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by: Johan Turcotte
Johan Turcotte
GPA 3.54


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This 6 page Class Notes was uploaded by Johan Turcotte on Sunday September 6, 2015. The Class Notes belongs to ORG 462 at University of Texas at Austin taught by Staff in Fall. Since its upload, it has received 40 views. For similar materials see /class/181398/org-462-university-of-texas-at-austin in Organ at University of Texas at Austin.


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Date Created: 09/06/15
E CD i gush Structure of Hexameric DnaB Helicase and Its Complex with a Domain of DnaG Primase Scott Bailey et al Science 318 459 2007 DOI 101126science1147353 Science The following resources related to this article are available online at wwwsciencemag org this information is current as of September 23 2008 Updated information and services including highresolution gures can be found in the online version of this article at httpwwwsciencemagorgcgicontentfull3185849459 Supporting Online Material can be found at httpwwwsciencemagorgcgicontentfull3185849459DC1 This article cites 34 articles 19 of which can be accessed for free httpwwwsciencemagorgcgicontentfull3185849459otherartices This article has been cited by 11 articles on the ISI Web of Science This article has been cited by 2 articles hosted by HighWire Press see httpwwwsciencemagorgcgicontentfull3185849459otherartices This article appears in the following subject collections Biochemistry httpwwwsciencemagorgcgicollectionbiochem Information about obtaining reprints of this article or about obtaining permission to reproduce this article in whole or in part can be found at httpwwwsciencemagorgaboutpermissionsdtI Science print ISSN 00368075 online ISSN 10959203 is published weekly except the last week in December by the American Association for the Advancement of Science 1200 New York Avenue NW Washington DC 20005 Copyright 2007 by the American Association for the Advancement of Science all rights reserved The title Science is a registered trademark of AAAS Downloaded from wwwsciencemagorg on September 23 2008 The 3 protrusion and orient The synapsis of The ends Fig 2D The 3 overhangs of The opposing Fig 2E and g S6 18 Each PolDom mono mer makes intimate contact with The 5 P on The downstream strand which is bound in a posi tively charged pocket formed by Lys16 and Lys26 Fig 3A Two residues absolutely conserved in NHEJ AEPs g S4 Notably The N Terminal PolDom region containing Lys16 is absent in AEPs from Archaea and Eukarya g S4 The Mt PolDom mutant Lys16 gt Ala was unable to bind to DNA and had very reduced polymerase activity g S5 whereas gap lling was normal OTher interactions wi D A are indicated in gs S4 and S6 The PolDom DNA interactions are reminiscent of The contacts ob served in The structure of The evolutionary un related NHEJ polymerase Pol 7t gapped DNA complex 18 19 Fig 3 The apical loop 1 BS 36 interacts wiTh The 3 protruding strand Thus constituting a poten tially important element for maintaining The syn apsis between two 3 protruding DNA ends Fig 2 C and D To analyze The functional impor Tance of These interactions we mutated loop 1 residues 83 to 85 to alanine and evaluated The DNA binding and polymerization capacity of The resulting mutant mm 0012 On a gapped DNA substrate The DNA binding potential of mm 0012 was equivalent to That of wild type PolDom g S7 Therefore The presence of a 5 P appears to be enough to ensure enzyme DNA stability in a gap and loop 1 is dispensable when The primer terminus the template and the 539 P are physically connected and not discontinuous However The integrity of loop 1 was critical to forming a synaptic complex of two 3 protruding DNAs Electrophoretic mobility shi and 3 extension assays showed That mm 0012 was very e i 39ent at forming a synaptic complex g S8 An analogous loop like structure may play a related role in eukaryotic NHEJ polymerases 20 21 The importance of PolDom and loop 1 in particular in mediating DNA synapsis was irther probed by uorescence resonance energy transfer FRET using DNA wiTh a 3 overhang identical to That present in The crystal structure The steady state uorescence spectra of doubly labeled 3 protruding DNA 339 uorescein donor and 3 rho amine acceptor wiTh increasing amounts of wild type Mt PolDom showed a marked concentration dependent increase in emission of The rhodamine uorophore at 605 nm Fig 4 due to FRET from uorescein The presence of a PolDom dependent FRET emission peak signi es a close approach of The 3 overhang with The duplex region of another DNA indicative of a stable protein mediated interaction between two DNA ends In contrast The mm 0012 mutant exhibited a markedly re duced FRET signal indicating Thatloop 1 plays a critical role in stabilizing The synaptic complex wwwtsciencemagprg SCIENCE VOL 318 This conclusion is further supported by protein cross linking studies g S2 e structure presented here establishes That NHEJ pol erases can promote The formation of end bridging complexes Thereby directing The break alignment process g S9 The limited number of contacts made between The enzyme and The 3 protrusions suggests That PolDom and presumably oTher olymerases allow a large degree of rotational freedom That enables The termini to search for sequence complemen tarities on The opposing break This homology searching process acts together with Ku to align The break by forming presynaptic bridging struc tures promoted by favorable microhomology directed base pairing That nucleate The formation of The synaptic complex g S9 Thus nal end synapsis like That shown in The crystal structure may require a certain degree of mispairing tem plate dislocation or realignment facilitated by base ipping and The eventual formation of hair pin structures at The terminal ends The hairpin like structures observed located in a large solvent accessible c annel wiThin The PolDom o 0 small 3 exonuclease domain o LigD NucDom 6 15 facilitating The controlled resectioning of The ends This may possibly explain The prefer ence of NucDom for recessed 3 ends 6 15 and sug ests That The nuclease resection process may be regulated by The conformation of The ends wiThin The synaptic complex References and Notes 1 as 2 n m 3 n P Sung A Tamkinsan 74 159 2003 1 M Daley P L Palmbas D Wu T E WilsanAnnU Rev N Genet 39 431 2005 3 G R Weller et al Science 297 1686 2002 4 R S Pitcher et al DNA Repair 6 1271 2007 5 R S Pitcher et al Mal Cell 23 743 2006 REPORTS 6 Ni Della et al Science 306 683 2004 7 K S Pitdier T E l on A Daherty Cell Cycle 4 675 2GB 8 R Bawater A Daherty P105 Genet 2 93 2006 9 C GangA Martins P Bangiarna Nl Glickman S Shuman Bial Chem 279 20594 2004 10 C Gang etal Nat Struct Mal Biol 12 304 2005 11 R S Pitcher L M Tankin A J Green A J Daherty Mal Bial 351 5312005 12 R S Pitcher et al Mal Bial 366 3912007 13 L Vakavleva S Shuman Biol Chem 281 25026 2006 14 H u etalPrac Natl Acad Sci USA 103 1711 2006 15 H Zhu S Shuman Bial Chem 281 13873 2006 16 L M lyer E V Kaunin D D Leipe L Aravind Nucleic Acids Res 33 3875 2005 17 S H Lap Sirieix L Pellegrini S D Bell Trends Genet 21 568 2005 18 See supporting material on Science Online 19 Ni Garcia Dial et al Cell124 331 2006 20 S A Nick NlcElhinny et al Mal Cell 19 357 2005 21 R Jua39rez J F Ruiz S Nick NlcElhinny D A Ramsden L Blanca Nucleic Acids Res 34 4572 2006 22 Abbreviations for amino acid residues F Phe H His K lys N Asn P Pm R Arg S Ser W Trp 23 We thankT Brown A Lebedev Nl Osbourne and D Thompson for technical advice and support T Wilson for valuable comments on the manuscript an the European Synchrotron Radiation Facility for provision at synchrotron radiation facilities War in AD s laboratory is supported by grants from the UK Biotechnology and Biological Sciences Research Council Cancer Research UK the Association for lnternatianal Cancer Research and the Royal Society Work in LB s laboratory was sup arted by Nlinisteria de Ciencia y Tecnalagla grant WC 2003 00186 Cansalider CSD2007 00015 and an institutional grant to Centre de Bialagla Nlalecular Severn Ochoa from Fundacia n Rama39n Areces ALP is a recipient of a fellowship from the Spanish Ministry of Science and Tec no agy a Supporting Online Material vwwvsciencemagargcgicantentfull3185849456DC1 Materials and Nletha s References 15 May 2007 accepted 14 September 2007 101126science1145 112 Structure of Hexameric DnaB Helicase and Its Complex with a Domain of DnaG Primase Scott Bailey1 William K Eliason1 Thomas A Steitzl zquot The complex between the DnaB helicase and the DnaG primase unwinds duplex DNA at the eubacterial replication fork and synthesizes the Okazaki RNA primers The crystal structures of hexameric DnaB and its complex with the helicase binding domain HBD of DnaG reveal that within the hexamer the two domains of DnaB pack with strikingly different symmetries to form a distinct twolayered ring structure Each of three bound HBDs stabilizes the DnaB hexamer in a conformation that may increase its processivity Three positive conserved electrostatic patches on the Nterminal domain of DnaB may also serve as a binding site for DNA and thereby guide the DNA to a DnaG active site ost DNA polymerases unlike RNA polymerases are una e to unwind duplex DNA and require a primed single stranded DNA ssDNA substrate to initiate DNA synthesis In eubacterial cells These functions are performed by a complex of the DnaB helicase and The D hydrolysis of nucleoside triphosphate NTP 2 whereas DnaG uses The newly formed 19 OCTOBER 2007 459 I RE PORTS 460 rw A L or by heavy atom derivative The two domains of the DnaB hexamers fw 39 t 39 39 39 L 4 l L had aked in form a distinct double layered ring structure in homo hexameric ring that has been observed solutions containing mercury chloride 17 whic the NTDs residues 1 to 152 pack into a by electron microscopy EM to form either Phasing of the dioraction from each crystal rigi triangul seated t p e six fold or three fold symmetry states 3 4 orm and the resulting electron density maps loosely packed ring of CTDS residues 186 to 39 39 39 at 454 1 B an C Adiac NTDs ado The DnaB ring s thought to unwind NA the replication fork by translocating along and encircling the 539 lagging strand w i the 339 leading strand is occluded 5 6 The crystal structure 0 a monomer 0 an extended helical hairpin 7 Although 39s required for helicase activity 8 10 and may define the direction ofmovement of the helicase on DNA 10 its precise function 39 DN un 39 ing is not clear The interaction between DnaB and DnaG stimulates both of their activities DnaG in creases both the NT1gtase and the helicase ac tivities ofDnaB 9 and DnaB both increases es the synthesis of RNA primers Nt in inding domain ZBD RNA polymerase do D an a termin HBD BD of DnaG whose tern cture consists of a helical bundle the Cl subdomain t 39 ate by a helical hairp e 2 s b omain is sufficient to both t and st the activities of DnaB 9 11 The tertiary struc D is h39 ly similar to the fold o th N o DnaB 7 13 The stability of the interaction between DnaB and DnaG varies species In Escherichia interaction is relatively weak and can only be detected by sensitive techniques run over a gel filtration column 9 Despite these differences in the stabili of their B1 andB and two crystal structures of in complex th HBD f D at forms BHI and H2 These four crystal fo diffr y tw 50 an 9 ately s fo either by e single wavelength anomalous diffraction method using selenomethionine 1Department ot Molemlar biophysics and biochemistry and Howard Hughes Medical Institute vale University New Haven CT 05520 USA Department ol chemistry vale University NEW Haven CT 05520 USA TD hom correspondence should be addressed E mail eathertonisbyaleedo 19 OCTOBER 2007 VOL 318 SCIENCE Fig 1A were substantially improved by al symmetry aver ing among 1 7 Data collection and stalf 39 as 3 i CTDs There otherwise stated discussion wiil focus on the 29 A resolution tructure aB complexed with HBD form BHI which has been refined to a free R factor of 293 to head dimers related by two fold symmetry which three of the alternately oriented NTDs the central channel of the ring Fig 1C This trimer of dimers is stabilized by the hydrophobic interface that buries 2300 A2 Table 1 Data Collection and Refinement Statistics Crystal Form 31 32 BH1 BHZ spat roup P21212 R32 P321 9321 Unit cell ahc A 371 no 113 zoo zoo 19s 229 229 193 23o 23o 193 Resolution it so 37 so so so 2 so 4o Rmerge Lay 31 s9o s 3 s33 7o gt1oo 1o3 so3 Completeness Lat 973 866 9s 3 74s 99 3 999 999 mom I l 1so 14 3s 9 3s 22 3 19 196 36 Rwork 3o3 3co 394 433 2s 9 324 32 o 34 free 323 3s3 397 491 4oo 344 334 RMSDT bond Alangle L ooo9141c ooo91339 oo1o1cs9 Fi 1 Architecture oi A t g DnaB hexamer A i 3 4 Ex erimentall hased 4 1 J p y p t e a scry al avers a F I e aged electron density 2 maps oi the tour DnaB quot a J crystal iornts Shawn a quot39 the foot of each ma is BHt 2 9A 525 0A the highsresulutiun limit at which eadi ma was MD We calculated B side B intertaoe a shown in a suriace representation the NTDs are shown as ribbons Those subunits whose NTDs lie on the inner iace oi the ring are colored white in Side view oi thetwo distinct contorniations oi the DnaB subunits within the hexamer colored as in a Adjacent Cios interacting with the linker region are shown as white suriace representations wwwsciencemagorg Downloaded from wwwsciencemagorg on September 23 2008 closed mp V Bm mgSlle 7 139 Fig 2 Slruclure ui he an ring A Suriare represenlaliun ui he an rings ui cryslaliurrns BH1le and BI nghl Allernale subunils are culured while and red The predicled DNA binding luups are ulured blue and he linker helices are shuwn as y slruclure ui he 17 gp splayed as in panel A C R D rings ui cryslal righl Allemale subunils are culured while and nk NTP mudeled al he six pulenlial NTP binding siles ui na are s uwn as green spheres he Arginine ngers Argm are isplayed as red spheres n The slruclure ui he 17 gp4 hexamerwilh iuur NTD binding siles uccupied displayed as in c is A mmiunei lnlenace NTD Dimer lnlenace Dimer lnlenace 02 lnleilac D NT Tvlmev lnleilace Fig 3 Slruclure ui he cumplex belween DnaB and HBD A Tup Tup view ui a ribbun represenlaliun ui he cumplex shuwing he hree HBDs green buund al he periphery ui he NTD llar igh blue and blue The cm and linker regiun are culured red and elluw respeclively a nd HBD uwn as nbbu s wllh a ransparenl su e B Sid ew s re represenlahun ui he cumplex rev ng nu inle cliun HBDs gre andl DnaB cm red ur linker regiun lluw Backbune rare ui he H DnaB culured spheres wwwsciencemagorg SCIENCE VOL 318 REPORTS I wihin he dimers and by he hydrophobic inerrace berween dimers ha buries 1 5 oa1 ion of e NTD collar appears o be highly cooperalive requiring he presen othe cm rolein are similar in all four Crystal arl o he slrucrures of he four DnaB hexamers shows ha heir C IDs adopt a yariey of differen orientations around he ring bu sdll bind he linker hel39cc residues 162 o 178 o he adjaoent subunit at he periphery of he amer and orient heir proposed DNA binding loops 18 oward he cenlral ch e Fig 2A he cm maehree fold s ry Fig 2A The hexameric rings of C IDs are held ogeher primaril b heir ons wihin e linker e area Fig 1D In addidon Io hese inleracdons he C39ID 39 appears o be addiiionally sabilized by he inleiacnons berween he NTDs because muans lacking he NTD have reduced hexamer stability 9 19 The aB hexamer assembly has an ouler diameer of 115 A and a height of 75 A The diameer oflhe cenual channelihr E1 helicase uses different regions of its RecA like domains or h annex formation and DNA 313 bound to ill dimensional 3D EM reconstructions of E coli 19 OCTOBER 2007 Downloaded from wwwsciencemagorg on September 23 2008 I RE PORTS DnaB 3 and GAOP 4 g 53 however they are consistent w39 EM projections used to generate the 3D reconstructions Therefore it seems likely that the diffeences between the crystal and 3D EM data could be due to distor tions generated by the negative staining process or to the methodolog39 al difficulties ofgenerating 3D reconstructions of molecules as flexible as DnaB discussed further in supporting online text The T7 gp4 protein ofphage T7 contains two I main 39 39 39 and the other for the primase activity which are necessary for the replication ofthe T7 phage 22 than those seen in DnaB These more extensive contacts are geneated by a rotation of the gp4 examer tral charnnel only wide enough to accommodate ssDNA Fig 2 The larger diameter of the central channels observed in the bound NTP supporting online text because a more extended conformation of DnaB has been observed by BM in the absence ofnucleotide 3 addition 39 h 39 39 fth DnaB CTDs most ofthe NTP binding sites are not near an arginine finger Fig 2C Only the B1 r r r r r r r r r r the DnaB NTD 7 23 Instead two domains re the N terminus of the protein The crystal struc ture of the hexameic CTD of gp4 bo d t nonhydrolysable NTP analog 18 shows that the NTP binding pockets are formed between adjacent CTDs Fig 2 B and D Themajority of each pocket is formed by one CTD whereas the adjacent CID provides an arginine residue the inine finger whose guanadini group con taets the 7 phosphate of the bound NTP The arginine finger is believed to stimulate NTP hy drolysis and to help modulate the relative orien 39 CTDs irnresponsetoNTlgt hydrolysis 1824 Comparison ofthe structures ofDnaB and the gp4 helicase shows that the oligomerization of the two proteins is facilitated by a similar linker ig 2 but the contacts between the c r r r Fig 4 DNAinteractiuns A A Left quotTu view of a DnaB cm red DnaG HBD green DnaG RPD pink and DnaG 25 the RNA pnnrer is Shawn in dark blue 19 OCTOBER 2007 VOL 318 SCIENCE with the 7 phosphate of a bound NTP Fig 2C bound state of DnaB However homology be en the CTDs ofDnaB and gp4 and the fact that superimposing the structure of the DnaB 39 CTDs ofthe nucleotide bound gp4 structure produces a model with no steric clashes 7 suggest that the complex with nucleotide will 39 a central channel that is only wide enough to accommodate ssDNA The ability of DnaB to modulate the diameter of its central charnnel is owever consistent with the observation that DnaB can translocate on ssDNA even when a complementary strand is present within the cen tral channel 25 filtration studies of the complex between Est 1136 which show that between two and three molecules ofDnaG bind to each DnaB hexame 9 This number is also consistent with tluorescence anisotropy and cross link39 p iments conducted using the complex ONE 901139 DnaB and DnaG 15 The bound HBDs do not 39 39h 39h theCTDorthelinkerregion of DnaB Fig 3B consequently there is no correlation between HBD binding and the po sition of the CTDs or the diameter ofthe CTD rings in the four structures presented here The 2 subdomains of the HBD k against the genesis studies ofE oozi DnaB 11 Overall the interface with the Cl subdomain is less tightly 39 39 nethzm explaining why the isolated 2 subdomains can form a gel frlterable complex with DnaB wheeas the isolated 11 subdomain cannot 12 The c2 subdomain ofE can is sm er than that of 3B which DnaB and a of the complex Fig 3A This stoichiomery Pro Twain Lagging strand DNA e gagedwun primase active Ste Lagging strand D A mm 0 ma proposed NTD ssDNA binding site Occiuded leading strand DNA Dn G than their E can counterparns for more details n W L A xes the three fold arrangement of the NTD collar Fin i 39 39 39 39 force microscopy results 2 Mutation ofresidues Tyr 5 119 or 119 in d ofthe equivalent residues in E coli 27 29 and Sazrrtortenrt ryphx39mwium 30 inhibits the formation ofa complex between H lies n the does not directly Fig 3C this mutation presumably disnrpts the tertiary structure ofth NTD helical bundle Residues Ile 9 and 119 are buried from solvent at the NTD dimer interface Fig 3C which suggests that their mutation would disrupt dimeriz ion e NTD Hence inhibition of it has already been suggested that helicases may have altered NTD positions 27 Mutation of Glu in BernaB has no etfect on 39 39 39 DnaG but does modulate the length 39 d by DnaG 16 The both at the 1 b site NTD trime int e rg 3C consistent with its having a rol on of a Dna is currently not clear wwwsc39lencemagorg Downloaded from wwwsciencemagorg on September 23 2008 The binding of DnaG to DnaB stimulates the activities of DnaB 1 and stabilizes the ee old conformation of the DnaB TDs s te r resents an activated form o therefore it seems doubtful that the DNA trans location mechanism of DnaB involves transitions between six and three fold symmetries Both DnaB and the T7 gp4 proteins require a stable hexamer for NlTase and helicase activity 9 18 Therefore the DnaG mediated stimulation of the activities of DnaB could also result from the increased stability of the hexamer produced by the binding of DnaG which is consistent with the observation that although the isolated C2 subdomain of the HBD can bind DnaB both subdomains of the HBD are required for the stimulation of the activities of DnaB 16 Although the presence of DnaG at the repli cation fork in E coli has been shown to be distributive 31 the binding of only one mol ecule of DnaG to DnaB would be sufficient to stabilize the three fold conformation of DnaB The closed circular structure of the NTD collar could also contribute to the stimulation of the helicase activity by keeping the two ssDNA strands topologically separated during unwind ing In addition the topological linking of DnaB to the DNA also would ensure that the two molecules could not easily disengage thus increasing the processivity of the reaction inetic analysis has shown that isolated Dn is only a moderately processive enzyme and it is assumed that it gains the processivity needed to replicate the enome from o er compo nents of the replication fork 32 A similar processivity role as also been suggested for the unrelated NTD of the papillomavirus E1 helicase 20 h T e T ollar may also provide an ad ditional binding site for ssDNA The interior o e N co lar exhibits three dis tinct sites of positive electrostatic potential separated by regions of negative electrostatic potential Fig 4A These positive sites are consistent with their bindin NA contain resi ues at are conserved across naB species and are well positioned for binding the ssDNA as it emanates from the CTD ring Fig 4 Nuclease protection and uorescence energy transfer studies have also suggested the presence ofa second ssDNA binding site at the N terminus of DnaB 33 It is now possible to construct a model of the complex between DnaB and DnaG that illumi nates how they cooperatively work together and stimulate each other s activities T e N terminus f e ch HBD is situated adjacent to the central channel of DnaB Fig 3 thereby positioning the N terminal ED and RPD of full length DnaG directly above the central channel Fig 4B Thus the structure of the RPD ZBD fragment 34 can be positioned relative to the HBD in a manner that orien primase active site with the proposed N terminal ssDNA binding site of wwwtsciencemagtorg SCIENCE VOL318 DnaB and is consistent with the structure of the truncated T7 gp4 helicase primase heptamer plex between the full length proteins is consist ent with the possibility that DnaB stimulates the activity of DnaG by increasing the local concentration of the ssDNA substrate and by ensuring that multiple DnaG subunits are in close proximity to each other 35 Fig 4B The latter is important because the RPD and ZBD function have been shown to function in trans with each domain provided by a separate subunit 35 References and Notes 1 J E Corn 1 Ni Berger Nucleic Acidg Reg 34 4082 2006 J H teBowitz R Nlchacken Biol Chem 261 4738 1986 S Vang etol Mol Biol 321 839 2002 4 R Nunez Ramirez etol Mol Biol 357 1063 2006 5 Ni 1 Jezewska S Rajendran D Bujalowski W Bujalowski Biol Chem 273 10515 1998 6 D t Kaplan Mol Biol 301 285 2000 7 S Bailey W K Eliason T A Steitz Nucleic Acidg Reg 35 4725 2007 8 N Nakayama N Arai V Kaziro K Arai Biol Chem 259 55 1984 9 t E Bird H Pan P Soultanas D B Wigley Biochemigtry 39 171 2000 Mesa 1 C Alonso S Ayora Mol Biol 357 1077 N w 10 2006 11 K Tougu K J Nlarians Biol Chem 271 21391 1996 12 K Syson J Thirlway A Ni Hounslow P Soultanas J P Waltho Structure 13 609 2005 13 A Oakley et ol Biol Chem 280 11495 2005 14 V B Lu 5 Bhattacharyya Ni A Griep Proc Natl Acod Sci USA 93 12902 1996 15 A V Niitkoya 5 Ni Khopde S B Biswas Biol Chem 278 52253 2003 16 J T irlway P Soultanas Bocteriol 188 1534 2006 REPORTS 17 Materials and methods are available as supporting material on Science Online 18 Ni R Singleton Ni R Sawaya T Ellenberger D B Wigley Cell 101 589 2000 19 S B Bisvvas P H C en E E Bisvvas Biochemigtry 33 11307 1994 20 E J Enemark t Joshua Tor Nature 422 270 2006 21 W Bujalowski Ni 1 Jezewska Biochemigtry 34 8513 1995 22 E A Toth V ti Ni R Sawaya V heng T Ellenberger Mol Cell12 1113 2003 23 Ni R Sawa a S Guo S Tabor C C Richardson T Ellenberger Cell 99 167 1999 D J rampton S Guo D E Johnson C C Richardson Proc Natl Acod Sci USA 101 4373 2004 D t Kaplan Mol Biol 301 285 2000 J Thirlway etol Nucleic Acidg Reg 32 2977 2004 rdal R Niaurer Bocteriol 178 4620 1996 P Chang K J Nlarians Biol Chem 275 26187 2000 N is NNNN m axm B Lu P V A t Ratnakar B K Nlohanty D Bastia Proc Natl Acod Sci USA 93 129 2 1996 R Nlaurer A Wong Bocteriol 170 3682 1988 C A Wu E t Zechner K J Nlarians Biol Chem 267 4030 1992 R Galletto Ni 1 Jezewska W Bujalowski Mol Biol 343 83 2004 33 Ni 1 Jezewska S Rajendran W Bujalowski Biol Chem 273 9055 1998 J E Corn P J Pease G t Hura 1 Ni Berger Mol Cell 20 391 2005 35 S Lee C C Richardson Proc Natl Acod Sci USA 99 12703 2002 This research was supported by NlH grant Gil57510 to thank P Soultanas for the expression plasmids Coordinates and structure factors of crystal forms BH1 BH2 B1 and B2 have been deposited under accession codes 2R6A 2R6C 2R6D and 2R6E respectively WW b O w w Jgt N w a Supporting Online Material vwwvsciencemagorgcgicontenttull3185849459DC1 Materials and Nietho s All Text Figs 51 to 57 Tables 51 and 52 References 3 July 2007 accepted 11 September 2007 101126science1147353 Network Analysis of Oncogenic Ras Activation in Cancer Edward C Stites1 2 3 Paul C Trampont1 Zhong Ma1 Kodi S Ravichandran To investigate the unregulated Ras activation associated with cancer we developed and validated a mathematical model of Ras signaling The modelbased predictions and associated experiments help explain why only one of two classes of activating Ras point mutations with in vitro transformation potential is commonly found in cancers Modelbased analysis of these mutants uncovered a systemslevel process that contributes to total Ras activation in cells This predicted behavior was supported by experimental observations We also used the model to identify a strategy in which a drug could cause stronger inhibition on the cancerous Ras network than on the wildtype network This systemlevel analysis of the oncogenic Ras network provides new insights and potential therapeutic strategies as is a small guanosine triphosphatase RGTPase that binds the guanine nu cleotides guanosine triphosphate GT and guanosine diphosphate GDP 1 2 Ras bound to GTP RasGTP is the active form with which downstream effector proteins spe 19 OCTOBER 2007 Downloaded from wwwsciencemagorg on September 23 2008


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