Base Flipping

Xiaodong Cheng, Emory University, Atlanta, Georgia, USA Richard J Roberts, New England Biolabs, Beverly, Massachusetts, USA
Base flipping involves rotation of backbone bonds in double-stranded DNA to expose an out-of-stack base, which can then be a substrate for an enzyme-catalysed chemical reaction. The phenomenon is fully established for DNA methyltransferases and for several key DNA repair enzymes, and is likely to prove general for enzymes that require access to unpaired, mismatched or damaged bases.

Secondary article
Article Contents
. Introduction . Examples of Known Base-flipping Systems . Probable Base-flipping Systems . Common Themes . Mechanism . Summary

Introduction
The binding of proteins to deoxyribonucleic acid (DNA) is crucial to life. While some proteins exert their eects simply through binding interactions, other proteins both bind to DNA and catalyse chemical reactions. These include polymerases, nucleases, glycosylases, methyltransferases (MTases) and various integrases and recombinases that rearrange DNA segments. DNA binding frequently deforms the usual B-helix somewhat and bending and kinking of DNA is common. Proteins that perform chemistry on the DNA bases have a dicult accessibility problem. This problem was resolved in 1994 when a structure was reported for the ternary complex of a cytosine-C5-specic DNA MTase, M.HhaI, its DNA substrate and the methyl donor S-adenosyl-l -methionine (Klimasauskas et al., 1994). M.HhaI needs to interact with the aromatic ring of its target cytosine base, which in B-DNA is buried by base pairing and stacking interactions. Surprisingly, the enzyme does not distort the DNA in some crude fashion, but rather the target cytosine undergoes a conformational shift of 1808, swinging completely out of the helix by

torsional rotation of backbone bonds and into the activesite pocket of the enzyme (Figure 1). The rst example of base ipping had been discovered. Klimasauskas et al. (1994) proposed that other classes of DNA MTases and some DNA glycosylases might also use base ipping to gain access to DNA bases. Both predictions proved accurate. This article summarizes current knowledge about base ipping, including all systems in which it is proven to occur and some more speculative possibilities. Some authors refer to this phenomenon as nucleotide ipping (Slupphaug et al., 1996; Lau et al., 1998).

Examples of Known Base-flipping Systems

Several examples of base ipping have now been observed in cocrystal structures of DNAprotein complexes (Table 1).

HhaI and HaeIII DNA methyltransferases

DNA MTases function as monomers, methylating only one strand at a time. The structure of M.HhaI has been characterized extensively by X-ray crystallography in complexes with various forms of its DNA substrate containing an unmethylated, hemimethylated or fully methylated target, containing a mismatch (G.A, G.U or G.AP) at the target base pair, and containing a modied nucleotide (5-uoro-2-deoxycytidine, 4-thio-2-deoxycytidine or 5,6-dihydro-5-azacytidine) as the target. Most interestingly, even an apurinic/apyrimidinic (abasic or AP) site is ipped out of the DNA helix and located in the enzymes active-site pocket (OGara et al., 1998). The ipped abasic sugar adopts the same conformation as is found in the ipped-out normal substrate. A similar conformation is also observed for the ipped-out abasic nucleotide in three glycosylaseDNA complexes (see below): UDG-DNA (Parikh et al., 1998), MUG-DNA
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Figure 1 M.HhaI complexed to its substrate DNA (Protein Data Bank code 1mht).

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Table 1 Known base-ipping systems Specic protein HhaI DNA MTase HaeIII DNA MTase T4 endonuclease V Human uracil DNA glycosylase Human 3-methyladenine DNA glycosylase E. coli mismatch-specic uracil DNA glycosylase E. coli endonuclease IV Catalytic reaction Forms 5-methylcytosine in DNA Forms 5-methylcytosine in DNA Removes pyrimidine dimers from DNA Removes uracil from DNA Removes 3-methyladenine from DNA Removes uracil or thymine from DNA containing G.T or G.U Cleaves the DNA backbone 5 of AP sites Reference Klimasauskas et al. (1994) Reinisch et al. (1995) Vassylyev et al. (1995) Slupphaug et al. (1996) Lau et al. (1998) Barrett et al. (1998) Hoseld et al. (1999)

AP, apurinic/apyrimidinic; MTase, methyltransferase.

(Barrett et al., 1998) and alkyladenine glycosylase (AAG)DNA (Lau et al., 1998). In the structure of M.HaeIII bound to hemimethylated DNA, the substrate cytosine is ipped out from the DNA helix (Reinisch et al., 1995), as observed for M.HhaI. The M.HaeIII structure is, so far, unique in having some rearrangement of the bases adjacent to the ipped cytosine.

E. coli mismatch-specific uracil DNA glycosylase (MUG)

MUG removes pyrimidines by a glycolytic mechanism from mismatches arising from deamination of cytosine or 5-methylcytosine and containing uracil or thymine opposite guanine. A crystal structure has been obtained for E. coli MUG bound to DNA (Barrett et al., 1998). The structure shows great similarity to UDG, especially around the active site. In the crystal, the glycosidic bond to the uracil base has been hydrolysed and a ipped-out abasic site is left in the enzymes active site.

Human uracil DNA glycosylase (UDG)

UDG, an enzyme that functions within the base excision repair pathway, removes uracil residues from either singleor double-stranded DNA. The crystal structures of human, herpes simplex virus and Escherichia coli enzymes have been solved and reveal an extraordinarily specic binding pocket that excludes normal DNA bases and uracil within ribonucleic acid (RNA) from binding. These interactions could not occur if the uracil was positioned inside a B-DNA helix. Base ipping was conrmed by the description of cocrystal structures for wild-type and two catalytically impaired mutant human UDGs complexed with a uracil-containing double-stranded DNA (Slupphaug et al., 1996; Parikh et al., 1998). The cocrystal structures show that the uracil and deoxyribose are rotated 1808 from their starting structure within DNA. Even though the glycosidic bond was cleaved, the uracil remained in the binding pocket of both the wild-type UDG and a double mutant UDG, while Leu272 penetrates into the DNA helix and occupies the space left by the ipped nucleotide. Parikh et al. (1998) also envisioned that the missing leucine side-chain of the L272A enzyme does not aect ipping (although the rate of ipping could have been aected). The mutant enzyme cleaved the glycosidic bond, freed the cleaved uracil and then rebound the AP-containing DNA prior to crystallization.

Human 3-methyladenine DNA glycosylase

The human 3-methyladenine DNA glycosylase, alternatively named alkyladenine glycosylase (AAG), removes 3methyladenine and a wide variety of other damaged bases from DNA. Lau et al. (1998) reported a crystal structure of AAG complexed to DNA containing a pyrrolidine abasic nucleotide, which is a potent inhibitor of excision-repair glycosylases. The AAG protein is a single domain structure, and the abasic pyrrolidine nucleotide is ipped into the enzymes active site (Figure 2).

T4 endonuclease V
T4 endonuclease V is a DNA glycosylase/AP lyase that can initiate repair of cis-syn cyclobutane pyrimidine dimers in DNA, cleaving the glycosidic bond of the 5 pyrimidine and then cleaving the phosphodiester backbone. T4 endonuclease V kinks the dimer-containing DNA, at an angle of approximately 608 (Vassylyev et al., 1995). Surprisingly, endonuclease V does not ip out the damaged bases, but rather it moves the nucleotide opposite the 5 pyrimidine of the dimer into a binding pocket on the surface of the enzyme (Figure 3). The ipped adenine is sandwiched between two layers of protein atoms, which are arranged in parallel with the base plane. Vassylyev et al. (1995) suggested that the adenine was stabilized by van der Waals

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Base Flipping

carry out a nucleophilic attack on C1 of the 5 pyrimidine of the dimer.

Probable Base-flipping Systems

Several crystal structures of DNA-binding proteins strongly imply that base ipping may be involved, because modelling of normal B-DNA into these structures fails to bring the target base close to the active site unless the base is ipped. Although denitive proof of base ipping for these enzymes (Table 2) awaits successful cocrystallization studies, their concave active-site pockets within deep clefts would require a simple base ipping in the DNA or a tremendous conformational change in protein.
Figure 2 AAG complexed to DNA containing a pyrrolidine abasic nucleotide (Protein Data Bank code 1bnk).

The amino-methyltransferases M.TaqI, M.PvuII and M.DpnII

Another class of DNA MTases, the amino-MTases, methylate the exocyclic amino groups of adenine (N6) or cytosine (N4). None of these amino-MTases have been structurally characterized in complex with DNA, but adenine-N6-specic M.TaqI and M.DpnII and cytosineN4-specic M.PvuII have a catalytic-domain structure with a concave active-site pocket very similar to that of M.HhaI. When these structures are modelled with normal B-DNA, the target base is far away from S-adenosyl-l methionine, unless the base is ipped out of the helix. Thus, the amino-MTases probably ip out their target nucleotides. In the adenine-N6-specic MTase, M.EcoRI, biochemical evidence in favour of base ipping has been obtained using a uorescence-based assay to detect conformational alterations in DNA induced by protein binding. This method, which uses 2-aminopurine to replace the substrate adenine in DNA, may have general applications to probe potential base ipping in other systems. Thus, when M.HhaI and M.TaqI bind to DNA containing 2-aminopurine as the target base within their recognition sequences, there is a great enhancement of the uorescence intensity, consistent with base ipping. Biochemical evidence in favour of base ipping has also been developed for another adenine-N6-specic MTase, M.EcoRV, that enhanced binding takes place when the target adenine is replaced by a modied base that weakens base pairing. These results parallel those found for M.HhaI. In all of these studies it is believed that destabilization of the base pair leads to an increased rate of base ipping and the concomitant appearance of enhanced binding.

Figure 3 T4 endonuclease V complexed to DNA containing a thymine cyclobutane dimer (Protein Data Bank code 1vas).

interactions. These interactions do not provide specic contacts that allow unambiguous recognition of the base. Biochemical evidence shows that the glycosylase activity is unaected by the nature of the base opposite the 5 lesion. The key feature associated with endonuclease V base ipping is that the hole in DNA, created by movement of the base, is lled by the enzyme inserting its active-site amino acids into that hole. Thus, through the change in the structure of the DNA, the enzyme is correctly positioned to

E. coli DNA photolyase

DNA photolyase also appears to incorporate base ipping in its mechanism of action. E. coli photolyase uses a blue3

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Table 2 Probable base-ipping systems Specic protein TaqI DNA MTase PvuII DNA MTase DpnII DNA MTase E. coli photolyase E. coli endonuclease III E. coli mutY catalytic core E. coli 3-methyladenine DNA glycosylase II E. coli exonuclease III Human AP endonuclease T4 b-glucosyltransferase E. coli Ada O6-methylguanine DNA MTase Catalytic reaction Forms N6-methyladenine in DNA Forms N4-methylcytosine in DNA Forms N6-methyladenine in DNA Converts pyrimidine dimers to TT Removes pyrimidine radiolysis products from DNA Removes adenine from mispairs with 8-oxoguanine and guanine Removes 3-methyladenine from DNA Cleaves 5 to AP sites Cleaves 5 to AP sites Transfers glucose residues to T4 DNA Removes methyl from O6-methyl-guanosine

light harvesting chromophore 5,10-methenyl tetrahydrofolylpolyglutamate (MTHF) to absorb a photon and transfer the excitation energy to a catalytic chromophore, avinadenine dinucleotide (FAD). The enzyme subsequently transfers an electron from FADH 2 to the cyclobutane pyrimidine dimer to catalyse its ssion, yielding the two original pyrimidines. Examination of the solvent-accessible surface of the photolyase revealed that the FAD cofactor is accessible to the pyrimidine dimer only by way of a cavity in the enzyme. The dimensions of the putative binding-site cavity are sucient to bind the bases of the dimer but to exclude the intradimer phosphate. The cavity is lined with polar amino acids on one side and hydrophobic amino acids on the other. The asymmetric polarity of the cavity matches well with the asymmetric polarity of the pyrimidine dimer, in which the cyclobutane ring is hydrophobic and the opposite edges of the thymine bases have nitrogens and oxygens capable of forming hydrogen bonds.

The DNA glycosylase MutY excises adenine from mispairs with guanine and 8-oxoguanine. The MutY catalytic core domain structure conserves the bilobal architecture of endonuclease III: the two helical domains form a positively charged groove with the adenine-specic pocket in their interface. This suggests that mutY also interacts with a ipped adenine. E. coli 3-methyladenine DNA glycosylase II (AlkA) eciently removes 3-methyladenine, 7-methylguanine, 3methylguanine, O2-methylcytosine and O2-methylthymine from double-stranded DNAs. The crystal structure of the free enzyme shows that AlkA is composed of three approximately equal-sized domains. Domain 1 has the topology and shape of the TATA-binding protein and appears to serve as a platform for the other two domains. The combination of domains 2 and 3 have the same topology and fold as E. coli endonuclease III and the mutY catalytic core, and thus AlkA probably also ips bases.

The helix hairpin helix (HhH) DNA glycosylase superfamily

E. coli endonuclease III, mutY and 3-methyladenine DNA glycosylase II are members of the HhH DNA glycosylase superfamily. The structure of endonuclease III showed that this enzyme possesses an elongated bilobal structure with a deep cleft separating two distinct domains. The cleft separating the two helical domains can accommodate Bform DNA, and thus it has been suggested that both domains are involved in DNA binding. Substrate binding was investigated by soaking the enzyme crystals in thymine glycol, a known inhibitor of the glycosylase activity. The thymine glycol-binding site was located within a waterlled pocket in the cleft. Thus, the damaged base is suggested to be ipped extrahelical and inserted into this pocket for catalysis to occur.
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E. coli exonuclease III and human AP endonuclease (HAP1)

E. coli exonuclease III and human HAP1 are the hydrolytic AP endonucleases that cleave the phosphodiester backbone 5 to the AP site leaving a 3-hydroxyl nucleotide and deoxyribose 5-phosphate as termini. The overall structures of the two enzymes are signicantly similar to bovine DNAase I; they consist of two b sheets, anked by a helices forming a four layered ab-sandwich motif. A ternary complex of E. coli exonuclease III with Mn2 1 and 2deoxycytidine 5-monophosphate (dCMP) has the dCMP bound at one end of the ab sandwich, a region of the enzyme that contains a high concentration of basic residues. The corresponding region of DNAase I interacts with DNA. A suggested mechanism for AP-site binding by HAP1 involves the recognition of the deoxyribose moiety in a

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Base Flipping

spontaneous extrahelical conformation; however, recent work has shown this suggestion to be incorrect, as the ring structure of an abasic site is not a critical element in target recognition. Alternatively, the nucleotide opposite the AP site might be ipped into a pocket on the surface of the protein. If this does occur, then exonuclease III would resemble T4 endonuclease V. However, as discussed for M.HhaI and three DNA glycosylases, an abasic sugar could also be actively ipped out.

T4 b-glucosyltransferase
Bacteriophage T4 contains 5-hydroxymethylcytosine in its DNA, which is further modied by the addition of glucose. This reaction is catalysed by the b-glucosyltransferase, which transfers glucose from uridine diphosphoglucose to 5-hydroxymethylcytosine. The structure of the enzyme has been solved both in the presence and absence of the glucose donor, uridine diphosphoglucose (UDP-glucose). The structure comprises two domains of similar topology which are separated by a cleft that contains a possible site for duplex DNA binding. The catalytic site can be inferred from the position of the UDP-glucose that is deeply bound in a pocket at the bottom of the cleft. When normal BDNA is modelled into the structure, the glucose donor and the target base are widely separated; however, base ipping of the 5-hydroxymethylcytosine would nicely juxtapose the two interacting moieties.

be discovered. Of particular interest will be studies of a family of newly discovered mammalian methyl-CpGdinucleotide DNA-binding proteins and the various mismatch repair systems, such as the methyl-directed system in E. coli, which might also use base ipping. One might anticipate the occurrence of base ipping in other processes where it might prove advantageous. One such process is the local unwinding in DNA that is needed during the initiation of transcription or replication. With one base ipped out of helix and the concomitant loss of both base-pairing and stacking interactions, it would be easier to disrupt an adjacent base pair and so begin the unzipping of the helix. While this is not the only mechanism that could be envisioned to initiate strand separation, it is a plausible possibility. All enzymes that perform chemistry on RNA must also be considered candidates to employ base ipping. The enzymes that modify transfer RNA (tRNA) or ribosomal RNA (rRNA) within base-paired regions, either by methylation or by introducing more complex side-chains, might use this mechanism. Another likely candidate is double-stranded RNA adenosine deaminase, which has been implicated in RNA editing. This enzyme converts adenosine residues in RNA into inosine.

Mechanism
At present we have little hard data about the mechanism of base ipping, although two basic models have been proposed: active or passive. Base ipping could be an active process in which the base is rst pushed out of the helix by appropriate amino acid(s) on the protein, and then pulled into the active-site pocket of the enzyme, where it remains trapped during the reaction. This push and pull mechanism has been suggested for human UDG (Slupphaug et al., 1996), although Parikh et al. (1998) suggested neither push nor pull is essential for ipping. Stivers (1998) suggested an enzyme-assisted active mechanism for uracil ipping by E. coli UGA. Panayotou et al. (1998) suggested a passive mechanism for viral UDG in which the enzyme traps a transient extrahelical uracil in the free substrate. Further work is needed to settle the issue. Cheng and Blumenthal (1996) have proposed a threestep active model pathway for the DNA MTases, in which the enzymes rst recognize the target base pair within the recognition sequence and increase the interstrand phosphatephosphate distance by phosphate binding primarily on one strand. Next, base ipping is initiated by protein invasion of the widened DNA, and nally the ipped base is trapped in a binding pocket. It would be useful to have the structure of a base-ipping protein in its starting state, complexed to nonspecic DNA, which might reect protein binding during its search for a recognition site.
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E. coli ada O6-methylguanine DNA methyltransferase

O6-methylguanine DNA MTase is a suicidal DNA-repair protein that removes the dangerous methyl group from the promutagenic lesion O6-methylguanine and transfers it to a cysteine in the protein. The resulting self-methylation of the active-site cysteine renders the protein inactive. A structure has been reported for the protein without DNA, but when B-DNA is modelled into the structure, a target away from the buried base would be more than 20 A catalytic cysteine that is the acceptor for the methyl group. While a model was proposed in which the protein underwent a drastic conformational distortion to bind DNA, it seems more likely that ipping the O6-methylguanine out of the DNA helix would solve the problem of accessibility.

Common Themes
The common theme among all current examples of base ipping is the requirement for enzymes to perform chemistry on bases normally embedded in a B-DNA helix. It is likely that as more proteins that perform chemistry on DNA are examined, further examples of base ipping will

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Base Flipping

The passive model makes use of the normal breathing of DNA, during which the bases naturally spend some time in a spontaneously ipped-out position. It is this transient conformation in DNA that is proposed to be recognized and trapped by the protein. Current data tend to favour the active theory. A nuclear magnetic resonance dynamics study of M.HhaI shows that the initial product of binding is a complex containing normal B-DNA. Only later does a conformational change take place that results in the ipped base. It appears that the base per se is not the target for the structural change in the DNA. Since abasic sugar is also ipped by M.HhaI, AAG, and possibly by UDG and MUG, there is clearly nothing special about the cytosine, 3methyladenine, uracil, or any of the bases, that is required for ipping. Thus, we conclude that it is the backbone that is targeted for rotation by the enzyme and the base is merely carried along with it.

Panayotou G, Brown T, Barlow T, Pearl LH and Savva R (1998) Direct measurement of the substrate preference of uracil-DNA glycosylase. Journal of Biological Chemistry 273: 4550. Parikh SS, Mol CD, Slupphang G et al. (1998) Base excision repair initiation revealed by crystal structures and binding kinetics of human uracil-DNA glycosylase with DNA. EMBO Journal 17: 52145226. Reinisch KM, Chen L, Verdine GL and Lipscomb WN (1995) The crystal structure of HaeIII methyltransferase covalently complexed to DNA: an extrahelical cytosine and rearranged base pairing. Cell 82: 143153. Slupphaug G, Mol CD, Kavli B et al. (1996) A nucleotide-ipping mechanism from the structure of human uracil-DNA glycosylase bound to DNA. Nature 384: 8792. Stivers JT (1998) Kinetic mechanism of damage site recognition and uracil ipping by Escherichia coli uracil DNA glycosylase. Biochemistry 38: 952963. Vassylyev DG, Kashiwagi T, Mikami Y et al. (1995) Atomic model of a pyrimidine dimer excision repair enzyme complexed with a DNA substrate: structural basis for damaged DNA recognition. Cell 83: 773782.

Summary
The phenomenon of base ipping is appearing in a number of systems following its initial discovery in the DNA MTases, and many additional enzymes are likely candidates for use of this novel mechanism. Unfortunately, detailed mechanistic information is lacking, and it remains to be proven whether base ipping is an active process in which the protein rotates the base out of the helix, or a passive one in which the protein binds to a transiently ipped base. If the process is active, then one might expect that the push take place not on the base, but rather on the sugarphosphate backbone.

Further Reading
Allan BW and Reich NO (1996) Targeted base stacking disruption by the EcoRI DNA methyltransferase. Biochemistry 35: 1475714762. Cal S and Connolly BA (1997) DNA distortion and base ipping by the EcoRV DNA methyltransferase. Journal of Biological Chemistry 272: 490496. Erzberger JP, Barsky D, Scharer OD, Colvin ME and Wilson DM III (1998) Elements in abasic site recognition by the major human and Escherichia coli apurinic/apyrimidinic endonucleases. Nucleic Acids Research 26: 27712778. Gong W, OGara M, Blumenthal RM and Cheng X (1997) Structure of PvuII DNA-(cytosine N4) methyltransferase, an example of domain permutation and protein fold assignment. Nucleic Acids Research 25: 27022715. Gorman MA, Morera S, Rothwell DG et al. (1997) The crystal structure of the human DNA repair endonuclease HAP1 suggests the recognition of extra-helical deoxyribose at DNA abasic sites. EMBO Journal 16: 65486558. Klimasauskas S, Szyperski T, Serva S and Wuthrich K (1998) Dynamic modes of the ipped-out cytosine during HhaI methyltransferaseDNA interactions in solution. EMBO Journal 17: 317324. Mol CD, Kuo C-F, Thayer MM, Cunningham RP and Tainer JA (1995) Structure and function of the multifunctional DNA-repair enzyme exonuclease III. Nature 374: 381386. Moore MH, Gulbis JM, Dodson EJ, Demple B and Moody PCE (1994) Crystal structure of a suicidal DNA repair protein: the Ada O6methylguanine-DNA methyltransferase from E coli. EMBO Journal 13: 14951501. Park HW, Kim ST, Sancar A and Deisenhofer J (1995) Crystal structure of DNA photolyase from Escherichia coli. Science 268: 18661872. Vrielink A, Ruger W, Driessen HPC and Freemont PS (1994) Crystal structure of the DNA modifying enzyme beta-glucosyltransferase in the presence and absence of the substrate uridine diphosphoglucose. EMBO Journal 13: 34133422. Yamagata Y, Kato M, Odawara K et al. (1996) Three-dimensional structure of a DNA repair enzyme, 3-methyladenine DNA glycosylase II, from Escherichia coli. Cell 86: 311319.

References
Barrett TE, Savva R, Panayotou G et al. (1998) Crystal structure of a G:T/U mismatch-specic DNA glycosylase: mismatch recognition by complementary-strand interactions. Cell 92: 117129. Cheng X and Blumenthal RM (1996) Finding a basis for ipping bases. Structure 4: 639645. Hoseld DJ, Guan Y, Haas BJ, Cunningham RP and Tainer JA (1999) Structure of the DNA repair enzyme endonuclease IV and its DNA complex: double-nucleotide ipping at abasic sites and three-metalion catalysis. Cell 98: 397408. Klimasauskas S, Kumar S, Roberts RJ and Cheng X (1994) HhaI methyltransferase ips its target base out of the DNA helix. Cell 76: 357369. Lau AY, Scharet OD, Samson L, Verdine GL and Ellenberger T (1998) Crystal structure of a human alkylbase-DNA repair enzyme complexed to DNA: mechanisms for nucleotide ipping and base excision. Cell 95: 249258. OGara M, Horton RJ, Roberts RJ and Cheng X (1998) Structures of HhaI methyltransferase complexed with substrates containing mismatches at the target base. Nature Structural Biology 5: 872877.

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