE QE NK K L PK Q K F K R Y R V VQ SCSQ THIV N VS N PA EL S T L . MA A . S. S T A

260 270 280 290 300 310 320 330 340 350 360 370 380

human_cdc7 VKR Y Q K G DGKE SVGLSVQRSV GERNFNI SS SHESPA KL KQ K D SRKL KK STK S R T SCPA TCDCY D VCS CLSRRQQVAPRAGTPGFR APEVL P TNA IQI Q K G F H I V M S TV VL AT KAI VMN VM K AS SL AT K I A Mouse_Cdc7 VKR Y Q K G DGKE SVGLSVQRSV GERNFNI SS SHESPA KL KQ K D SRKL KK STK S R T SCPA TCDCY D VCS CLSRRQQVAPRAGTPGFR APEVL T V I Q K F H I I S TV II AT AI AMN VM A L S R V S .. H R E T . E R V G Dog_Cdc7 VKR Y Q K G DGKE SVGLSVQRSV GERNFNI SS SHESPA KL KQ K D SRKL KK STK S R T SCPA TCDCY D VCS CLSRRQQVAPRAGTPGFR APEVL P TNA QI Q K G F H I V M S TM VL AT KAI VMN GVM K AS SL AT K I T Horse_Cdc7 VKR Y Q K G DGKE SVGLSVQRSV GERNFNI SS SHESPA KL KQ K D SRKL KK STK S R T SCPA TCDCY D VCS CLSRRQQVAPRAGTPGFR APEVL P TNA IQI K G F H I V M S TV VL AT KAV VMN GVM K AS SL AT K I H Cow_Cdc7 VKR Y Q K G DGKE SVGLSVQRSV GERNFNI SS SHESPA KL KQ K D SRKL KK STK S R T SCPA TCDCY D VCS CLSRRQQVAPRAGTPGFR APEVL P TN Q K G F H I V M S TV LL T K I VMN GVM K AS TL AT K I T T T H G T Pig_cdc7 VKR Y Q K G DGKE SVGLSVQRSV GERNFNI SS SHESPA KL KQ K D SRKL KK STK S R T SCPA TCDCY D VCS CLSRRQQVAPRAGTPGFR APEVL P T A IQI Q G F H V V M S TV LL AT KAI VVN GVM K AS SL AT K I H Q Panda_Cdc7 VKR Y Q K G DGKE SVGLSVQRSV GERNFNI SS SHESPA KL KQ K D SRKL KK STK S R T SCPA TCDCY D VCS CLSRRQQVAPRAGTPGFR APEVL P TNA QI Q K G F H I V M S TV VL AT KAV VMN GVM K AS SL AT K I S Elephant_Cdc7 VKR Y Q K G DGKE SVGLSVQRSV GERNFNI SS SHESPA KL KQ K D SRKL KK STK S R T SCPA TCDCY D VCS CLSRRQQVAPRAGTPGFR APEVL P TSA IQI Q K G F H I V M S TV I AT KAI VMN GVM K T N AT K I F T P Marsupial_cdc7 VKR Y Q K G DGKE SVGLSVQRSV GERNFNI SS SHESPA KL KQ K D SRKL KK STK S R T SCPA TCDCY D VCS CLSRRQQVAPRAGTPGFR APEVL P S A IQ Q K G I A I M I A KVI VT IV K AS NL AT V H T L Q P L T A T N Q

390 400 410 420 430 440 450 460 470 480 490 500 510

human_cdc7 TKCP QTTAIDMWSAGVIFLSLLSGRYPFYKASDDLTALAQIMT RG RETIQAAK FGKS LCSKEVPAQ LR LCE LRG S N I S T I D K R M SSTPKL SDIQG ASH P EKTD HKA LV TP Q SGNS D T H Q AI . SC Q PG Y FKMouse_Cdc7 TKCP QTTAIDMWSAGVIFLSLLSGRYPFYKASDDLTALAQIMT RG RETIQAAK FGKS LCSKEVPAQ LR LCE LRG S I S V D R L STTPR S G AS DP TD H V QAQ S SL D A A D SA GPP N Y AA KN .. KASR QAA H ED YDog_Cdc7 TKCP QTTAIDMWSAGVIFLSLLSGRYPFYKASDDLTALAQIMT RG RETIQAAK FGKS LCSKEVPAQ LR LCE LRG S N I S T I D K K I SNTP L SD QG ASHDP EKTD HKA HLI TPQA SGNSL N Q T T P SF . S Q LP CHorse_Cdc7 TKCP QTTAIDMWSAGVIFLSLLSGRYPFYKASDDLTALAQIMT RG RETIQAAK FGKS LCSKEVPAQ LR LCE LRG S N I S T I D K K I S T KL SDIQG ASHDP EKTD HKA HLI TPQAQ SGTSV D K S T P TV . S Q P CCow_Cdc7 TKCP QTTAIDMWSAGVIFLSLLSGRYPFYKASDDLTALAQIMT RG RETIQAAK FGKS LCSKEVPAQ LR LCE LRG S N I T T I D K K I STTPKL SDIQ SHDP EKTE HKI H I TPQAQ SGN N I ECS AF N V F Q P PSYPig_cdc7 TKCP QTTAIDMWSAGVIFLSLLSGRYPFYKASDDLTALAQIMT RG RETIQAAK FGKS LCSKEVPAQ LR LCE LRG S N I S T I D K K V SSTPRL DV AS DP EK D RKA LL TPSAQ G SV N AG REL P SF P . AD L AW P CPanda_Cdc7 TKCP QTTAIDMWSAGVIFLSLLSGRYPFYKASDDLTALAQIMT RG RETIQAAK FGKS LCSKEVPAQ LR LCE LRG S N I S T I D K K I SNTP L DIQG ASHDP EKTD HRA HL TPQAQ SGSSL D Q TG P TF . A KH H YElephant_Cdc7 TKCP QTTAIDMWSAGVIFLSLLSGRYPFYKASDDLTALAQIMT RG RETIQAAK FGKS LCSKEVPAQ LR LCE LRG S N V S T I D R I S TPKL S IQG VS DP E TD HKA HLM TPQAQ SGSSL Q D K T G T L AF T . A P R LMarsupial_cdc7 TKCP QTTAIDMWSAGVIFLSLLSGRYPFYKASDDLTALAQIMT RG RETIQAAK FGKS LCSKEVPAQ LR LCE LRG S N I S T V R P L E Q HE RTD KL QV G S N T P.... D AA K EDYV SIL L ....Q KLPL APA R FE

520 530 540 550 560 570

human_cdc7 GW VPDEAYDLLDKLLDLNPA RITA AL H F DM KGDSN C E TN LE E S EE L PF K SL S EHCFD YN . N Mouse_Cdc7 GW VPDEAYDLLDKLLDLNPA RITA AL H F DM K DN G D SN E D S E L F K R D YWSHPK CT .S S A A CS Dog_Cdc7 GW VPDEAYDLLDKLLDLNPA RITA AL H F DM KGDSNGCG LE E S EE L PF K SL RGLNMDTAD. N Horse_Cdc7 GW VPDEAYDLLDKLLDLNPA RITA AL H F DM KGDSNGCG TN LE DE S E L PF K SL GHFDVCS . G Cow_Cdc7 GW VPDEAYDLLDKLLDLNPA RITA AL H F DM KGD NGCG E TN LE DE S EE L PF K SL G GNFE SA . Pig_cdc7 GW VPDEAYDLLDKLLDLNPA RITA AL H F DM K CG E T DE S EE L PF SL EGGDS AHSD TAA SGG X Panda_Cdc7 GW VPDEAYDLLDKLLDLNPA RITA AL H F DM GDSNGCG TN E E S E L P K SL E GSLNTDA .S N G L Elephant_Cdc7 GW VPDEAYDLLDKLLDLNPA RITA AL H F DM KGDSNGCG E TN LE D S E L PF K SL NRFE DT . K G Marsupial_cdc7 GW VPDEAYDLLDKLLDLNPA RITA AL H F DM G D L D T M PF R SL EDGRG SEAHTS HRAG. R Q QQ

Supplementary Figure 1b | Amino acid sequence alignment of mammalian CDC7 orthologs.

CDC7 Homo sapiens Mus musculus Canis familiaris Equus caballus Bos taurus Sus scrofa Ailuropoda melanoleuca Loxodonta africana Monodelphis domestica

Homo sapiens Mus musculus Canis familiaris Equus caballus Bos taurus Sus scrofa Ailuropoda melanoleuca Loxodonta africana Monodelphis domestica

Homo sapiens Mus musculus Canis familiaris Equus caballus Bos taurus Sus scrofa Ailuropoda melanoleuca Loxodonta africana Monodelphis domestica

N (1–36)

Homo sapiens Mus musculus Canis familiaris Equus caballus Bos taurus Sus scrofa Ailuropoda melanoleuca Loxodonta africana Monodelphis domestica

Homo sapiens Mus musculus Canis familiaris Equus caballus Bos taurus Sus scrofa Ailuropoda melanoleuca Loxodonta africana Monodelphis domestica

N 1 N 2 1 2 3 C 4

5 D E 6 7 KI2 1

G EF

F G KI3 1 KI3 1 KI3 2

H I

DFG

P loop

RD N182

K90 E104

APE

Q391

2q (228–359)

3b(484–529)

Kinase insert 2

Kinase insert 3

N lobe C lobe

Nat

ure

Str

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Mol

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iolo

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oi:1

0.10

38/n

smb.

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human_MC 220 230 240 250

human_MC P D P P RLK FVKVE YR YL L N N S PF K MSQL F Q T MP.FI YS.IQK C ........ Dgallus_MC P D P P RLK FVKVE YR YL L S N S PF N RSRS F Q P FP.SL YC.VPK C ........ EAnolis_MC P D P P KLK FLKVE YR YI L S N S PF N RRCH F Q S FP.VI YS.NPR C ........ DXlaevis_MC P D P P KLK YIKVE YR YL L Q N S S CSCQ L V P FRSFQ ...... V ......NYSVEDaniorer_MC P D P P RIR FVKVE YR YL L N S PF R SSRH I P S MPVCNLRS..FP C ........ LScer_MC P D P P K V L W N PF YF Y H Y Y LWQT A IITLEWKPQELT LDELPY ILKIGSFGRC I

human_MC 290 300 310 320 330 340

human_MC GYCE C K L H S H F D K KKK C YE L EQ N S Y VV IV D V EK .. LQ D ET L R AQ. NQ Q D SKLVF F EYEgallus_MC GYCE C K L H S H F D K KRR C YE L EQ N S Y VV II E V DK .. GK D QT E K AQ. AQ Q D SKFVY F EYKAnolis_MC GYCE C K L H S H F D K KRK C YD V EQ N S Y VV II D L QK .. LK D QA E Q AQ. LH Q D SKVSC F EFRXlaevis_MC GYCE C K L H S H F D K KKH C YD I Q N S Y VV LI D V EQ .. LK D ES L P K SE. AY Q D STFDF F DWSDaniorer_MC GYCE C K L H S H F D KRK C FE L EQ S Y VV V D V GRE .G EV N KA D QA SK. NE G R TAGLTC L NISScer_MC GYCE C K L H S H F D K K YE I E S F AI LI KETV NS N RV S EQ V K L AENDLN E S ENLRFQI....

Mammal (H. sap. 214-254) Avian (G. gal. 238-278) Reptile (A. car. 218-258) Amphibian (X. lae. 194-232) Fish (D. rer. 209-249) Yeast (S. cer. 260-310)

Mammal (H. sap. 294-342) Avian (G. gal. 319-367) Reptile (A. car. 298-346) Amphibian (X. lae. 274-322) Fish (D. rer. 292-340) Yeast (S. cer. 659-704)

β1 β2

β3 β4 α1 α2 α3

DBF4 Motif–M

DBF4 Motif–C C296/299 H309/315

Supplementary Figure 1c | Amino acid sequence alignment of motif–M and –C regions from distal DBF4 orthologs.

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Human_Dbf4 1 10 20 30 40 50 60 70 80 90 100 110

Human_Dbf4 M MRI GGIQ NEKNR K K D E K KPLWGK FY DL S E L KD LGGRVEEFLSKDI Y SNKKEAK AQTL SP PSPES TSP N GA HSKGH Q VK PSL SL T N RP KS V L P VTIS K Q IKD S LI F GRI V AYTAET S F . C Mouse_Dbf4 M MRI GGIQ NEKNR K K D E K KPLWGK FY DL S E L KD LGGRVEEFLSKDI Y SNKKEAK AQTL SP PSPES TSP N HSK R PSL SL N R KS Y I L P ITI K Q IKE S V Y GRV V AYTAET LET APLP D A . L C F Dog_Dbf4 M MRI GGIQ NEKNR K K D E K KPLWGK FY DL S E L KD LGGRVEEFLSKDI Y SNKKEAK AQTL SP PSPES TSP N GA HSKGH Q VK SM SM T N KP K Y I I P VTVS K Q I D S LI F GRI V VYTAET A L . . Y Q Horse_Dbf4 M MRI GGIQ NEKNR K K D E K KPLWGK FY DL S E L KD LGGRVEEFLSKDI Y SNKKEAK AQTL SP PSPES TSP N GA HSKGH Q VK PSL SL T N KP KS Y V I P VTIS K Q IKD S LI F GRI V AYTAET S F . Cow_Dbf4 M MRI GGIQ NEKNR K K D E K KPLWGK FY DL S E L KD LGGRVEEFLSKDI Y SNKKEAK AQTL SP PSPES TSP N GV HSKGH Q VK PSL SL T N KP KS Y V I P VT S K Q IKD S LI F GRI I A TAET S F . T N Pig_Dbf4 M MRI GGIQ NEKNR K K D E K KPLWGK FY DL S E L KD LGGRVEEFLSKDI Y SNKKEAK AQTL SP PSPES TSP N GA HSRGH Q VK PSL SL T N KP KS Y V I P LS K Q IKD N LI F GRI V AY AET S F . GA A Panda_Dbf4 M MRI GGIQ NEKNR K K D E K KPLWGK FY DL S E L KD LGGRVEEFLSKDI Y SNKKEAK AQTL SP PSPES TSP N GA HSKGH Q VK SL SM T N KP KS Y I I P VTIS K Q I D S LI F GRI V AYTAET S F . . Q Elephant_bf4 M MRI GGIQ NEKNR K K D E K KPLWGK FY DL S E L KD LGGRVEEFLSKDI Y SNKKEAK AQTL SP PSPES TSP N GA H KGH Q V PSL SL T S KP KS Y V L P VSIS K Q IKD S LI F RI V AYNAET S G F N N S Opposum_Dbf4 M MRI GGIQ NEKNR K K D E K KPLWGK FY DL S E L KD LGGRVEEFLSKDI Y SNKKEAK AQTL SP PSPES TSP G RSKGH IK PTL TV N N K Y V L V IS R LKE S LV Y G L V AYT N KA T HP . S NP T V E C GG

Human_Dbf4 120 130 140 150 160 170 180 190 200 210 220 230

Human_Dbf4 PS DGSSFKS D VC SRGKLL EKA K HDFIP NSILSNALSWGVKILHIDDI YY EQ KK L KKSS S GK QK LKKPF KVED YRPFYLQL H H P T L V I D S R I K E YLL T VRD RVG G RTGR V MSQ TNM G S A T L Mouse_Dbf4 PS DGSSFKS D VC SRGKLL EKA K HDFIP NSILSNALSWGVKILHIDDI YY EQ KK L KKSS S GK QK LKKPF KVED YRPFYLQL H H L A V D R I K AL KDA KAG G RTGR L VN SL Q R A A S A G P I T RC P Dog_Dbf4 PS DGSSFKS D VC SRGKLL EKA K HDFIP NSILSNALSWGVKILHIDDI YY EQ KK L KKSS S GK QK LKKPF KVED YRPFYLQL H H P S L V I D S R I K E YLL T IKDA RVG G RTGR V MSQ TNM I T A L Horse_Dbf4 PS DGSSFKS D VC SRGKLL EKA K HDFIP NSILSNALSWGVKILHIDDI YY EQ KK L KKSS S GK QK LKKPF KVED YRPFYLQL H H P S L V I D S R I K E YLL T VRDV RVG G RTGR V M Q TNM I T A G L Cow_Dbf4 PS DGSSFKS D VC SRGKLL EKA K HDFIP NSILSNALSWGVKILHIDDI YY EQ KK L KKSS S GK QK LKKPF KVED YRPFYLQL H H P T L V I D S R I R E YLL T ARDV RVG G KS V MSQ TNM V T T R. L Pig_Dbf4 PS DGSSFKS D VC SRGKLL EKA K HDFIP NSILSNALSWGVKILHIDDI YY EQ KK L KKSS S GK QK LKKPF KVED YRPFYLQL H H P S L V I D S R I K E YLL T LRDV RVG G RTG V MSQ TNM V A G G L Panda_Dbf4 PS DGSSFKS D VC SRGKLL EKA K HDFIP NSILSNALSWGVKILHIDDI YY EQ KK L KKSS S GK QK LKKPF KVED YRPFYLQL H H P S L V I D S R I K E YLL T VRDV RVG G RTGR V MSQ TNM I T A L Elephant_bf4 PS DGSSFKS D VC SRGKLL EKA K HDFIP NSILSNALSWGVKILHIDDI YY EQ KK L KKSS S GK QK LKKPF KVED YRPFYLQL H H P S L V I D S R M K E YLL T VRDV RV KTGR V M Q TNM CT.. T C F Opposum_Dbf4 PS DGSSFKS D VC SRGKLL EKA K HDFIP NSILSNALSWGVKILHIDDI YY EQ KK L KKSS S GK QK LKKPF KVED YRPFYLQL P M V I E S K I K E YLI T LREA R G KTGR V AS SNIQ C P F I.. T HH

.. Human_Dbf4 240 250 260 270 280 290 300 310 320 330 340 350

Human_Dbf4 P INY KP SPFD K QK Q K R L E KKGY CECC QKYE L HLL E HR FA YQVVDDI S L D VE R S G SIQ VD PSSM QT V L IQTDGDK GG IQLQ K KK L D ET S Q N QSN V K VF F YEKD PKKK IKY V F C Y TS .. Q . T Mouse_Dbf4 P INY KP SPFD K QK Q K R L E KKGY CECC QKYE L HLL E HR FA YQVVDDI S L D VE R S G LQ IE SSV Q L IN DGDK G PVQLQ K KR L D ET S N QSN V VF F Y RD P KK IRY V C F C S A P M C .T .. K Q Q . G T Q Dog_Dbf4 P INY KP SPFD K QK Q K R L E KKGY CECC QKYE L HLL E HR FA YQVVDDI S L D VE R S G SIQ VD PSSI QT V L IQTEG K GG PVQLQ K KK L D ET S Q N QSN V K IF F YERD PKKK IKY I L S N G I .. H . M Horse_Dbf4 P INY KP SPFD K QK Q K R L E KKGY CECC QKYE L HLL E HR FA YQVVDDI S L D VE R S G SVQ VD PSSI QT V L IQTDGDK GG PIQLQ K KK L D ET S Q N N V K I F YERD PKKK IKY V F S C I .. PR P R G M Cow_Dbf4 P INY KP SPFD K QK Q K R L E KKGY CECC QKYE L HLL E HR FA YQVVDDI S L D VE R S G SIQ AE PSS QT V L IQTDGDK GG PVQ Q K KK L D D S Q N QSN I K VF F YERD PKKK IKY V F S T C I F ..I H . M Pig_Dbf4 P INY KP SPFD K QK Q K R L E KKGY CECC QKYE L HLL E HR FA YQVVDDI S L D VE R S G SIQ AD P N QT V L IQTDGDK GG PVQLQ K KK L D DS S Q N QSN V K VF F YERD PKKK IKY V F S P T C V VN H . T Panda_Dbf4 P INY KP SPFD K QK Q K R L E KKGY CECC QKYE L HLL E HR FA YQVVDDI S L D VE R S G SIQ VD PSSI QT V L IQTDG K GG PIQLQ K KK L D ET S Q S QSN V K IF F YERD PKKK IKY I F S N C I .. H . M Elephant_bf4 P INY KP SPFD K QK Q K R L E KKGY CECC QKYE L HLL E HR FA YQVVDDI S L D VE R S G SVQ VD PSSI QT V L IQTDGDK G PIQL K KK L D ET Q N QSN I K VF ERD PKKK IKY V F C QD T H .. C Q . L C T Opposum_Dbf4 P INY KP SPFD K QK Q K R L E KKGY CECC QKYE L HLL E HR FA YQVVDDI S L D VE R S G SV LD S L T A E G A R K S Q N QST I K VY F R VK I L P C T A P Q NRVGC AP ....P R S S A QA.. Q . SKSGIS .. R

Human_Dbf4 360 370 380 390 400 410 420 430 440 450 460

Human_Dbf4 S Q E E SE E LP LSPVS SVLKKT K VEL H S KD V EQ LYK E E K L P PSNELR L EK KCSMLS A DI QNFT HKNKQ I DISE A EQ EK Q I CQ DD.TT K NF TQ .T K L FI .......PI H G N MSN. T D R Q L EC L Mouse_Dbf4 S Q E E SE E LP LS VS NVLK T PK L KD S LL Y PE K S LK K SM N D Q Y R S E S A N A EKPL EPNF VG S.GH KPNSQ E TQK. E HGFA .......PTTYS AG GCDR ....PV F AS P PE E AQ L D .......TP Dog_Dbf4 S Q E E SE E LP LSPIT SVLRKS PK LE H S KD N VIEQ LYK E PE K V P PSSELR L EK KCS LN A DI QNFT HKNKQ I DVS A E EK SQ I FK N.VQ RF IQ . Q F FI .......CV S R D VTS. T P D G H L EC L KHorse_Dbf4 S Q E E SE E LP LSPIT NVLKKS PK LEL H S KD N VIEQ LYK E E K V P PSSELR L EK KCSVLN A DI NFT HKNKQ I DVSE A Q EK Q I FR N.VQ SF TK .L Q F FI .......CI Y G H TTD. P N KD H L GY L Cow_Dbf4 S Q E E SE E LP LSPI NVVKRS L L H S KD N MMEQ LYK PE K V SSELR L DK KCSMLN A V QNFT KSKQ I DVSE IA G.TKE K L N SR ..VH GF PVRE Q F SS .......HTLRR E D TSH. P NN K P PC EY L Pig_Dbf4 S Q E E SE E LP LSPIT N RKS PK LEV H S KD N VMEQ L K E PE R V P PSSELR L EK KCSMLN A DI QNFT HKNK I DVSE T FP E KK Q N SR N.IP TL H TL . K F ST .......HI H G . TSN. P N K P P KEC L Panda_Dbf4 S Q E E SE E LP LSPIT SVLKKS PK LEL H S KD N VIEQ LYK E PE K L P PSSELR L EK KCSMLN A DI QNFT HKNKQ I DVSE A E EK Q I FK N.VQ HF IQ . Q F FI .......CI C G D VTS. R D K H P EC L Elephant_bf4 S Q E E SE E LP LSPLT NVLKKT PK LEL K N AVEQ LYK E PE V P PS E R L E KCSILN A DI QNFT H NKQ I DVSE T K EQ QQIP NFR DIIQ SL TQ . QEL LI .......PI F D P E S EKMD. T N K Q S E EC F Opposum_Dbf4 S Q E E SE E LP PI N KRN P LEL R S S VI YR D P R A P P N A L E K L N A E K Q EV E FI IG TF E EKQ H I NGPW TTKE KHDFQ SR . AQ L CS PSFSFASCN Y Y S SK R QLDCR SY T P K SALDLVQ PQ DS GCSP A

Human_Dbf4 470 480 490 500 510 520 530 540 550 560 570 580

Human_Dbf4 E V Q D K E T E RKV H LSE DL LRVD Q SV S S DNS S KQKS TVLFPAKDLKE HSIF HDS LI INSSQ HL V AK P TPP E P CD N D LP SGKI KII ..T N E HYKCNI A H DF .T G P DL T G T Q A FH . NE FK M S . H Mouse_Dbf4 E V Q D K E T E RKV H V D RVD Q Q S S ESNL AKDL E H V H S LVALNTS L M AR P SP P CD N E LP GKI RML Q TEGRN G. Q PAPGVS SCG HL .T P PQLAA ITQLS Q GF V IG A D K Q K T PC ..Q HE TE M N .C Q Dog_Dbf4 E V Q D K E T E RKV H L NE DL LRVD Q SV S S DNTAS KQKS TVLF AKDLKE HSIF HDS LAVNS Q HL I AK PS SPP E P C N D L SG I KIL K MT K K HCPRRL T K NV SM P S DL G GR L R T R . NE NFS M G QT M H Horse_Dbf4 E V Q D K E T E RKV L INE EL LKVD Q SIQ N DNSAS KQKS TVLFPAKDLKE HSVF HES LLAINSSQ HL V AK S SPP E P CD N D LP SGKI KILQK I D . HCPFKL A ANF .M P DH G G Q TV R . RE IK I S . H Cow_Dbf4 E V Q D K E T E RKV H L LNE DL VKVD Q SIQ S N D SAS KQK TVLFPAK LKE SVF RD LLAINSSQ HL I A PS SPP E P CD S D LP SGKI KI K I S . HCPCRP T NF .T D W L H NLY H CD W EI E . NT IK T S . C PPig_Dbf4 E V Q D K E T E RKV L INE DL VKVD Q SIQ S N DNT S KQKS TVL PAKDLKE HSVF HDS LL VNSSQ HL I AK PS SPP E P CD N D P SGKI KILQK I K E HRPCRP I NF .T T P L DF C D G Q T Q . NA IK M SS . H Panda_Dbf4 E V Q D K E T E RKV H L ANE DL LRVD Q SVQ S S DNTAS KQKS TVLFPAKDLKE SIF HDS LLAINSSQ HL I AK PS SPP E P CD N D LP SG I KIL K M S E QCQCRL T SV .M P DLY G G Q T P . NA FN T G . T H Elephant_bf4 E V Q D K E T E RKV H L VNE L LKV Q TIQ S N DNSAS KQKS TILFPA DLK HSIF HDS LIAMNSSQ HL I AK PS NPP V KI KII K I SN E NRCQCRL T SF .K L E V DL G G Q T H .R SQXMQPQEIWI YFLV H Opposum_Dbf4 E V Q D K E T E RKV K I LK E MQ S D NVS K KS S LFP K RS DS L VM SS V S N P E P D S E LP SGKL KIV QGT KKKNSE T CCPYILETF DFDET K L R T DTCQ K GT PHDY S R G P QP GQ QVV H H G KEA VE S S . Q

Human_Dbf4 590 600 610 620 630 640 650 660 670

Human_Dbf4 K N E TE E C SP SL LF TS E SEFLGFT Y E C EE N LL F SSPS S F GF LGRNR E LEPNA DKR FI Q NRI S VQ LD Q E K S T SGI VLDIW E S N TA F T T T F .. T E . K N D . Mouse_Dbf4 K N E TE E C SP SL LF TS E SEFLGFT Y E C EE N LL F SSPS S F GF LG AEPSA LDKKR YL R VQ LD Q E K T SGI DVLDIW E SST S F T Q.Q A . PAH .D T G G N T . A V Dog_Dbf4 K N E TE E C SP SL LF TS E SEFLGFT Y E C EE N LL F SSPS S F GF LGRNK E LEPNV LDKKR FV T NRI S VQ LD Q E K S T SGI DV DIW D SNN SM F T T T . I Q R N F . Horse_Dbf4 K N E TE E C SP SL LF TS E SEFLGFT Y E C EE N LL F SSPS S F GF LGRNK E LEPNV LDKKR FL T RI S VQ LD Q E K S T SGI DVLDIW E SNN SV F T T T . I Q EK N . Cow_Dbf4 K N E TE E C SP SL LF TS E SEFLGFT Y E C EE N LL F SSPS S F GF LGRNK E LEPNV LDKKR FL T NRV S V LD Q E K S T N I DVLDI E SN TM F T T T . T Q . K K N L . . Pig_Dbf4 K N E TE E C SP SL LF TS E SEFLGFT Y E C EE N LL F SSPS S F GF LGRNK E LEPNV L KKR L T NRI S LQ D Q E K S T SGI DVLDIW E SN SM F T T T N . C T Q E R K . . Panda_Dbf4 K N E TE E C SP SL LF TS E SEFLGFT Y E C EE N LL F SSPS S F GF L RNK E LEPNV LDKKR FL T NRI S VQ LD Q E K S T SGI DVLDIW E SNN SV F T T T E . I Q G N . Elephant_bf4 K N E TE E C SP SL LF TS E SEFLGFT Y E C EE N LL F SSPS S F GF I RNK E LEPNV LDKKR FL T NRI S VQ VD Q E K S SG DALDIW E SNS SM F N T T E . I Q E P N T . Opposum_Dbf4 K N E TE E C SP SL LF TS E SEFLGFT Y E C EE N LL F SSPS S F GF LGR K NV L T N I S Q VE S S G DVLEVW E NS SM T T T . R QKK KVENK VPA K E S T K G N DC S E L S

Supplementary Figure 1d | Amino acid sequence alignment of mammalian DBF4 orthologs.

C296/299 H309/315

1 2

3 4 1 2 3 Motif–M

Motif–C

Motif–N

Homo sapiens Mus musculus Canis familiaris Equus caballus Bos taurus Sus scrofa Ailuropoda melanoleuca Loxodonta africana Monodelphis domestica

Homo sapiens Mus musculus Canis familiaris Equus caballus Bos taurus Sus scrofa Ailuropoda melanoleuca Loxodonta africana Monodelphis domestica

Homo sapiens Mus musculus Canis familiaris Equus caballus Bos taurus Sus scrofa Ailuropoda melanoleuca Loxodonta africana Monodelphis domestica

Homo sapiens Mus musculus Canis familiaris Equus caballus Bos taurus Sus scrofa Ailuropoda melanoleuca Loxodonta africana Monodelphis domestica

Homo sapiens Mus musculus Canis familiaris Equus caballus Bos taurus Sus scrofa Ailuropoda melanoleuca Loxodonta africana Monodelphis domestica

Homo sapiens Mus musculus Canis familiaris Equus caballus Bos taurus Sus scrofa Ailuropoda melanoleuca Loxodonta africana Monodelphis domestica

disordered loop

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Supplementary Figure 1e | Amino acid sequence alignment of MC regions from human DBF4 and DRF1 proteins.

Dbf4 220 230 240 250 260 270 280 290 300 310 320 330

Dbf4 RLK PF K ED S RPF Q P I K SPF T D KKGY CECC E L HL S QHR FA Y VD I V V Y N NY DV K G L KK Y D N V K M QL YL LT M F SIQ PC DKPSSMQ Q QVKLRIQTDG KY GTSIQ QLKE LQK ET L E QS.NQ QV D SDrf1 RLK PF K ED S RPF Q P I K SPF T D KKGY CECC E L HL S QHR FA Y VD I L I F S SF EA H P A RR F E S I A E RK HH FK F E LGP DA PTTLGSM H RESK...... GE SPRSA HTMP QEA HV Q A LEAHL AE R A

Motif–M (45% seq. identity)

Motif–C (53% seq. identity)

1 2 3 4 1 2 3

Dbf4 (214-332) Drf1 (225-338)

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Supplementary Figure 2 Limited proteolysis of CDC7–DBF4 and construct optimization for crystallography (next page). (a) Limited proteolysis of full-length CDC7–DBF4 with trypsin. The gel image on the top shows separation of undigested (left lane) and trypsin-digested (right lane) full-length CDC7–DBF4 by tricine SDS PAGE electrophoresis. Migration positions of molecular mass markers (kDa) are shown to the left; identities of the protein bands (CDC7, DBF4, A–E) along with their N–terminal sequences and apparent molecular masses are indicated with arrowheads. Locations of the proteolytic fragments within CDC7 and DBF4 amino acid sequences are indicated on the diagrams to the right of the gel image; positions of the C–termini of the fragments were estimated based on their apparent molecular masses. (b) Examples of size exclusion chromatography and 1H NMR spectroscopy of CDC7–DBF4 deletion constructs. Elution profile from a Superdex-200 column (top) and 1H NMR spectra (bottom) of CDC7(Δ(1–36)(221–339)(469–529))–MC (CDC7 missing residues 1–36, 221–339 and 469–529 in complex with DBF4(210–350), containing motifs –M and –C) and CDC7(Δ(1–36)(228–359)(484–529))–MC (CDC7 missing residues 1–36, 228–359 and 484–529 in complex with DBF4(210–350)). During size exclusion chromatography of each construct, both subunits co-eluted in the major peaks (data not shown). Predicted molecular masses for the heterodimeric constructs are indicated (55 and 57 kDa). Note that although the constructs have similar masses, CDC7(Δ(1–36)(228–359)(484–529))–MC elutes later, indicating absence of aggregation (confirmed by multiangle laser light scattering, data not shown) and/or a more compact structure. 1H NMR spectra of the methyl group region is shown for each construct. The large peak at 0.8 ppm is dominated by methyl groups that are poorly structured. The peaks at around ~0 ppm represent high field shifted methyl groups (structured methyl groups) that are present in the hydrophobic core of the protein.

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A (37~213) B (341~475) D (515~574)

CDC7

1 433 574 58 203 375 538

KI-2 KI-3

E (210~266)

1 48 92 214

253 291 331

674

N M CDBF4

- + trypsin

36.5 31.0

21.5

14.4

6.0

kDa

116.3 97.4 66.3 55.4

A: CDC7, 37LAGVK… (20 kDa)

B: CDC7, 341TASSCP… (15 kDa)

C: CDC7, 126KNDHV… (10 kDa) D: CDC7, 515KGDSN… (8 kDa) E: DBF4, 210TRTGR… (6 kDa)

CDC7

DBF4

C (126~213)

Manual run 9:10_UV Manual run 9:10_Logbook

0.0

10.0

20.0

30.0

40.0

50.0mAU

0 20 40 60 80 100 120

Manual run 6:10_UV Manual run 6:10_Logbook

0

50

100

150

mAU

0 20 40 60 80 100 120

CDC7(Δ(1–36)(228–359)(484–529))–MC

57 kDa 76 ml

150

100

50

0 100 0 20 40 60 80 100

a

70 ml

CDC7(Δ(1–36)(221–339)(469–529))–MC

55 kDa

40

20

10

0

0 20 40 60 80 100

Elution volume (ml)

A28

0 (m

Au)

30

b

‘unstructured’ methyl groups

‘unstructured’ methyl groups

‘structured’ methyl groups

‘structured’ methyl groups

A28

0 (m

Au)

Elution volume (ml)

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Supplementary Figure 3 Examples of electron density maps (next page). (a-c) Stereo views of the final 2Fo–Fc electron density map for three regions of the structure: active site (a), Dbf4 motif–M (b) and Dbf4 motif–C (c). Weighted 2Fo–Fc map contoured at 1σ is shown as blue mesh; protein chains are shown as sticks, with carbon atoms colored by chain, as in Fig. 2. The nucleotide is shown in sticks with carbon atoms in blue. Red spheres are water molecules, and gray spheres are metal atoms. Positions of the nucleotide, metal atoms and selected amino acid residues are indicated on the contour images. (d) Validation of the presence and the identity of the metal atom associated with Dbf4 motif–C by anomalous scattering. The protein is shown as sticks and the final weighted 2Fo–Fc map (contoured at 1σ) as blue mesh. Positions the metal atom and selected amino acid residues are indicated on the contour images to the right. Anomalous difference maps calculated from diffraction data acquired using X-ray energy of either 9,665.9 or 9,656.1 eV are shown as green (contoured at 10σ) and red (contoured at 3σ) mesh, respectively. A single peak of >10σ coinciding with the assumed Zn atom position is observed in the higher energy anomalous map, while the latter (based on data collected at an X-ray energy 5 eV below the Zn K edge) shows only noise. (e-f) Stereo views of the initial unbiased Fo–Fc omit and the final 2Fo–Fc maps for the active site region in PHA767491 (e) and XL413 (f) bound structures. The protein chain and inhibitor molecules are shows as sticks. The omit maps (green mesh) are contoured at 3σ and the final 2Fo–Fc maps (blue mesh) at 1σ.

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H139

P-loop

Mg

ADP*

T68

D177 I64

Y73

L184

Y233

L234

L421

F253

R176 Q391

β2

P231 β1

Zn

F253

V326

I330

α3

αC L210

H97

E297

Y295

KI-2

C298

H309 C296

H315 C299 Zn

PHA-767491

M118

Y136 V72

I64

P135 V195

D196

L74

M134

XL413

M118

Y136 V72

I64

P135 V195

D196

L74

M134

S70

S70

d

a

b

c

e

f

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E E K K

N N Dm Dm Dn Dn R R

TPO TPO

KI2α1 KI2α1 αC

E217 Q391 Q391

β1 β2 β3

β1 β2 β3

αC Mg Mg

DBF4–C

CycA N lobe

C lobe DBF4–M

CDC7–DBF4 CDK2–CycA

a

b

Supplementary Figure 4 Comparison of the CDC7–DBF4 structure with activated CDK2–Cyclin A. (a) Stereo view of a superposition of the CDC7–DBF4 and CDK2–Cyclin A (PDB ID 1QMZ) active sites. The protein chains are shown as cartoons and selected amino acid residues as sticks and indicated: E (Glu104 and Glu51 of CDC7 and CDK2, respectively), K (Lys90/Lys33), N (Asn182/Asn132), Dm (Mg2+-coordinating Asp196/Asp145), Dn (Asp177/Asp127), R (Arg176/Arg126), TPO (CDK2 phospho-Thr160). The N–lobes of CDC7 and CDK2 are shown in green and brown, the C–lobes in magenta and gray, carbon atoms of bound nucleotides (sticks) in light and dark blue, and Mg2+ ions (spheres) in green and brown, respectively. For clarity, the DBF4 chain is hidden fro view. Locations of β1, β2, β3 and αC of the kinase N–lobes are indicated in black print; (c) Comparison of DBF4 (left) and Cyclin A (right) structures on their complexes with CDC7 and CDK2, respectively. The catalytic subunits are shown in space fill mode, DBF4 and Cyclin A as cartoons.

E217

N lobe: CDK2, CDC7

C lobe: CDK2, CDC7

N lobe: CDK2, CDC7

C lobe: CDK2, CDC7

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97 55

31

21

14

6

2.5

97

55

31

21

14

6

250

130 100

70

55

35

M,C

MC, His6MC

ΔN2q3b

kDa:

MC

ΔN2q3b, ΔN2aa3b

MC

CDC7

kDa:

kDa:

25

15

Supplementary Figure 5 Purified CDC7–DBF4 heterodimeric constructs resolved in SDS-PAGE gels. The proteins (10–15 µg) were resolved in SDS-PAGE gels and detected with Sypro Orange or Coomassie Blue. Identities of individual constructs are indicated above the gel. The construct Migration of full length CDC7 (CDC7), CDC7 deletion mutants (CDC7(ΔN2q3b) and CDC7(ΔN2aa3b), lacking KI2α1), DBF4(210–350) with or without a hexa-histidine tag (His6-MC and MC), DBF4 motif–M (M; 210-266) and DBF4 motif–C (C; 288-350) are indicated with arrowheads.

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CDC7–DBF4 MAPK (Erk2)

Supplementary Figure 6 Comparison of the CDC7–DBF4 (left) with MAPK Erk2 (right, PDB ID 2ERK). The structures are shown as cartoons. CDC7–DBF4 is colored as in Fig. 2. The canonical N– and C–lobes structures of Erk2 are shown in green and purple, respectively. MAPK insert and C–terminal extension are colored yellow and orange, respectively. Locations of CDC7 insert 3, MAPK insert, DBF4 motifs –M and –C, C–termini of DBF4 and Erk2, and the secondary structure elements discussed in the text are indicated. Gray sphere is a Zn atom.

DBF4–C

DBF4–M

CDC7 insert 3 MAPK insert

MAPK C–terminal extension α3

αC αC

αL16

C–term

KI2α1 310L16

N lobe

C lobe

C–term

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PHA767491 XL413CDC7-DBF4 ≤30%Abl >30%, ≤60%ALK >60%, ≤90% Aurora-A >90% inhibitionBrSK2BTKc-RAFCaMKIIβCDK1/cyclinBCDK2/cyclinACDK2/cyclinECDK5/p35CDK6/cyclinD3CDK7/cyclinH/MAT1CDK9/cyclin T1CHK1CHK2CK1δCK2CK2α2cKitcKitDAPK2eEF-2KEphA3EphB1FerFGFR1FGFR2Flt1GCKGRK6GSK3αGSK3βIKKαIRIRAK4JAK2JAK3KDRLckLynMAPK1MAPKAP-K2MAPKAP-K2MELKMetMKK7βMRCKβNEK2NEK6PAK3PAK4PDGFRαPDGFRα(V561D)PDK1Pim-1Pim-2PKCαPKCεPKCζPlk1Plk3PRK2RetRIPK2Rsk3SAPK2aSAPK3SRPK1TAO1TAO3TrkATrkATSSK1WNK3ZAP-70ZIPK

PHA-767491 Compound 507Cdc7-Dbf4 ≤30%Abl >30%, ≤60%ALK >60%, ≤90% Aurora-A >90% inhibitionBrSK2BTKc-RAFCaMKIIβCDK1/cyclinBCDK2/cyclinACDK2/cyclinECDK5/p35CDK6/cyclinD3CDK7/cyclinH/MAT1CDK9/cyclin T1CHK1CHK2CK1δCK2CK2α2cKitcKitDAPK2eEF-2KEphA3EphB1FerFGFR1FGFR2Flt1GCKGRK6GSK3αGSK3βIKKαIRIRAK4JAK2JAK3KDRLckLynMAPK1MAPKAP-K2MAPKAP-K2MELKMetMKK7βMRCKβNEK2NEK6PAK3PAK4PDGFRαPDGFRα(V561D)PDK1Pim-1Pim-2PKCαPKCεPKCζPlk1Plk3PRK2RetRIPK2Rsk3SAPK2aSAPK3SRPK1TAO1TAO3TrkATrkATSSK1WNK3ZAP-70ZIPK

Supplementary Table 1 Inhibitory activities of PHA767491 and compound XL413 on a panel of divergent human kinases. All compounds were assayed at 1 µM concentration. Level of inhibition is color-coded as indicated in the inset.

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