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1 BRC repet RPA DSB RAD52 DSB Repir doi:1.138/nture9399 Gp Repir ssdna/dsdna junction ssdna/dsdna junction RPA Binding Resection RPA Binding Filment Formtion or or Filment Formtion DNA Piring DNA Piring Dissocition? Supplementry Informtion 2nd End Cpture / Invsion Brnch Migrtion Brnch Migrtion Purified stimultes medited recomintion DNA humn Synthesis / Extension DNA Synthesis / Extension Holidy Junction Formtion Ryn B. Jensen, Aur Crreir, nd Stephen C. KowlczykowskiHolidy Junction Formtion Resolution / Dissolution Resolution / Dissolution REPAIRED CHROMOSOME Supplementl Figure Legends REPLICATION FORK RESTART Supplementry Figure 1. Model depicting the functions of in recomintionl DNA repir. () A DSB generted y either exogenous (e.g., ionizing rdition) or endogenous (e.g., metolic yproducts) sources is resected to revel ssdna tils which in vivo re immeditely coted y the ssdnainding protein, RPA. As more clerly illustrted in pnel, Supplementry Figure 1 my then ind either t the ss/dsdna junction or long the ssdna region. promotes filment formtion y loding onto the RPAcoted ssdna til nd lso y limiting the ssemly of onto dsdna. Becuse inhiits the ATPse ctivity of, the filment on ssdna is stilized s the ctive ATPound form of, llowing susequent filment extension. The nucleoprotein filment finds DNA sequence homology in donor duplex DNA nd promotes DNA strnd invsion to form joint molecule. Completion of DNA DSB repir is then fcilitted y multiple proteins cting ww w. n tt u r e. c o m / NATURE 1

2 RESEARCH sequence homology in donor duplex DNA nd promotes DNA strnd invsion to form joint molecule. Completion of DNA DSB repir is then fcilitted y multiple proteins cting t severl susequent steps, resulting in repired chromosome with genetic informtion intct. () Results from this study lso revel potentil role for in ssdna gp repir, involving gps such s those encountered during DNA repliction of dmged templte. In this scenrio, RPA would ind the ssdna gp flnked y the two regions of dsdna. could then ind t either DNA junction or long the ssdna, nd deliver to the ssdna gp. Formtion of continuous filment y growth in either the or direction would then llow DNA piring into the homologous dsdna templte, with possile dissocition of from the ssdna region, followed y the susequent steps descried in () resulting in DNA gp repir nd repliction restrt. 2

3 RESEARCH Trnsfect 293T cells M MBP TCL B FT E 2XMBP TCL B FT E Hrvest cell lystes 31 hrs posttrnsfection 2 Amylose HiTrp Q 12 1 c M TCL Western Anti Q Step Elutions Amylose Elutes FT NCl (M) Western Anti Supplementry Figure 2. Purifiction of MBP tgged full length. () Schemtic of strtegy used to purify full length humn with two tndem repets of MBP tg t the N terminus (2XMBP). () Western lot (6% SDSPAGE) using n ntiody specific for the croxyterminus of showing mylose resin purifiction of either single MBP tg fused to the Nterminus of (lnes 14) or doule MBP tg t the Nterminus of (lnes 58). Lnes 1 & 5: TCL = totl cellulr lyste. Lnes 2 & 6: B = eds. Remining ound to mylose eds fter mltose elution. Lnes 3 & 7: FT = flowthrough (unound). Lnes 4 & 8: E = elute. Mltose elutes demonstrte the incresed yield of with tndem MBP tg. (c) Amylose resin purifiction of 2XMBP, followed y NCl step elutions off the HiTrpQ column. Western lot (6% SDSPAGE) using the sme ntiody s in (). TCL = totl cell lyste. Amylose resin elutes (lnes 36) were pooled efore loding onto the HiTrpQ column. 3

4 RESEARCH N MBP Full Length C rc2 / MBP rc2 / MBPFL (3) Cterm rc2 / rc2 / MBPFL (3) 2XMBPFL clones (11) (15) MBP Cterm MBP Cterm RTPCR c M () rc2 / MBPFL rc2 / 2XMBPFL IP/Western Westerns Anti Supplementry Figure 3. Confirmtion of stle MBP expression in rc2 mutnt (VC8) cells y RTPCR nd western lotting. () Two sets of primer pirs designed to trget nd mplify either the MBP tg on the Nterminus of or the Cterminl sequence. () Totl RNA ws isolted from G418resistnt rc2 mutnt cells stly trnsfected with MBP, 2XMBP, or empty vector nd utilized in n RTPCR strtegy s depicted in () to screen for clones expressing only full length MBPtgged. The ethidium romide stined gel on the right demonstrtes one clone (11) positive for PCR mplifiction t oth ends of the cdna, while clone (15) is negtive. (c) Clones deemed positive y RTPCR screening were further tested for protein expression y immunoprecipittion of from cellulr lystes (using A1), followed y western lotting to detect recominnt expression (using A2). Lne 1 () represents the rc2 mutnt cells (VC8) trnsfected with empty vector. Clone 3 (lne 2) ws positive for recominnt MBP expression while clone 4 (lne 3) ws negtive. Clone 11 (lne 4) ws positive for 2XMBP expression while clone 15 (lne 5) ws negtive, s expected from the RTPCR results. Clones 3 (MBP) nd 11 (2XMBP) were used in the clonogenic survivl ssy to ssess complementtion in Figure

5 RESEARCH [/] DNA Strnd Exchnge Buffer Buffer B Totl (µm) Supplementry Figure 4. The inding of to under conditions used for DNA strnd exchnge ssys. 2XMBP ws incuted with incresing mounts of purified nd the complexes were nlyzed with the pulldown ssy s descried for Figures 1e & f, using either DNA strnd exchnge uffer ( ) or uffer B ( ). The dt were fit to segmentl liner regression (Prism 5.), s descried in Figure 1f. For the experiments in DNA strnd exchnge uffer, the error rs represent S.D. for two independent experiments. For the control, in uffer B, one experiment ws conducted nd no error rs re shown; however, the reproduciility of similr experiments (e.g., Figure 1), the vrition is pproximtely ±15%. The preprtion used in this pulldown nlysis is slightly less ctive thn the preprtions used in the min ody of the pper, ccounting for the minor decrese in inding demonstrted in uffer B. The inding stoichiometry t the intersection of the two lines is 3.5 ± 1. per for oth solution conditions. 5

6 RESEARCH / DNA Complex (µm) / DNA Complex (µm) / DNA Complex (µm) Free DNA Free DNA Free DNA.22 µm.44 µm.22 µm.44 µm.22 µm.44 µm ()/ DNA Complex (nM) c Free DNA ()/ DNA complexes, (%) * * * Tiled ssdna Tiled 1 Tiled DNA 1 Tiled DNA 1 Tiled DNA Tiled DNA Tiled DNA Tiled DNA 75 ssdna 75 ssdna 75 ssdna 5 25 ()/ DNA complexes, (%) 5 25 ()/ DNA complexes, (%) (nm) (nm).22 µm (nm).44 µm Supplementry Figure 5. The inding of to DNA is unltered y preincution with. () EMSA nlyses of complexes inding (from left to right): Tiled DNA, ssdna, or Tiled DNA. Turnover y results in less thn 1% of the DNA persisting s complex. () EMSA nlyses of complexes inding to: Tiled DNA, ssdna, or Tiled DNA. (c) Quntifiction of the EMSA results in (): Tiled DNA ( ), ssdna ( ), or Tiled DNA ( ). These results re sed on single experiment nd the differences for ny of the DNA complexes re not considered to e sttisticlly significnt sed on typicl gel to gel vrition oserved (e.g., Figure 2). 6

7 RESEARCH No Protein ATP Mg 2 RPA (µm) (µm) Product *dsdna c 75 d 75 Product (%) 5 25 Product (%) (µm) Supplementry Figure 6. Optimiztion of the DNA strnd exchnge rection. () Autordiogrm of DNA strnd exchnge rections utilizing the tiled DNA sustrte t different concentrtions of (lnes 15). () Autordiogrm showing DNA strnd exchnge rections using the tiled DNA sustrte in the presence of.22 µm (lnes 27). Stndrd DNA strnd exchnge uffer, which contins 2 mm CCl 2, ws used. Lne 1: No protein control. Lne 2: ATP omitted. Lne 3: Mg 2 omitted. Lnes 47: incresing mounts of RPA were incuted with the DNA sustrte for 5 minutes t 37 C prior to the ddition of. (c) Quntifiction of the dt in () indicting tht optiml exchnge occurs t.22 µm. Error rs represent the S.D. (d) Quntifiction of the dt in (). 7

8 RESEARCH cold oligo Hetero. dsdna ATP RPA Product (%) cold Oligo Heterologous dsdna ATP Supplementry Figure 7. DNA strnd exchnge controls. () In ll DNA strnd rections shown: RPA is.1 µm, is.22 µm, nd is 4 nm. Lne 1: no protein control. Lne 2: RPA lone control. Lne 3: lone control. Lne 4: lone. Lne 5: nd. Lne 6: RPA nd. Lne 7: RPA first, second. Lne 8: RPA first, nd second. Lne 9: RPA first, nd second, with 1 fold excess cold oligonucleotide complementry to the leled piring strnd in the donor dsdna present in the deproteiniztion step. Lne 1: RPA first, nd second using heterologous leled donor dsdna. Lne 11: RPA first, nd second. Lne 12: Sme rection s in lne 11 with ATP omitted. () Quntifiction of the dt in the utordiogrm in (). 8

9 RESEARCH Tiled DNA / *dsdna 3.4 µm (nm) Product *dsdna Supplementry Figure 8. stimultes DNA strnd exchnge in the presence of excess. () Scheme for DNA strnd exchnge rections in the sence of RPA. The tiled DNA sustrte ws incuted first with the indicted nd for 5 minutes t 37 C, nd then the rdioleled dsdna ws dded. () Autordiogrm of DNA strnd exchnge rections, performed s descried in (), contining excess (.4 µm) in the presence of incresing concentrtions of. 9

10 RESEARCH ()/ dsdna Complex /dsdna Complex Free dsdna (nm) dsdna complex, (%) (nm) c d B=Biotin S=Streptvidin / B Tiled DNA 9.p. Heterologous dsdna SB Mgnetic Bed 1 Cpture Tiled DNA Wsh & Elute dsdna dsdna Tiled DNA Elute (Beds) Western Anti e f (% Bound) dsdna Tiled DNA Elute (Beds) Western Anti Beds Beds Tiled DNA (nm) dsdna Tiled DNA Supplementry Figure 9. prevents inding of onto duplex DNA nd trgets onto Tiled DNA. () EMSA showing effect of on inding to dsdna. Incresing concentrtions of were preincuted with.48 µm for 15 minutes t 37 C prior to mixing with 4.p. duplex DNA (4 nm molecules; the sme dsdna RJOligo1/RJOligo2 used in the previous DNA strnd exchnge nd EMSA nlysis). The proteindna complexes were incuted for further 3 minutes t 37 C nd resolved on 6% TAE polycrylmide gel. () Quntifiction of the nd for the dsdna complex detected in (). (c) Digrm of edcpture experiment to mesure inding of to 1

11 RESEARCH Tiled DNA in the presence competing heterologous dsdna; proteins were mixed prior to their ddition to the iotinylted Tiled DNA nd 9.p. dsdna. (d) (.6 µm) inding to iotinylted Tiled DNA (.2 nm molecules) either in the sence (lne 2) or presence (lnes 35) of excess dsdna (1, 4, or 4 nm molecules respectively). (e) (.6 µm) preincuted with or without incresing concentrtions of for 15 minutes t 37 C. The proteins were then incuted with the two DNA sustrtes for 5 minutes t 37 C. (f) Quntifiction of the mount of ound to the iotinylted Tiled sustrte. The mount of ound to the in the sence of dsdna (pnel e, lne 2) ws set to vlue of 1%. 11

12 RESEARCH Tiled DNA RPA / *dsdna 3 1,, 1, 3, 6 Product *dsdna c Product (%) Time efore *dsdna ddition (min) d Tiled DNA RPA / *dsdna 1,, 1, 3, 6 e Product *dsdna f Product (%) Time fter *dsdna ddition (min) Supplementry Figure 1. Kinetic nlyses. () Scheme for DNA strnd exchnge rections indicting tht ssys were performed s in Figure 4 except tht, fter the ddition of nd, the proteins were incuted with the tiled sustrte from 16 minutes efore ddition of the leled dsdna to initite the 3 minute rection. () Autordiogrm of DNA strnd exchnge ssys with 4 nm ( ) or without ( ). (c) Quntifiction of (). (d) In this rection scheme, time course from 16 minutes ws performed fter the ddition of the leled dsdna. (e) Autordiogrm of the gel from rections following the time course s descried in (d) with 4 nm ( ) or without ( ). (f) Quntifiction of the gel in (e). Error rs represent the S.D. from three independent experiments. A comprison of the liner regions of the time courses shows tht presynptic complex formtion on RPAssDNA is incresed ~2fold y. In contrst, when the time for presynptic complex formtion is held constnt, ut the time fter ddition of homologous dsdna is vried, stedy differentil throughout the time course of the rections is seen. These kinetic nlyses show tht ccelertes the rte of nucleoprotein filment formtion on ssdna tht is complexed with RPA, confirming the conclusions of the prior section. 12

13 RESEARCH Tiled DNA RPA or SSB / RecA or *dsdna 3 RPA RecA SSB h Tiled DNA Tiled DNA (nm) Product *dsdna RecA h Supplementry Figure 11. Species specificity of DNA strnd exchnge. () Scheme for the DNA strnd exchnge rections used in (). () Left pnel depicts n utordiogrm of ssys performed s in () utilizing the tiled sustrte, except E. coli RecA (.22 µm) ws sustituted for. The right pnel depicts n utordiogrm of rections performed s in () except E. coli SSB (.1 µm) ws sustituted for RPA. 13

14 RESEARCH Tiled DNA RPA / / hrad52 / yrd52 *dsdna 3 Product *dsdna hrad52 yrd (nm) c Product (%) yrd52 hrad Protein (nm) Supplementry Figure 12. Neither humn RAD52 nor yest Rd52 stimultes the DNA strnd exchnge ctivity of humn in the presence of RPA. () Scheme for the DNA strnd exchnge rections used in () (c). The tiled DNA sustrte ws incuted first with RPA for 5 minutes, followed y, humn RAD52, or yest Rd52. ws then dded, followed y 5 minute incution, nd finlly the rdioleled dsdna ws dded to strt the 3 minute rection. () Autordiogrms of the ssys compring (left to right):, humn RAD52, or yest Rd52. (c) Quntifiction of the gels shown in (): ( ), humn RAD52 ( ), or yest Rd52 ( ). Error rs represent the S.D. 14