别再提起(C)
F igure 5.3: E xperimental diagram (A) shows how a single DNA molecule is pulled into protamine solution using an optical trap. The DNA is stained with the dye YOYO-1 at low concentrations so that binding of the sperm nuclear proteins is not affected and illuminated with light from an argon-ion laser ( λ ? 488 nm) (B). Fluorescence from the YOYO-1 allowed the DNA molecule to be imaged using an image-intensi? ed CCD camera. An image of how
DNA toroids are thought to form is shown in (C) ( B rewer et al., 1999 )
148 C hapter 5
wa s s taine d u s ing low c on c entration s of the inter c alating d ye, YOYO-1. F luore sc en c e fro m the d ye wa s s ti m ulate d u s ing an argon-ion la s er (wavelength, λ ?488 n m ) an d the s tru c tural c hange s to the DNA m ole c ule were i m age d u s ing an i m age-inten s i? e d CCD c a m era. U s ing thi s te c hnique, we were able to m ea s ure the rate s of bin d ing an d d i ss o c iation of s per m nu c lear protein s an d d ire c tly m ea s ure the equilibriu m c on s tant for a s pe c i? c protein –D NA c o m plex without nee d ing to ? t the d ata to a theoreti c al m o d el. F igure 5.4 s how s a three-c hannel ? ow c ell. Gla ss frit wa s u s e d to bon d a gla ss c over s lip to a m i c ro sc ope s li d e at high te m perature. The m i c ro sc ope s li d e ha d c hannel s et c he d into it u s ing a “ d ry-et c h ”te c hnique. U s e of the s e fabri c ation m etho ds c reate s a very s trong an d m e c hani c ally s table ? ow c ell that i s i m perviou s to s olvent s s u c h a s m ethanol, u s e d for relea s ing bubble s fro m the ? ow c ell, an d d enaturant s s u c h a s guani d iniu m hy d ro c hlori d e, u s e d for c leaning DNA an d protein s fro m the c ell after ea c h u s e .
T he Protamines P1 and P2 T he pri m ary fun c tion of the prota m ine s i s to re d u c e the volu m e of the s per m geno m e ( D ooher an d Bennett, 1973 ) by c o m pa c ting it into DNA – p rota m ine toroi ds (
F igure s 5.1 an d 5.2 ) (
G agnon, 1999 ). P1 i s pre s ent in all vertebrate s an d P2 i s a dd itionally pre s
ent F igure 5.4: A three-channel ? ow cell was fabricated using a glass microscope cover slide, a glass microscope slide into which channels have been formed, and glass frit (manufactured by MMR Technologies). The three parts form a sandwich and were bonded together in an
oven at high temperature ( B rewer and Bianco, 2008 )
S ingle Molecule Studies of Chromatin Structure and Dynamics 149
in s o m e m a mm alian s pe c ie s , in c lu d ing hu m an an d m ou s e ( O liva, 2006 ). Both P1 an d P2 are arginine-ri c h, highly ba s i c protein s ( B rewer et al., 2002 ) that bin d to DNA in a non s equen c e d epen d ent m anner. P2 i s s ynthe s ize d a s a pre c ur s or (preprota m ine 2, F igure
5.1)an d pro c e ss e d after it bin ds to DNA to for m P2 ( E ven s on et al., 2002 ). The P1/P2 ratio i s u s e d exten s ively a s a m etri c for hu m an m ale fertility. In c rea s e d P1/P2 ratio s are s trongly c orrelate d with m ale infertility an d low level s of prota m ine 2 ( C ho et al., 2003 ), abnor m al s per m m orphology in c lu d ing roun d hea d e d s per m , an d high level s of DNA frag m entation ( O liva, 2006 ; T orregro s a et al., 2006 ).
W e were the ? r s t group to s tu d y the c on d en s ation of s ingle DNA m ole c ule s by s al m on prota m ine (s al m ine) ( B rewer et al., 1999 ) a s well a s P1 an d P2 in the bull an d ha ms ter ( B rewer et al., 2002, 2003 ). We were able to m ea s ure that the off-rate of prota m ine fro m
F igure 5.5: R eal-time formation of a DNA – p rotamine toroid starting at the left end of a 50 μ m concatemer of λ-phage DNA. The DNA molecule was extended by ? owing buffer and attached to a 1 μ m bead (right end) held by an optical trap. The protamine
concentration is 1.1 μ M ( B rewer et al., 1999 )
150 C hapter 5
DNA wa s le ss than one m ole c ule per s e c on d, in d i c ating that the geno m e woul d take approxi m ately 6 year s to c o m pletely d e c on d en s e in the ab s en c e of other m e c hani sms. However, on c e the s per m c ell fertilize s the egg, the s per m geno m e m u s t be unpa c kage d fro m it s very d en s e s tate within 10 m in or s o. We c on c lu d e d that prota m ine m u s t be
a c tively re m ove d fro m DNA on c e s per m c hro m atin enter s the egg’s c ytopla sm, in agree m ent with the work of other s( R uiz-Lara et al., 1996 ).
T he Transition Proteins TP1 and TP2
A nu m ber of group s have s tu d ie d the ability of the tran s ition protein s TP1 an d TP2 to
c on
d en s
e DNA in vitro ( C aron et al., 2001;K un d u an d Rao, 1995, 1996;L eve s que et al., 1998;M eetei et al., 2002;S ato et al., 1999). However, interpretation o
f their re s ult s wa s ha m pere d by the for m ation of DNA –p rotein aggregate s. We repeate d the s e experi m ent s on s ingle DNA m ole c ule s, a dd itionally s tu d yin
g the bin d ing kineti cs of Syrian ha ms ter TP1 an d TP2 ( B rewer et al., 2002 ). In c ontra s t to the m ea s ure m ent s of K un d u an d Rao (1995, 1996)an d M eetei et al. (2002), we foun d that bot
h TP1 an d TP2 c on d en s e d DNA with rate s s
i m ilar to the prota m ine s, P1 an d P2. Mea s ure m ent s of the DNA c on d en s ation rate u s ing a 25-re s i d ue pepti d e s equen c e c orre s pon d ing to the C-ter m inal of TP2’s bin d ing d o m ain were i d enti c al to the DNA c on d en s ation rate u s ing the entire protein an d allowe d u s to d eter m ine that thi s portion of the protein wa s re s pon s ible for c on d en s ing DNA.
E xperi m ent s perfor m e d by Z hao et al. (2001, 2004a,b) s howe d that kno c kout m i c e
la c king either TP1 or TP2 re m aine d fertile. They hypothe s ize d that ea c h protein c oul d
c o m pen s ate if the other wa s ab s ent an
d that ther
e wa s a re d un d an c y in the fun c tion
of the s e protein s. K no c kout m i c e la c king both protein s were s terile, an d c hro m atin
c on
d en s ation wa s abnor m al in all s per m ati ds (F igur
e 5.1,s tep 8) an d m any late
s per m ati ds s howe d DNA s tran d break s. The tran s ition protein s are require d for nor m al
c hro m atin c on
d en s ation in m a mm al s in or d er to m ini m iz
e the nu m ber o
f DNA s tran d break s an d m aintain the integrity of the geno m e. Intere s tingly, hi s tone s were s till
d i s pla c
e d fro m nu c leo s o m e s d uring s per m iogene s i s for m i c e la c king both TP1 an d
TP2. It i s s till not un d er s too d how hi s tone s are d i s pla c e d fro m nu c leo s o m e s d uring
s per m iogene s i s.
S haping of the Sperm Head and the Role of the Manchette
T he m e c hani sm for s haping the s per m hea d d uring s per m iogene s i s–re d u c ing c ell
volu m e by a fa c tor of 40 an d altering it s m orphology –ha s long been a c ontrover s ial
s ubje c t. Stu d ie s of s per m c ell d evelop m ent via m i c ro sc opy by K ier s zenbau m an d other s
S ingle Molecule Studies of Chromatin Structure and Dynamics 151 (C ole et al., 1988;K ier s zenbau m et al., 2003;K ier s zenbau m an d Tre s, 2004;M ei s tri c h
et al., 1990;R u ss ell et al., 1991) have hypothe s ize d that the m an c hette, a s leeve-like
c olle c tion of m i c rotubule s exterior to the s per m c ell, i s re s pon s ible for c hanging the c ell’s s hape
d uring s per m iogen
e s i s( F igure 5.1 ). However, F aw c ett et al. (1971)argue d that
the s per m hea d s hape i s a d ire c t re s ult of the c on d en s ation an d c o m pa c tion of the s per m geno m e. They note d that if the m an c hette were re s pon s ible for the large variation in
s per m hea d s hape s, then there woul d be equally large variation s in m an c hette m i c rotubule arrange m ent between s pe c ie s, whi c h ha d not been ob s erve d. They al s o argue d that the
c ir c ular s hape of the m an c hette c oul
d not pro d u c
e the high d egree o
f ? attenin
g ob s erve d in m o s t m a mm alian s per m, parallel to the long axi s of the c ell. D ooher an d Bennett (1973)c on c lu d e d that the s hape of the s per m nu c leu s i s d ue to a s pe c ie s-s pe c i?c lo c ation, atta c
h m ent an d c on d en s ation of c hro m o s o m e s within the nu c leu s. Single m ole c ule exper
i m ent s will be i m portant for un d er s tan d ing if bio m e c hani c al for c e s play a role in both repa c kaging the s per m geno m e an d s haping the s per m hea d d uring s per m iogene s i s. P osttranscriptional Modi? cations of Sperm Nuclear Proteins
P ho s phorylation –d epho s phorylation c y c le s are thought to regulate the c orre c t bin d ing
of s per m nu c lear protein s to DNA a s well a s to initiate DNA c on d en s ation d uring
s per m iogene s i s( D’O cc hio et al., 2007 ). A nu m ber of d ifferent lab s have reporte d that P1, P2, an d TP2 are all s trongly pho s phorylate d in vivo an d in vitro ( G reen et al., 1994;L eve s que
et al., 1998;M eetei et al., 2002), however, it i s not c lear what fun c tion thi s s erve s. M eetei
et al. (2002)have propo s e d that the pho s phorylation of TP2 inhibit s it s ability to c on d en s e DNA, allowing it to bin d opti m ally, while s ub s equent d epho s phorylation initiate s DNA
c on
d en s ation. Pho s phorylation of th
e prota m ine s o cc ur s at s erine an d threonine re s i d ue s an d i s thought to en s ure the proper for m ation o
f DNA –p rota m ine toroi ds( L ewi s et al., 2003 ).
T he internal s tru c ture of DNA –p rota m ine toroi ds, although never d ire c tly ob s erve d, ha s been hypothe s ize d to c on s i s t of a s erie s of loop s of DNA that are s ta c ke d on top of ea c h other ( H u d et al., 1995 ). Di s ul?d e bon ds for m between c y s teine re s i d ue s in a d ja c ent m a mm alian prota m ine m ole c ule s( R auka s an d Mikel s aar, 1999 ) after toroi d for m ation an d m ay greatly in c rea s e the toroi d’s s tru c tural s tability. It i s un c lear whether d i s ul?d e bon ds for m only between prota m ine m ole c ule s within the s a m e loop of DNA, or if they c an al s o for m between prota m ine m ole c ule s on a d ja c ent loop s of DNA, ? r m ly lo c king the toroi d together. Z in c i s e ss ential for m ale fertility ( A bba s i et al., 1980;D in c er an d Oz, 1990).
