C r y s t a l S t r u c t u r e o f t h e H o m o l o g o u s - p a i r i n g D o m a i n o f t h e H u m a n R a d 5 2 P r o t e i n C r y s t a l S t r u c t u r e o f t h e H o m o l o g o u s - p a i r i n g D o m a i n o f t h e H u m a n R a d 5 2 P r o t e i n A DNA break that remains unrepaired is lethal fo r an y ce ll . Do ub le st ra nd br ea ks oc cu r in ce ll s exposed to ionizing radiation and chemotherapeutic a g e n t s , o r n a t u r a l l y b y r e p l i c a t i o n e r r o r s a n d o x y g e n r a d i c a l s g e n e r a t e d f r o m r e s p i r a t i o n . T o repai r such lesio ns, cells have devel oped effic ient h o m o l o g o u s r e c o m b i n a t i o n a l r e p a i r s y s t e m s . I n bu dd in g ye as t, th e RA D5 2 ep is ta si s gr ou p ge ne s ( RAD51 , RAD52 , RAD54 , RAD55 , RAD57 , RAD59 ) play central roles in homologous recombination and ar e co ns er ve d am on g eu ka ry ot es . Ra d5 2, a ke y member of this group, displays two critical activities that have been demonstrated in vitro : homologous pa ir in g an d in te ra ct io n wi th th e Ra d5 1 re co mb in as e. T h e s e a c t i v i t i e s a r e l i k e l y t o b e e s s e n t i a l f o r t h e i n i t i a t i o n o f h o m o l o g o u s r e c o m b i n a t i o n , a l t h o u g h n o d e t a i l e d m o l e c u l a r m e c h a n i s m s a r e c u r r e n t l y available. T h e h u m a n R a d 5 2 p r o t e i n c o n s i s t s o f 4 1 8 a m i n o a c i d r e s i d u e s , a n d w e , a s w e l l a s o t h e r s , have found that the N- and C-terminal halves of this pr ot ei n ha ve di st in ct ro le s in re co mb in at io n. Th e N - t e r m i n a l h a l f o f R a d 5 2 ( a b o u t 2 0 0 a m i n o a c i d r e s i d u e s ) i s h i g h l y c o n s e r v e d a m o n g R a d 5 2 homologs, and this region composes a structurally st ab le do ma in as su gg es te d fr om li mi te d pr ot eo ly si s exp eri men ts [1] . The iso lat ed, N-t erm ina l dom ain of Ra d5 2 bi nd s to si ng le -s tr an de d DN A ( ss DN A ), an d ha s ho mo lo go us -p ai ri ng ac ti vi ty eq ui va le nt to t h a t o f t h e f u l l - l e n g t h p r o t e i n [ 1 ] . I n t e r e s t i n g l y , a l t e r n a t i v e - s p l i c i n g v a r i a n t s c o n t a i n i n g o n l y t h e N - t e r m i n a l h a l f o f R a d 5 2 w e r e f o u n d i n h u m a n s , i n d i c a t i n g t h a t t h e i s o l a t e d , N - t e r m i n a l d o m a i n o f Rad 52 has a rol e in rec omb ina tio n [2] . The C- te rm in al h al f, b y co nt ra st , is p oo rl y co ns er ve d am on g Rad52 homologs. This region physically interacts Fig. 1. The structure of Rad52 1-212 . (a) Ribbon diagram of the undecameric ring of Rad52 1-212 , viewed down the central channel from the top of the domed cap. (b) The ring viewed from the side. The domed cap region is coloured in blue and magenta, and the stem region is coloured in grey. The diameter of the ring is about 120 Å, and that of the central channel is about 50 Å at the narrowest point. 17 ( a ) ( b ) Fig. 1 Fig. 2 F i g . 3 ( a ) Fig. 3 ( b ) with the Rad51 recombinase [1], and may have an i m p o r t a n t r o l e i n t h e r e c o m b i n a t i o n m e d i a t e d b y Rad52 and Rad51. Therefore, the N-terminal half is present in both the longer (full-length) and sh or te r ( a l t e r n a t i v e s p l i c i n g v a r i a n t ) f o r m s o f R a d 5 2 , w h e r e a s t h e C - t e r m i n a l h a l f i s o n l y p r e s e n t i n t h e l o n g e r form of Rad52. T o g a i n i n s i g h t i n t o t h e m e c h a n i s m o f h o m o l o g o u s - pa ir in g pr om ot ed by Ra d5 2, th e N- te rm in al fr ag me nt ( R a d 5 2 1 - 2 1 2 ) c o n t a i n i n g t h e h o m o l o g o u s - p a i r i n g domain was designed, and crystals of this fragment wer e pre par ed for str uct ura l ana lys es. Dat a fro m b o t h t h e n a t i v e a n d s e l e n o m e t h i o n i n e - s u b s t i t u t e d R a d 5 2 1 - 2 1 2 c r y s t a l s w e r e c o l l e c t e d a t t h e R I K E N S t r u c t u r a l B i o l o g y b e a m l i n e I ( B L 4 5 X U ) , a n d t h e str uct ure was det erm ine d by the mul ti- wav ele ngt h a n o m a l o u s d i s p e r s i o n ( M A D ) m e t h o d a t 2 . 8 5 Å res olu tio n [3] . The cry sta l str uct ure of Rad 52 1-21 2 r e v e a l e d a n u n d e c a m e r i c r i n g , in di ca ti ng th at th e sh or te r fo rm of Ra d5 2 is un de ca me ri c. The overall structure resembles a mushroom with a stem and a domed cap region ( ). Rad52 1-212 has an exposed groove around the e n t i r e r i n g s t r u c t u r e . T h i s g r o o v e e n c i r c l e s t h e s t e m r e g i o n , a n d i s h i g h l y p o s i t i v e i n c h a r g e , a s dete rmin ed from the surf ace pote ntia l calc ulat ions ( ). The groove has an approximate width of 1 0 Å a n d a n a p p r o p r i a t e s i z e t o a c c o m m o d a t e ssDNA. To identify the precise DNA binding site, the basic and aromatic amino acid residues on the positively charged surface, which have the potential to interact directly with ssDNA, were replaced with a l a n i n e b y s i t e - d i r e c t e d m u t a g e n e s i s ( ) . Th es e mu ta nt s we re th en te st ed fo r ss DN A bi nd in g. T h e m u t a t i o n s o f A r g 5 5 , T y r 6 5 , L y s 1 5 2 , A r g 1 5 3 , a n d A r g 1 5 6 , w i t h s i d e c h a i n s i n s i d e t h e g r o o v e , clearly decreased the ssDNA binding activity ( ) . T h e r e f o r e , t h e s h o r t e r f o r m o R a d 5 2 f i t s s s D N A i n s i d e t h e e x p o s e d g r o o v e , a n d w r a p s ssDNA around the stem region. In co nt ra st to th e sh or te r fo rm , ou r se di me nt at io n e q u i l i b r i u m s t u d i e s o n t h e l o n g e r f o r m o f R a d 5 2 d e m o n s t r a t e d t h a t t h e p r o t e i n i s h e p t a m e r i c i n s o l u t i o n . P r e v i o u s e l e c t r o n m i c r o s c o p i c s t u d i e s also concluded that Rad52 forms a heptameric ring [4 ]. In te re st in gl y, th e tw o ri ng fo rm s ha ve si mi la r Fig. 2. Side (a) and bottom (b) view of the surface of Rad52 1-212 . The surface is coloured according to the electrostatic potential. (red) –12 k B T -1 to (blue) 12 k B T -1 . 18 ( a ) ( b ) Fig. 3. Mutagenesis and ssDNA binding. (a) Amino acid residues ( coloured in yellow) essential for ssDNA binding mapped on the Rad52 1-212 monomer. (b) The residues essential for ssDNA binding are located inside the groove. (c) Gel shift assay of the Rad52 1-212 -ssDNA complexes. The ssDNA binding was analyzed by 1% agarose gel electrophoresis. Fig . 4 d i a m e t e r s , w h i c h a l l o w e d u s t o m o d e l t h e h e p t a m e r i c rin g usi ng the cry sta l str uct ure of Rad 52 1-21 2 ( ) . In th e r e s u l ti n g h e p ta m e r m o d e l , a p o s i ti v e l y c h a r g e d D N A g r o o v e i s p r e s e n t , s u g g e s t i n g t h a t t h e g r o o v e f o r D N A b i n d i n g i s c o n s e r v e d i n b o t h ri ng fo rm s. Th is is co ns is te nt wi th th e si mi la r ss DN A b i n d i n g a n d h o m o l o g o u s - p a i r i n g a c t i v i t i e s o f t h e longer and shorter forms of Rad52 [1]. In conclusion, the crystal structure of Rad52 1-212 r e v e a l e d t h a t t h e h o m o l o g o u s - p a i r i n g d o m a i n o f Ra d5 2 is or ga ni ze d in to a ri ng wi th a ci rc ul ar gr oo ve essential for ssDNA binding. Mutagenesis studies d e m o n s t r a t e d t h a t s s D N A b i n d s t o R a d 5 2 b y wrapping around the ring structure. Both ring forms a r e c a p a b l e o f p r o m o t i n g h o m o l o g o u s p a i r i n g , su gg es ti ng th at ri ng fo rm at io n is es se nt ia l fo r th e a c t i v i t y . O n t h e o t h e r h a n d , o n l y t h e h e p t a m e r i c f o r m o f R a d 5 2 c a n i n t e r a c t w i t h t h e R a d 5 1 re co mb in as e through the C-terminal half, which wa s predic ted to be located between the monomer s i n the heptamer model. The two ring forms of Rad52 c o u l d b e i n v o l v e d i n d i f f e r e n t r e c o m b i n a t i o n p a t h w a y s . Th e sh or te r fo rm , wh ic h la ck s th e Ra d5 1- i n t e r a c t i n g r e g i o n , m a y f u n c t i o n i n r e c o m b i n a t i o n p a t h w a y s t h a t a r e i n d e p e n d e n t o f R a d 5 1 . B y co nt ra st , th e lo ng er fo rm co ul d fu nc ti on in Ra d5 1- dependent recombination pathways. In both cases, o u r r e s u l t s s u g g e s t t h a t t h e N - t e r m i n a l h a l f o f R a d 5 2 p r o m o t e s h o m o l o g o u s p a i r i n g i n t h e D N A binding groove around the ring structure. 19 ( c ) ( a ) ( b ) Rad52 R55A Y65A Y104A K141A E145A K152A R153A R156A K169A R173A K177A R180A 1-212 Fig. 4. Heptamer model of Rad52. The heptameric ring of the longer form was modeled b y s i m p l y r e m o v i n g f o u r m o n o m e r s f r o m t h e u n d e c a m e r i c r i n g ( a ) , a n d s p a c i n g t h e remaining seven monomers evenly, without changing the diameter of the ring (b) . Wataru Kagawa a,c , Hitoshi Kurumizaka a,b and Shigeyuki Yokoyama a,b,c (a) RIKEN Genomic Sciences Center (b) RIKEN Cellular Signaling Laboratory (c) The University of Tokyo E-mail: kurumi @ jota.gsc.riken.go.jp References [1 ] W. Ka ga wa et al . , J. Bi ol . Ch em . 27 6 (2 00 1) 35201. [ 2 ] K . K i t o e t a l . , B i o c h i m . B i o p h y s . A c t a 1 4 8 9 (1999) 303. [ 3 ] W . K a g a w a , H . K u r u m i z a k a , R . I s h i t a n i , S . F u k a i , O . N u r e k i , T . S h i b a t a , a n d S . Y o k o y a m a , Mol. Cell 10 (2002) 359. [4] A. Z. Stasiak et al. , Curr. Biol. 10 (2000) 337. (a) (b) 20
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