17. Nguyen, V. Q., Co, C. & Li, J. J. Cyclin-dependent kinases prevent DNA re-replication through

multiple mechanisms. Nature 411, 1068–1073 (2001).

18. Labib, K., Diffley, J. F. X. & Kearsey, S. E. G1-phase and B-type cyclins exclude the DNA-replication

factor Mcm4 from the nucleus. Nature Cell Biol. 1, 415–422 (1999).

19. Nguyen, V. Q., Co, C., Irie, K. & Li, J. J. Clb/Cdc28 kinases promote nuclear export of the replication

initiator proteins Mcm2–7. Curr. Biol. 10, 195–205 (2000).

20. Elsasser, S., Chi, Y., Yang, P. & Campbell, J. L. Phosphorylation controls timing of Cdc6p destruction:

A biochemical analysis. Mol. Biol. Cell 10, 3263–3277 (1999).

21. Drury, L. S., Perkins, G. & Diffley, J. F. X. The cyclin-dependent kinase Cdc28p regulates distinct

modes of Cdc6p proteolysis during the budding yeast cell cycle. Curr. Biol. 10, 231–240 (2000).

22. Calzada, A., Sanchez, M., Sanchez, E. & Bueno, A. The stability of the Cdc6 protein is regulated by

cyclin-dependent kinase/cyclin B complexes in Saccharomyces cerevisiae. J. Biol. Chem. 275,

9734–9741 (2000).

23. Din, S., Brill, S. J., Fairman, M. P. & Stillman, B. Cell-cycle-regulated phosphorylation of DNA

replication factor A from human and yeast cells. Genes Dev. 4, 968–977 (1990).

24. Foiani, M., Liberi, G., Lucchini, G. & Plevani, P. Cell cycle-dependent phosphorylation and

dephosphorylation of the yeast DNA polymerase a-primase B subunit. Mol. Cell. Biol. 15, 883–891

(1995).

25. Pearson, R. B. & Kemp, B. E. Protein kinase phosphorylation site sequences and consensus specificity

motifs: Tabulations. Methods Enzymol. 200, 62–81 (1991).

26. Pondaven, P., Meijer, L. & Beach, D. Activation of M-phase-specific histone H1 kinase by modification

of the phosphorylation of its p34cdc2 and cyclin components. Genes Dev. 4, 9–17 (1990).

27. Schwob, E., Bohm, T., Mendenhall, M. D. & Nasmyth, K. The B-type cyclin kinase inhibitor p40SIC1

controls the G1 to S transition in S. cerevisiae. Cell 79, 233–244 (1994).

28. Christianson, T. W., Sikorski, R. S., Dante, M., Shero, J. H. & Hieter, P. Multifunctional yeast high-

copy-number shuttle vectors. Gene 110, 119–122 (1992).

29. Sikorski, R. S. & Hieter, P. A system of shuttle vectors and yeast host strains designed for efficient

manipulation of DNA in Saccharomyces cerevisiae. Genetics 122, 19–27 (1989).

30. Zachariae, W. et al. Mass spectrometric analysis of the anaphase-promoting complex from yeast:

Identification of a subunit related to cullins. Science 279, 1216–1219 (1998).

Supplementary Information accompanies the paper on Nature’s website

(http://www.nature.com).

AcknowledgementsWe thank T. Iida, J. F. X. Diffley, K. Labib, H. Wong, S. Elledge, K. Sugimoto andH. Masukata for strains and plasmids; T. Kishi and H. Seino for PSTAIRE antibody anddiscussions; P. Russell for information regarding the unpublished results; and J. F. X.Diffley, K. Labib, H. Masukata, H. Takisawa and A. Sugino for critical reading of themanuscript. This study is partially supported by grants-in-aid from the Ministry ofEducation, Culture, Sports, Science and Technology, Japan, to H.A. H.M. was apostdoctoral fellow in the National Institute of Genetics.

Competing interests statement

The authors declare that they have no competing financial interests.

Correspondence and requests for materials should be addressed to H.A.

(e-mail: hiaraki@lab.nig.ac.jp).

..............................................................

An exonucleolytic activity of humanapurinic/apyrimidinic endonucleaseon 3 0 mispaired DNAKai-Ming Chou & Yung-Chi Cheng

Department of Pharmacology, Yale University School of Medicine, 333 CedarStreet, Sterling Hall of Medicine B-315, New Haven, Connecticut 06520, USA.............................................................................................................................................................................

Human apurinic/apyrimidinic endonuclease (APE1) is an essen-tial enzyme in DNA base excision repair that cuts the DNAbackbone immediately adjacent to the 5 0 side of abasic sites tofacilitate repair synthesis by DNA polymerase b (ref. 1). Micelacking the murine homologue of APE1 die at an early embryonicstage2. Here we report that APE1 has a DNA exonuclease activityon mismatched deoxyribonucleotides at the 3 0 termini of nickedor gapped DNA molecules. The efficiency of this activity isinversely proportional to the gap size in DNA. In a base excisionrepair system reconstituted in vitro, the rejoining of nicked

mismatched DNA depended on the presence of APE1, indicatingthat APE1 may increase the fidelity of base excision repair and

may represent a new 3 0 mispaired DNA repair mechanism. The

exonuclease activity of APE1 can remove the anti-HIV nucleoside

analogues 3 0 -azido-3 0 -deoxythymidine and 2 0 ,3 0 -didehydro-2 0 ,3 0 -dideoxythymidine from DNA, suggesting that APE1 might

have an impact on the therapeutic index of antiviral compounds

in this category.

The DNA base excision repair (BER)3 system contributes tomaintenance of the genetic integrity of the cell. BER is initiated by

the action of DNA glycosylases on damaged bases generating

apurinic/apyrimidinic (AP) sites3. These non-instructional lesions,

which block replication and cause cell death4, can also be generatedby non-enzymatic hydrolytic depurination. APE1 is the key enzyme

that repairs AP sites through endonuclease action immediately

adjacent to the 5 0 side of AP sites, generating 5 0 deoxyribose-5-phosphate and nucleotide 3 0 hydroxyl-terminal residues1.

Two pathways have been described for completing the BER

process: long-patch and short-patch repair3. In short-patch repair,

which is considered to be the dominant pathway for BER, DNApolymerase b inserts a nucleotide to fill in the gap and releases 5 0

deoxyribose-5-phosphate through its intrinsic lyase activity5; DNA

ligase I (ref. 3) or DNA ligase III/XRCCI (ref. 3) then seals the nick.

However, DNA polymerase b lacks a proofreading 3 0 ! 5 0 exo-nuclease activity6 and is prone to error, misincorporating 1 nucleo-

tide in 4,000 (ref. 3). The misincorporation of nucleotides during

DNA replication or repair processes results in mutagenesis and

carcinogenesis7. The relatively low fidelity of DNA polymerase b,combined with a conservative estimate of 20,000 AP sites produced

per cell per day3, can result potentially in several mutations per day

per genome.

In addition to its endonuclease activity, APE1 has 3 0 ! 5 0 DNAexonuclease, 3 0 phosphatase, 3 0 phosphodiesterase and RNaseH

activities. The 3 0 ! 5 0 exonuclease was considered to be insigni-

ficant biologically, because it is 2–4 orders of magnitude less activethan the AP endonuclease8,9. However, we previously identified10

APE1 as the principal exonuclease that removes the stereochemi-

cally unnatural L-configuration anticancer nucleoside analogue, b-

L-dioxolane-cytidine (L-OddC, BCH-4556; Troxacitabine)11, andother L-configuration nucleoside analogues from the 3 0 terminus

of DNA in leukaemic cell nuclei.

To examine the effect of base–base hydrogen bonding on the

exonuclease activity of APE1, we carried out a time course experi-ment using various combinations of recessed DNA with a 3 0 mispair

as substrates (Fig. 1a; and Supplementary Information Fig. 1b, d).

The initial rates of the 3 0 mispair removal are summarized in Table 1.

APE1 removed 3 0 mismatched nucleotides at least 50 times moreefficiently than those matched correctly. The same result was

observed with APE1 purified from the nuclei of acute lymphocytic

leukaemia cells10.As APE1 has been proposed to have a key role in coordinating the

serial steps of BER12,13, we studied whether the APE1 exonuclease

activity might have a role in correcting mispairs introduced by the

error-prone DNA polymerase b (ref. 3) during BER by usingdouble-stranded DNA oligonucleotides containing a mispair at

the 3 0 terminus of a single-strand break as substrates. The mis-

matched nucleotide was removed more efficiently from nicked

DNA than from recessed DNA (Fig. 1b; and Supplementary Infor-mation Fig. 1c, e). More notably, the reaction initial rates of APE1

exonuclease showed a significant preference (160-fold) for mis-

paired substrates than for matched nucleotides (Table 1).

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The 3 0 mispair exonuclease activity and the endonuclease activityon furan DNA (an abasic DNA analogue) substrate chromato-graphically co-eluted during APE1 purification, which, togetherwith the finding that the double site-directed APE1 mutant lacksboth AP endonuclease and exonuclease activity, confirmed thatthese two activities are properties of APE1 (Supplementary Infor-mation Figs 2 and 3).

Previous studies have indicated that APE1 requires a minimum offour base pairs 5 0 to the AP site, and three base pairs 3 0 to the site,for endonuclease activity1. Thus, the difference in mismatchednucleotide removal efficiencies between the nicked and recessedDNA might be due to differences in the stability of APE1–DNAbinding. We therefore tested mispairs at the 3 0 end of a nick or gapsof variable sizes as substrates. APE1 showed similar efficiency withnicked DNA and with DNA containing a single nucleotide gap, andshowed the lowest efficiency for recessed DNA (Fig. 2; and Sup-plementary Information Fig. 4). The efficiency of 3 0 mispairremoval decreased as the gap size increased. By contrast, DNAsubstrates with different combinations of internal mismatches (thatis, in the absence of a nick or a gap) were not substrates of APE1(data not shown).

We used an in vitro reconstituted system to investigate further thepotential role of APE1 in correcting DNA polymerase b errorsduring BER. It has been reported that DNA ligase I is inefficient inrejoining nicked DNAwhen the nucleotide pair at the 3 0 terminus ofthe nick is mismatched14. In the absence of APE1, we observed lessthan 10% of ligated product with mispaired DNA, compared with atleast 98% of product with the correct pair at the 3 0 terminus(Fig. 3a). The addition of APE1 (up to 270 pM) had no impact onthe ligation of correctly paired DNA substrates, but increased the

efficiency of ligating the 3 0 mispaired substrates in a concentration-dependent manner (Fig. 3a). These data support a role for APE1 inremoving 3 0 mispaired nucleotides at the nick site during short-patch BER.

We used the antiviral nucleoside analogues 3 0-azido-3 0-deoxy-thymidine (AZT) and 2 0 ,3 0 -didehydro-2 0 , 3 0-dideoxythymidine(D4T) as substrates to investigate the influence of sugar geometryon the APE1 exonuclease. Because all three analogues lack a 3 0

hydroxyl group, no ligation product formed in the presence of DNAligase I alone, but the addition of APE1 removed all three analoguesfrom the 3 0 terminus of a nick (Fig. 3b). In the presence of APE1,ligated products from polymerase b and ligase I were observed inproportion to the amount of APE1 used (Fig. 3b). Repair efficiencyvaried slightly among the three nucleoside analogues, perhapsowing to the different sugar geometry.

Previous studies have indicated that both D4T and AZT areincorporated into DNA by cellular DNA polymerases15, and over-expression of DNA polymerase b enhances the cytotoxicity ofAZT16. In contrast to cytosolic exonucleases, which remove AZTand D4T poorly from DNA17, APE1 acted efficiently against bothnucleoside analogues when present in nicked DNA or gapped DNA.Given that APE1 is expressed constitutively in the cell and isdistributed primarily in the nucleus, APE1 might be involved inreducing the cytotoxicity18 and improving the therapeutic index ofanti-HIV compounds that belong to the chain terminator categorysuch as AZT and D4T.

Using an APE1 “trap”19, we examined the relative contribution ofAPE1 to the total exonucleolytic activity on 3 0 mispairs of nickedDNA in crude nuclear extracts of human cells (Fig. 4a). The trapeliminated both the 3 0 mispair exonuclease and AP endonuclease

Recessed DNA substrates

Time (min)

Pro

duct

(%)

0 5 10 15 20 25 30 350

10

20

30

40

50

60

70A/G

T/G

G/G

C/G

Nicked DNA substrates

Time (min)

Pro

duct

(%)

0 5 10 15 20 25 30 350

10

20

30

40

50

60

70

a

b A/G

T/G

G/G

C/G

Figure 1 Rate of APE1 exonucelase activity on different DNA substrates. In each reaction,

1 nM DNA substrate was incubated with purified recombinant APE1 for various amounts

of time under the standard assay conditions. For the A/G, T/G and G/G DNA substrates,

5 pM APE was used. For the C/G DNA substrate, 125 pM APE was used. The enzyme

specificity of APE1 on F15 DNA was 313. a, APE1 exonuclease activity on 3 0 mispaired

recessed DNA. b, APE1 exonuclease activity on 3 0 mispaired nicked DNA.

Table 1 Specific exonucelase activities of APE1 on different DNA substrates

Base pairs Nicked DNA Recessed DNA.............................................................................................................................................................................

A/G 10.0 4.8T/G 32.0 8.0G/G 10.4 8.0C/G 0.2 0.1.............................................................................................................................................................................

The assay conditions are the same as Fig. 1. Reaction initial rates (DNA removed in nM min-1) fromthe time course experiments (Fig. 1) were used to determine the specific activity of APE1exonuclease on different DNA substrates (DNA removed in pM per min per pM protein).

100

80

60

40

20

0Nick 1 2 3 6 Recessed

32P TG

1 2 3 6DNA substrates

Pro

duc

t (%

)

Gap sizes (no. of nucleotides)

Figure 2 Activity of APE1 mismatch repair on DNA with different gap sizes. Arrows

indicate the position and sizes of the gaps. For each reaction, APE1 (13.5 pM) was added

to different substrates (1 nM) under the standard exonuclease assay condition at 37 8C for

30 min. The efficiencies of 3 0 mispair removal of different DNA substrates were adjusted

using the efficiency of nicked DNA as a 100% standard. The specific activity of APE1 on

F15 DNA was 257.

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Figure 3 In vitro reconstitution of the BER system. a, Sequences of the nicked DNA

substrates are indicated. Lanes 1–5, T/A; lanes 6–13, T/G. DNA substrates with different

nucleoside analogues at the 3 0 termini of a nick site are indicated. Lanes 1–2, T/A; lanes

3–14, ddT/A; lanes 15–26, AZT/A; lanes 27–38, D4T/A. In lanes 10–13 of a and lanes

7–10, 11–14, 19–22, 23–26, 31–34 and 35–38 of b, increasing concentrations of

APE1 were used (0, 2.7, 13.5, 67.5 and 270 pM, respectively). The enzyme specific

activity of APE1 on the F15 DNA was 257.

Figure 4 The contribution of APE1 exonuclease to removal of DNA 3 0 mispairs in nuclear

extracts of human cells. a, Trapping 3 0 mispair exonuclease and AP endonuclease

activities in the nuclear extract. For each reaction, 1 nM 32P-labelled T/G mispaired nicked

DNA or 32P-labelled F15 DNA were incubated in the presence or absence of the trap (6.1

mM heat degradation product and 2 mg ml-1 heparin) before the addition of 62.5 ng

nuclear extract at 37 8C under the standard exonuclease assay conditions. b, Inhibition of

3 0 mispair exonuclease activity in nuclear extract by F15 DNA. For each reaction, 62.5 ng

nuclear extract, 1 nM 32P-labelled T/G mispaired nicked DNA, 1 nM 32P-labelled F15 DNA

or undamaged T15 DNA, and increasing concentrations of unlabelled F15 or T15 DNA

were incubated for 5 min at 37 8C under the standard exonuclease assay conditions.

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activities in the nuclear extract. We then carried out a competitionassay in which increasing concentrations of an oligonucleotidecontaining the AP site analogue tetrahydrofuran (F15) wereadded to a fixed concentration of a crude nuclear extract (Fig. 4b).Both the endonucleolytic action on F15 DNA and exonucleolyticaction on a 3 0-terminal mispair were monitored simultaneously. Wechose F15 DNA as a competitor because it is a specific substrate forAP endonucleases, unlike aldehydic AP sites (which are also cleavedby AP lyases such as Nth1 and OGG1; ref. 20), and it is very similaror identical to natural aldehydic AP sites. The observed rate of the 3 0

mispair removal was inversely proportional to the concentration ofF15 DNA and the rate of the AP endonuclease activity (Fig. 4b). TheF15 DNA could inhibit most 3 0 mispair removal (,50% at 500 nM),whereas an undamaged oligonucleotide (T15) with identicalsequence to the F15 DNA substrate competed very poorly (lessthan 3% at 1,000 nM). Similar results were observed when the assaywas performed using purified APE1 instead of a nuclear extract(data not shown). Given that no other human enzyme has beenshown to possess both AP-specific and exonuclease activities, andthat APE1 is responsible for more than 95% of the AP endonucleaseactivity in the cell21, these data indicate that APE1 may be respon-sible for most of the 3 0 mispair exonuclease activity in the nucleus.

We found that in the reconstituted BER system, the rejoiningreaction is dependent on the presence of APE1. In addition, thelower efficiency of DNA ligase I in rejoining mispaired DNA createsa window of time for APE1 to remove the mismatched nucleotide; asimilar phenomenon has also been observed with DNA ligase III(ref. 22). Although other 3 0 ! 5 0 DNA exonucleases such as TREX1 have been proposed to provide a proofreading function for DNApolymerase b (refs 23, 24), APE1 is conceptually more suitable forthis task as its physical interaction with DNA polymerase b hasalready been shown13. The intracellular localization of TREX 1 hasnot been determined clearly, and a study has indicated that TREX 1has only a minor preference for DNA with a 3 0 mispair comparedwith its preference for matched DNA25. In contrast, APE1 localizesin the nucleus (data not shown), where BER takes place.

These results have expanded the functional scope of APE1, andthis versatile and important enzyme may prove to be a majorcontributor to the observed high fidelity of DNA repair andreplication. Our study provides a new perspective from which toview the role of APE1 during early embryonic development. More-over, the 3 0 ! 5 0 DNA exonuclease activity of APE1 provides anappropriate pharmacological target for the development of antiviraland anticancer nucleoside analogues. A

MethodsProtein purificationFor expression and purification of the wild-type and mutant APE1 proteins, seeSupplementary Information.

Oligonucleotide substratesSequences of DNA substrates used were as follows. Tetrahydrofuran DNA (F15):

½32P�50-TGA GCA AFA ACT AGC-3

0

30-ACT CGT TAT TGA TCG-5

0

T15 DNA:½32P�5

0-TGA GCA ATA ACT AGC-3

0

30-ACT CGT TAT TGA TCG-5

0

Recessed mismatched DNA (N/G, N = A, T or G):

½32P�50-GTG GCG CGG AGA CTT AGA GAN-OH

30-CAC CGC GCC TCT GAA TCT CTG TAA ACC GCG CCC CTT AAG G-5

0

Nicked mismatched and matched DNA (N/G, N = A, T, G or C):

½32P�50-GTG GCG CGG AGA CTT AGA GANtOH PsATT TGG CGC GGG GAA TTC C-3

0

30-CAC CGC GCC TCT GAA TCT CTG TAA ACC GCG CCC CTT AAG G-5

0:

Oligonucleotides with ddT-MP, AZT-MP and D4T-MP were synthesized, and their puritywas confirmed as described10. The purified single-stranded oligonucleotides containingnucleoside analogues at the 3 0 termini (21-base) and the downstream single-stranded

oligonucleotide (19-base) were then annealed to a 40-base template to form nickeddouble-stranded DNA substrates.

Exonuclease/endonuclease assaysThe standard exonuclease/endonuclease reaction contains 1 nM 32P 5 0 -end-labelled DNAsubstrate and 20 mM Tris-HCl (pH 7.4) 2 mM MgCl2, 0.5 mM EDTA, 30 mM KCl and0.1 mg ml-1 bovine serum albumin. The reactions were stopped by adding loading solution(90% foramide, 1 mM EDTA, 0.1% xylene cyanole and 0.1% bromophenol blue), andwere heated at 80 8C for 3 min. We analysed samples by a 12.5% polyacryamide gelcontaining 8 M urea. The gel was then dried under vacuum and subjected to autoradi-ography and phosphorimaging for quantification (Biorad). We calculated the percentageof removal efficiency of APE1 exonuclease by dividing the product radioactivity by thetotal radioactivity of substrate and product. One unit of enzyme activity is defined as 1 nMof F15 DNA cleaved in 30 min at 37 8C under the assay conditions.

In vitro reconstitution of the BER systemThe reconstitution reaction was carried out under the standard exonuclease assaycondition with the supplement of 30 mM dNTP, 2 mM ATP, DNA polymerase b (0.5 nM),DNA ligase I (20 nM) and increasing concentrations of APE1. We incubated the reactionmixtures for 30 min at 37 8C. The efficiency of ligation was calculated by dividing theradioactivity of rejoined product by the total radioactivity of substrate and product.

Cell cultureWe grew HepG2 cells in RPMI-1640 medium (Sigma) supplied with 10% fetal bovineserum (FBS) at 95% humidity with 5% CO2 in air at 37 8C.

Nuclear extracts preparationCells were collected and incubated with PBS (pH 7.4) containing 0.5% Nonidet-P40 on icefor 30 min, followed by a 10-min centrifugation at 2,000g. The nuclear pellet was collectedand frozen at -70 8C. Frozen nuclei were lysed by resuspending in a 100 ml lysis buffer(10 mM Tris-HCl, pH 7.4, 4 mM EDTA, 30 mM KCl, 1% Nonidet-P40 and a 100-folddilution of protease and phosphatase inhibitor cocktails; Sigma) and incubated on ice for30 min. The debris was removed by centrifugation at 10,000g for 30 min at 4 8C. Thesupernatant was collected and the protein amount was determined by Bradford assay.

Heat degradation product of abasic DNAThe heat degradation product from a double-stranded uracil-containing DNAoligonucleotide was prepared as described19. The sequence of the DNA oligonucleotideis 5 0 -ATTCCAGAGTGTCAATAACACGGUGGACCAGTCGATCCTGGGCTGCAGGAATTC-3 0 .

Received 14 August; accepted 19 November 2001.

1. Wilson, D. M., Takeshita, M., Grollman, A. P. & Demple, B. Incision activity of human apurinic

endonuclease (Ape) at abasic site analogs in DNA. J. Biol. Chem. 270, 16002–16007 (1995).

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USA 88, 11450–11454 (1991).

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blocking deoxyribose fragments from oxidized DNA. Nucleic Acids Res. 19, 5907–5914 (1991).

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ligases. Nucleic Acids Res. 27, 4028–4033 (1999).

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protein. EMBO J. 18, 3868–3875 (1999).

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Supplementary Information accompanies the paper on Nature’s website

(http://www.nature.com).

AcknowledgementsWe thank Z. Hatahet and J. B. Sweasy for discussion; M. Kelley and B. Demple forproviding the APE clones; A. Tomkinson for human DNA ligase I; and J. Sweasy for humanDNA polymerase b.

Competing interests statement

The authors declare that they have no competing financial interests.

Correspondence and requests for materials should be addressed to Y.C.-C.

(e-mail: Cheng.lab@Yale.edu).

..............................................................

Mechanism of force generation bymyosin heads in skeletal muscleGabriella Piazzesi*, Massimo Reconditi*, Marco Linari*, Leonardo Lucii*,Yin-Biao Sun†, Theyencheri Narayanan‡, Peter Boesecke‡,Vincenzo Lombardi* & Malcolm Irving†

* Universita di Firenze, Viale G.B. Morgagni 63, I-50134 Firenze, Italy† Randall Centre, School of Biomedical Sciences, King’s College London, LondonSE1 1UL, UK‡ European Synchrotron Radiation Facility, F-38043 Grenoble Cedex, France.............................................................................................................................................................................

Muscles generate force and shortening in a cyclical interactionbetween the myosin head domains projecting from the myosinfilaments and the adjacent actin filaments. Although manyfeatures of the dynamic performance of muscle are determinedby the rates of attachment and detachment of myosin and actin1,the primary event in force generation is thought to be a confor-mational change or ‘working stroke’ in the actin-bound myosinhead2 – 8. According to this hypothesis, the working stroke is muchfaster than attachment or detachment, but can be observeddirectly in the rapid force transients that follow stepdisplacement of the filaments3. Although many studies of themechanism of muscle contraction9 – 13 have been based on thishypothesis, the alternative view—that the fast force transients arecaused by fast components of attachment and detachment14 – 17 —has not been excluded definitively. Here we show that measure-ments of the axial motions of the myosin heads at angstromresolution by a new X-ray interference technique18 rule out therapid attachment/detachment hypothesis, and provide compel-ling support for the working stroke model of force generation.

When the length of an active muscle fibre is decreased suddenly,so that each set of myosin filaments slides by a few nanometres along

the neighbouring actin filaments, the force decreases during thelength change (Fig. 1a). This reflects the undamped elasticity of themuscle fibre, and is called phase 1 of the force transient3,19. After thelength change, force recovers at about 1,000 s21 (phase 2). Our aimwas to determine whether the phase 2 force recovery is caused by aworking stroke in the actin-attached myosin heads2 – 8, or by rapiddetachment of heads followed by rapid attachment to different actinmonomers14 – 17.

We measured the axial motion of the myosin heads during theforce transients using X-ray interference between the two arrays ofmyosin heads in each filament (Fig. 1c). Each half of the myosinfilament (magenta) contains an array of 49 layers of heads (orange)with a regular spacing d (,14.5 nm). These arrays give rise to anaxial X-ray reflection called the M3, with intensity distributionsin2(49pRd)/sin2(pRd) (Fig. 1d, orange), where R is the reciprocalspace parameter. The two arrays are separated by a ‘bare zone’ oflength B (,160 nm), so that their centres are a distance L = B þ 48dapart (Fig. 1c). X-ray interference between the two arrays effectivelymultiplies the intensity distribution by cos2(pRL) (Fig. 1d,magenta). The resulting fine structure of the X-ray reflection(Fig. 1d, green) provides an extremely sensitive measure of theaxial motion of the myosin heads18.

We recorded the intensity profile of the M3 reflection in a 2-msperiod before the length step (Fig. 1a, b, T0, green), in a 100-msperiod close to the end of phase 1 (T1, red), and in a 2-ms periodnear the end of phase 2 (T2, blue). The length step caused a decreasein the relative intensity of the higher angle peak of the M3 reflection,and shifted both peaks to a higher angle (Fig. 1b). These changes arein the direction expected for a decrease of the interference distance(L) between the two arrays of actin-attached myosin heads when thefibre shortens and the actin filaments move towards the centre of themyosin filament (Fig. 1c). Most of the change in L occurred duringphase 1 of the force transient (Fig. 1b, green to red).

The changes in interference fine structure of the M3 reflectionwere expressed as the ratio of the intensity of the higher angle peak

0

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–5

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2 ms Reciprocal spacing (nm–1)1/15 1/14.5 1/14

Reciprocal spacing (nm–1)1/15 1/14.5 1/14

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nsity

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ForceT0

T1

T2

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L

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Actin

Figure 1 Changes in interference fine structure of the M3 X-ray reflection produced

by rapid shortening. a, Length change in nanometres per half-sarcomere (hs), and

force normalized by isometric force T0. Fibre cross-sectional area, 27,300 mm2; T0, 293

kN m22; sarcomere length, 2.13 mm. b, Axial X-ray intensity distribution of the M3

reflection normalized to that of the lower angle peak. Colours denote the periods T0, T1

and T2 shown in a; the total exposure times (equivalent unattenuated beam) were 52,

158, and 105 ms, respectively. c, The two arrays of myosin heads (orange) in each

myosin filament (magenta). d, Axial X-ray intensity distribution (green) calculated as the

product of the diffracted intensity from an array of 49 heads with d = 14.57 nm (orange)

and the interference function for L = 865.92 nm (magenta).

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NATURE | VOL 415 | 7 FEBRUARY 2002 | www.nature.com 659© 2002 Macmillan Magazines Ltd