INTRODUCTION Genetic switches: mechanismand function R.H.A, PLASTERK DMSION OF MOLECUlaRBIOLOGY,NETHERLANDSC~CER INSTITUTE, PLESmaNIA~ 121, 1066 CXAMs~m~, T.E Nt"mE~C~'~DS.
DNA rearrangements can be responsible for relapsing fevers caused by unicellular parasites, can lead to cancer or AIDS as the result of retrovirus integration, can give pretty colour pattems to flowers and can change the sex of yeasts. No living organism seems to exist without them, just as no word processing progtmn exists without the options to move, delete and copy text. This issue of T/G brings together vertebrates, yeasts, nematodes, protozoa and bacteria. When these creatures are discussed together in reviews on biology, it is usually because of an apparent case of homology, that is, similarity thought to result from common ancestry. Here, on the other hand, we see analogy: they all do something similar for the same purpose. They all rearrange DNA (perhaps to switch gene expression, or to resolve products of DNA replication), not accidentally, but as a result of the programmed ability to switch. The distinction between programmed and incidental DNA rearrangements is somewhat arbitrary, as it all depends on the point of view. Integration of temperate bacteriophages ~ or Mu is a prerequisite for the establishment of immunity, and therefore a bonafide genetic switch from the point of view of the phage; for the host cell it is incidental. For transposable elements and retroviruses, transposition or integration is a programmed part of the lifestyle of the element, but as far as the host is concerned the event may appear random and unprogrammed. The points of view of parasite and host can coincide: transposons are thought to be 'selfish DNA', performing no necessary function for the host organism, but the somatic excision of a plant transposon can make the petals particularly attractive, and perhaps confer a selective advantage on the plant. Though lambdoid phages, retroviruses and other transposons are not reviewed in any detail in this T/G issue on genetic switches, a summary of the reaction mechanism of transposition is included in Fig. 1.
In this introduction, I will briefly describe and compare the mechanisms that are used for genetic switches, and then discuss their function: what are the apparent advantages and disadvantages, and why do most metazoan animals seem to switch genes only in their immune system and in no other tissue?
Mechanisms A previous classification of switching mechanisms focused on the outcome of a switch, and is based on the comparison of the situation before and after the switch1: does a gene transpose, replicate, invert with respect to flanking genes, and so on. The classification set out here emphasizes the molecular mechanism of the switch. Figure 1 shows a very simplified version of the reactions discussed below.
Homologous recombination and gene conversion The initial event in the homologous systems is a cut in DNA. Subsequently one or both free ends search the genome for homologous sequences, and initiate an interaction with them. Although these systems essentially employ general recombination for the exchange of DNA sequences, the result can nevertheless be site- or regionspecific. Mating-type switching in the yeast Saccharomyces cerevisiae (see Haber 2) and the establishment of diversity in the chicken B cell compartment (see Thompson3) both involve a sequence- or regionspecific cut in DNA that is followed by what seems to be general double-strand break-repair. (In the chicken immune system the doublestrand break has not actually been proven to initiate the process, but is inferred from analysis of the products.) It is the specific cut that makes a general recombination system a useful vehicle for a sitespecific switch. The process achieves additional specificity by 'donor preference' in repair of the break made by the HO endonuclease in TIG DECEMBER
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yeast: the cell preferentially uses as repair template the cassette that was not active before the initiation of the switch. The mechanism for this is still unknown. Antigenic variation in the African trypanosome also involves homologous events (see Van der Ploeg et al.4). In this case it is not known whether a sequence-specific DNA break initiates the recombination. It is conceivable that there is random exchange of homologous sequences between homologous genes and pseudogenes, in which case all the organism has invested in antigenic variation is the maintenance of a large repertoire of silent genes and an active general recombination machinery. High switching frequencies have been observed in vivo however 5, which suggests that some site- or region-specificity must be involved. The situation for the spirochaete Borrelia hermsii is similar to that in the eukaryotic trypanosomes 6,7.
Site-specific recombination At the other extreme are the precise site-specific recombination systems. Van de Putte and Goosen discuss bacterial inversion#, while Stark et al. include these in a wider discussion of site-specific recombination 9. Several, but not all, pathogenic bacteria employ similar mechanisms to switch genes (either with the aim of escaping the host immune response, or to switch on different adhesion structures on the cell surface; see Robertson and MeyerT). Here, synapsis precedes all cuts in DNA, and is brought about by recognition of specific DNA sequences by specialized proteins, followed by protein-protein recognition between the partners. Once a functional synapse is formed, the DNA is cut, and strands can be exchanged. It is essential for this mechanism that the proteins bound to the two recombination sites can sense each other's presence, and then cut the DNA in a concerted fashion. All site-specific
BNTRODUCTION (a)
FIGlil
o.g
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donor cut
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, • ....
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51 ~
DNA double strand break
9
- N template l
. . . . .
~
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-
-
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(c) subunit e.~hange
to protein
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signal joint RAG1? RAG2? nuclease terminal transferase gp SCID?
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A 'tourist's version' of the reaction mechanisms that are discussed in more detail later in this issue and in the literature cited. Many interesting aspects of these reactions are simplified or omitted from the figure for reasons of clarity. The reactions are described in the text (with the exception of transposition). (a) Transposition. Transposition is included in this figure to allow comparison with the other mechanisms, and also because (as argued in the text) its exclusion from this issue on programmed DNA rearrangements is somewhat arbitrary. The scheme is based primarily on what is known about the bacterial transposons Mu (Ref. 20) and Tnl0 (Ref. 21), about retrovims integration22, 23, and about the P element of the fruit fly24; in all likelihood, it applies to all of these transposons. Transposase recognizes transposon ends, and after the two ends are brought together, protein-protein interactions trigger the first chemical reaction: a nick at the 3' ends of the transposon. In concert with this, a nick can also be made in the opposite strand (e.g. in the cases of P or Tnl0), but this is not always the case (e.g. not for Mu or linear retrovirus DNA). This reaction is referred to as the donor cut. The second chemical reaction is a concerted nucleophilic attack of both 3' hydroxyl groups at the ends of the transposon on two phosphodiester bonds in opposite strands of the target DNA. This is referred to as strand transfer. Note that the high energy of the bonds in the target DNA is almost certainly directly transferred to the new DNA bonds, without a covalent intermediate between protein and DNA. Nothing is known about the number of transposase molecules involved in the reaction; only two are shown here. General cellular repair mechanisms are thought to remove loose ends, fill up single-stranded regions, and ligate single-strand nicks. The result is precise integration of the transposon, with a duplication at the target as a result of the 5' stagger in the target in the strand transfer reaction. (b) Homologous recombination and gene conversion. The initial event is a double-strand break in DNA, in the region that will receive new sequences. Site- or region-specificity in the switching reaction depends primarily on this break, and for the yeast HO enzyme it is clear that the break is precise and sequence-specific. A general double-strand break-repair process follows: a 5' to 3' exonuclease removes sequences from each end, and the remaining 3' single-stranded ends search the genome for homologous partners. (c) Site-specific recombination. After site-specific binding of the enzyme to the recombination sites, a synapse is formed between two sites. The figure drawn here shows the situation for integrases and resolvases; phage ~ Int and yeast FLP proteins have a different mechanism of strand exchange (see text and the review by Stark et a/.9). In many cases accessory sites also play a role in the synaptic complex (see Stark et al.9). (d) v-J joining. Most of the enzymology here is unknown. The two recombination sites are presumably brought together by specialized enzymes. Several proteins that specifically bind to recombination sites have been isolated, but for none of them has a role in the recombination reaction been proven. Whether RAG1 and RAG2 proteins are themselves the recombinases, or are regulators of them, is still an open question. Double-strand DNA breaks are made, and probably the two inside ends are quickly ligated together in a precise fashion (signal joint). After processing of the outside ends (involving hairpin formation, trimming by exonucleases and addition by terminal transferase), the two coding ends are joined.
T1G DECEMBER1992 VOL. 8 NO. 12
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~]~NTRODUCTION
recombination mechanisms analysed to date involve a covalent intermediate between a hydroxyl residue on the recombination enzyme and phosphates in the DNA backbone; this bond conserves the high energy of the phosphodiester bond, so that subsequent ligation to 3' hydroxyls in the DNA can proceed without exogenous energy carriers such as ATP. For the invertases and resolvases, the step following strand transfer of DNA to protein is the exchange of the four partners. The four DNA ends and the proteins covalently attached to each of them form a (probably twofold) symmetric complex, held together by interactions between the proteins. One 180 ° rotation could exchange the position of the subunits in this complex. What is not yet understood in structural terms is how the free rotation can occur without dissociation of the complex. The phage ~, Int protein and the yeast FLP protein exchange strands in a different fashion: in these cases Holliday junctions are formed (four-branched DNA structures, which result because only one pair of DNA strands has been exchanged9). The last step in the reaction is the precise reverse of the first step: the phosphodiester bonds are transferred back from protein to DNA again. Since the cuts in the recombination sites are made in a staggered fashion, correct base pairing between the protruding singlestranded ends of the partners is necessary before all new phosphodiester bonds can be made. In certain cases, some phosphodiester bonds can be made without proper base pairing, for example the phage )~ Int protein can ligate the first strand in the absence of base pairing, and Tn3 resolvase has been reported to ligate substrates that have imperfect complementarity in their extended single-strand ends. Invertases show the interesting mechanistic phenomenon of 'selectivity', that is, they sense whether their two recombination sites are in the correct (in this case inverted) orientation, and will not act efficiently on direct repeats. The opposite preference is found for resolvases: these proteins are 30% identical in sequence to the inver-
tases, but will only act efficiently on direct rather than inverted repeats. The explanation for the difference lies in the role that accessory sites play in the establishment of a synaptic complex (see Stark et al.9). Those recombinases that do not depend on accessory sites for synapsis (e.g. yeast FLP, or phage P1 Cre) do not show selectivity.
v-J joining and related systems Many switching systems have not been studied in vitro in any detail, and consequently not much is known about the switching mechanism. In most of these cases all that has been done is 'book keeping' of sequences present before and after the switch. In addition, in some cases (e.g. that of V-J joining) artificial substrates were tested in vivo to determine substrate requirements, and to study how substrate structure determines product structure. The review by Gellert 1° demonstrates that this type of book keeping, in combination with careful genetic analysis, can lead to a good picture of several steps in the switching reaction. In principle, the recombination removes a stretch of DNA between the V and J segments. The intervening sequence is released as a circle; the signal joint in the circle is precise. The coding joint between the V and J segments is imprecise. Mutant analysis also points to a difference between the two types of joints: the scid mutation of the mouse does not affect the formation of the signal joint, but impairs the joining of the coding ends. The controlled imprecision at the coding joint is one of the most interesting aspects of this mechanism. The imprecision introduces an extra possibility for sequence variation, and thus enhancement of the repertoire of rearranged genes. One of the enzymes that is probably responsible for the imprecision has been identified: terminal deoxynucleotidyl transferase (TdT) was shown to be capable of adding nontemplated nucleotides at the junction. Forced activation of v - J joining in cell lines that do not express TdT results in coding joints without nontemplated sequences, and additional forced expression of TdT results in joints with these TI6 DECEMBER 1 9 9 2 VOL. 8 NO. 12
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sequences 11. A beginning has recently been made on the biochemical analysis of this remarkable switching system. An important advance is the demonstration of a double-strand DNA break that occurs in T cell receptor DNA only in cells in which switching occurs (in the thymus) 12. In DNA from wild-type animals only the broken signal ends could be found, and the corresponding coding ends were not detectable; interestingly, the coding ends could be observed in DNA isolated from scid mutants, and they have an unusual hairpin structure 13. It is possible that the scid gene product plays a role in the processing of these hairpins.
Other mechanisms Chromosome diminution in nematodes may be the first genetic switch described, but only recently has its mechanism been studied in some detail (see Tobler et al.l~). The germ-line configuration of the genome differs considerably from that of the somatic cells, and the process of removal of a considerable part of the genome is referred to as diminution. The nematode Parascaris removes 85% of its germ-line DNA (mainly repetitive sequences) in the soma. Ascaris removes 25%. Cuts are apparently made in a region-specific not a sitespecific manner and the broken ends are elongated by a telomerase. Ciliates (unicellular protozoa) show a similar phenomenon. One nucleus is kept apart for the transmission of DNA to the next generation; this DNA is not transcribed, and the nucleus is small (micronucleus). Two other nuclei (the macronuclei) contain many copies of what could be referred to as 'messenger DNA', each containing one gene. These nuclei do not contribute to the genome of subsequent generations of cells. The conversion from germ-line to somatic configuration can be quite spectacular, as illustrated by the alignment of the micronuclear and macronuclear versions of the actin I gene in the ciliate Oxytricha (see Prescottl5). Not only are the intervening sequences removed from the gene, but the order of the coding sequences after the switch is different from that before the
~'~NTRODUCTION switch. The information for unscrambling seems to reside at least partially in short stretches of homology at the recombination sites, but since these are sometimes only 3 bp long, more information be hidden somewhere to guide precise unscrambling.
must
Why they do it The advantages of DNA rearrangements as a molecular basis for" choice of cell program are obvious: they allow efficient use of the genetic material, control over the rate of switching between types, control of the repertoire, and, in many cases, reversibility. Therefore, one of the most striking aspects of DNA rearrangements is their apparent absence in most multicellular animals and plants (with the exception of the immune system). The discovery of the expression of the RAG genes (see Oettinger 16) in cells of the nervous system has led to speculations about possible roles for gene switching in the establishment of nerve cell function 17. Experimental support for this notion seemed to come from the detection of a V-J joining event in the brain of transgenic mice TM, but a recent interpretation of these experiments suggests that they may have been based on artifacts 19. At present, it seems fair to say that in all likelihood cell lineage in multicellular organisms is not determined by DNA sequence alterations. To venture a few speculations as to why that might be: (1) It may not be necessary. Several alternative modes of epigenetic inheritance may be sufficient for cell determination (phosphorylation of proteins, chromatin structure, methylation of DNA, etc.). (2) It may not be possible. Most of the switching systems that are described here involve a chanceand-selection mechanism, where many cells switch, and only the successful survivors make it. This is true for parasites that escape the immune system (illustrated by the African trypanosome, where only a few out of many millions of cells survive a fever peak, to cause the next). This is also the case for their opponents, maturing pre-B,or pre-T cells, where one successful switch activates the next step. The presence of abundant clones allows this
'wasteful' strategy. In a spatially ordered solid tissue, the option of continuing with only the productively switched cells from a random population would be more problematic. (3) DNA shuffling may be too risky. In animals, one incorrectly switched cell may be fatal for the whole organism. The only vertebrate tissue in which DNA rearrangements seem to play an integral role in cell determination, the immune system, shows this quite well: many leukemias result from incorrect programmed DNA rearrangements (v-J joining). An enhanced risk of cancer is the price leukocytes pay for the ability to change their genomes. The 'Why?' question is always dangerous in biology. The nematode Ascaris reshuffles its whole genome during development, whereas the related nematode Caenorhabditis elegans does not. The spirochaete B. hermsii can persist in the mammalian host by antigenic variation, but the highly related spirochaete B. burgdorferi seems to be doing pretty well without it (and is currently causing a major epidemic of Lyme disease in the western world). If one were not aware of Ascaris or B. hermsii, one might have wasted effort on futile arguments to explain w h y nematodes do not rearrange chromosomes, and Borrelia does not vary antigens. Likewise we should be cautious not to overinterpret the absence of DNA rearrangements in animals. We do not live in the best of all worlds, but in the world as we find it, and no laws of physics or chemistry would be broken if one day we were to find a new switching system that determines a step in animal development.
silencing of transcription (see Haber2). Studying structure still seems the best way to study function, perhaps because it is the least influenced by the bias and preconceived ideas of the researcher.
Acknowledgements I have kept literature references to a minimum by referring as much as possible to the reviews in this issue; my apologies to those whose work has not been cited directly. I thank Scan Colloms, Gloria Rudenko and Piet Borst for comments on the manuscript. Research in my lab is supported by a Pioneer grant from the Dutch Science Foundation NWO.
References 1 Borst, P. and Greaves, D.R. (1987) Science 235, 658-667 2 Haber, J.E. (1992) Trends Genet. 8, 446-452 3 Thompson, C.B. (1992) Trends Genet. 8, 416-422 4 Van der Ploeg, L.H.T., Gottesdiener, K. and Lee, M.G-S. (1992) Trends Genet. 8, 452-457 5 Turner, C.M. and BarryJ.D. (1989) Parasitology 99, 67-75 6 Plasterk, R.H.A., Simon, M.I. and Barbour, A.G. (1986) in Antigenic Variation in Infectious Diseases
7 8 9 10 11 12
13 14 15
Study structure!
16
Although the systems described in the following articles are brought together because they share a function, most of the emphasis in each of the articles is on structure and mechanism. The continuing story of mating-type regulation in yeast is a beautiful illustration of h o w detailed analysis of a mechanism, employing biochemical as well as genetic techniques, has led to new insights into other areas: the regulation of gene expression in the cell cycle, or the role of chromatin structure in local
17
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18 19 20 21
22 23 24
(Birkbeck, T.H. and Penn, C.W., eds), pp. 127-146, IRL Press Robertson, B.D. and Meyer, T.F. (1992) Trends Genet. 8, 422-427 van de Putte, P. and Goosen, N. (1992) Trends Genet. 8, 457-462 Stark, W.M., Boocock, M.R. and Sherratt, D.J. (1992) Trends Genet. 81 432-439 Gellert, M. (1992) Trends Genet. 8, 408-412 Kallenbach, S., Doyen, N., Fanton d'Andon, M. and Rougeon, F. (1992) Proc. Natl Acad. Sci. USA 89, 2799-2803 Roth, D.B. etal. (1992) Ce1169, 41-53 Roth, D.B. et al. Cell (in press) Tobler, H., Etter, A. and Mfiller, F. (1992) Trends Genet, 8, 427-432 Prescott, D.M. (1992) Trends Genet. 8, 439-445 Oettinger, M.A. (1992) Trends Genet. 8, 413-416 Chun, J.J. etaL (1991) Ce1164, 189--200 Matsuoka, M. et al. (1991) Science 254, 81-86 Abeliovich, A. et al. (1992) Science 257, 404-407 Mizuuchi, K. and Adzuma, K. (1991) Cell66, 129-140 Benjamin, H.W. and Kleckner, N. (1992) Proc. Natl Acad. Sci. USA 89, 4648-4652 Engelman, A., Mizuuchi, K. and Craigie, R. (1991) Ce1167, 1211-1221 Vink, C. et al. (1991) Nucleic Acids Res. 19, 6691-6698 Kaufman, P.D. and Rio, D.C. (1992) Cell 69, 27-39
DNA rearrangements can be responsible for relapsing fevers caused by unicellular parasites, can lead to cancer or AIDS as the result of retrovirus integration, can give pretty colour pattems to flowers and can change the sex of yeasts. No living organism seems to exist without them, just as no word processing progtmn exists without the options to move, delete and copy text. This issue of T/G brings together vertebrates, yeasts, nematodes, protozoa and bacteria. When these creatures are discussed together in reviews on biology, it is usually because of an apparent case of homology, that is, similarity thought to result from common ancestry. Here, on the other hand, we see analogy: they all do something similar for the same purpose. They all rearrange DNA (perhaps to switch gene expression, or to resolve products of DNA replication), not accidentally, but as a result of the programmed ability to switch. The distinction between programmed and incidental DNA rearrangements is somewhat arbitrary, as it all depends on the point of view. Integration of temperate bacteriophages ~ or Mu is a prerequisite for the establishment of immunity, and therefore a bonafide genetic switch from the point of view of the phage; for the host cell it is incidental. For transposable elements and retroviruses, transposition or integration is a programmed part of the lifestyle of the element, but as far as the host is concerned the event may appear random and unprogrammed. The points of view of parasite and host can coincide: transposons are thought to be 'selfish DNA', performing no necessary function for the host organism, but the somatic excision of a plant transposon can make the petals particularly attractive, and perhaps confer a selective advantage on the plant. Though lambdoid phages, retroviruses and other transposons are not reviewed in any detail in this T/G issue on genetic switches, a summary of the reaction mechanism of transposition is included in Fig. 1.
In this introduction, I will briefly describe and compare the mechanisms that are used for genetic switches, and then discuss their function: what are the apparent advantages and disadvantages, and why do most metazoan animals seem to switch genes only in their immune system and in no other tissue?
Mechanisms A previous classification of switching mechanisms focused on the outcome of a switch, and is based on the comparison of the situation before and after the switch1: does a gene transpose, replicate, invert with respect to flanking genes, and so on. The classification set out here emphasizes the molecular mechanism of the switch. Figure 1 shows a very simplified version of the reactions discussed below.
Homologous recombination and gene conversion The initial event in the homologous systems is a cut in DNA. Subsequently one or both free ends search the genome for homologous sequences, and initiate an interaction with them. Although these systems essentially employ general recombination for the exchange of DNA sequences, the result can nevertheless be site- or regionspecific. Mating-type switching in the yeast Saccharomyces cerevisiae (see Haber 2) and the establishment of diversity in the chicken B cell compartment (see Thompson3) both involve a sequence- or regionspecific cut in DNA that is followed by what seems to be general double-strand break-repair. (In the chicken immune system the doublestrand break has not actually been proven to initiate the process, but is inferred from analysis of the products.) It is the specific cut that makes a general recombination system a useful vehicle for a sitespecific switch. The process achieves additional specificity by 'donor preference' in repair of the break made by the HO endonuclease in TIG DECEMBER
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yeast: the cell preferentially uses as repair template the cassette that was not active before the initiation of the switch. The mechanism for this is still unknown. Antigenic variation in the African trypanosome also involves homologous events (see Van der Ploeg et al.4). In this case it is not known whether a sequence-specific DNA break initiates the recombination. It is conceivable that there is random exchange of homologous sequences between homologous genes and pseudogenes, in which case all the organism has invested in antigenic variation is the maintenance of a large repertoire of silent genes and an active general recombination machinery. High switching frequencies have been observed in vivo however 5, which suggests that some site- or region-specificity must be involved. The situation for the spirochaete Borrelia hermsii is similar to that in the eukaryotic trypanosomes 6,7.
Site-specific recombination At the other extreme are the precise site-specific recombination systems. Van de Putte and Goosen discuss bacterial inversion#, while Stark et al. include these in a wider discussion of site-specific recombination 9. Several, but not all, pathogenic bacteria employ similar mechanisms to switch genes (either with the aim of escaping the host immune response, or to switch on different adhesion structures on the cell surface; see Robertson and MeyerT). Here, synapsis precedes all cuts in DNA, and is brought about by recognition of specific DNA sequences by specialized proteins, followed by protein-protein recognition between the partners. Once a functional synapse is formed, the DNA is cut, and strands can be exchanged. It is essential for this mechanism that the proteins bound to the two recombination sites can sense each other's presence, and then cut the DNA in a concerted fashion. All site-specific
BNTRODUCTION (a)
FIGlil
o.g
\a, strand transfer
donor cut
5t
, • ....
31 exonuclease
51 ~
DNA double strand break
9
- N template l
. . . . .
~
r
-
-
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A 'tourist's version' of the reaction mechanisms that are discussed in more detail later in this issue and in the literature cited. Many interesting aspects of these reactions are simplified or omitted from the figure for reasons of clarity. The reactions are described in the text (with the exception of transposition). (a) Transposition. Transposition is included in this figure to allow comparison with the other mechanisms, and also because (as argued in the text) its exclusion from this issue on programmed DNA rearrangements is somewhat arbitrary. The scheme is based primarily on what is known about the bacterial transposons Mu (Ref. 20) and Tnl0 (Ref. 21), about retrovims integration22, 23, and about the P element of the fruit fly24; in all likelihood, it applies to all of these transposons. Transposase recognizes transposon ends, and after the two ends are brought together, protein-protein interactions trigger the first chemical reaction: a nick at the 3' ends of the transposon. In concert with this, a nick can also be made in the opposite strand (e.g. in the cases of P or Tnl0), but this is not always the case (e.g. not for Mu or linear retrovirus DNA). This reaction is referred to as the donor cut. The second chemical reaction is a concerted nucleophilic attack of both 3' hydroxyl groups at the ends of the transposon on two phosphodiester bonds in opposite strands of the target DNA. This is referred to as strand transfer. Note that the high energy of the bonds in the target DNA is almost certainly directly transferred to the new DNA bonds, without a covalent intermediate between protein and DNA. Nothing is known about the number of transposase molecules involved in the reaction; only two are shown here. General cellular repair mechanisms are thought to remove loose ends, fill up single-stranded regions, and ligate single-strand nicks. The result is precise integration of the transposon, with a duplication at the target as a result of the 5' stagger in the target in the strand transfer reaction. (b) Homologous recombination and gene conversion. The initial event is a double-strand break in DNA, in the region that will receive new sequences. Site- or region-specificity in the switching reaction depends primarily on this break, and for the yeast HO enzyme it is clear that the break is precise and sequence-specific. A general double-strand break-repair process follows: a 5' to 3' exonuclease removes sequences from each end, and the remaining 3' single-stranded ends search the genome for homologous partners. (c) Site-specific recombination. After site-specific binding of the enzyme to the recombination sites, a synapse is formed between two sites. The figure drawn here shows the situation for integrases and resolvases; phage ~ Int and yeast FLP proteins have a different mechanism of strand exchange (see text and the review by Stark et a/.9). In many cases accessory sites also play a role in the synaptic complex (see Stark et al.9). (d) v-J joining. Most of the enzymology here is unknown. The two recombination sites are presumably brought together by specialized enzymes. Several proteins that specifically bind to recombination sites have been isolated, but for none of them has a role in the recombination reaction been proven. Whether RAG1 and RAG2 proteins are themselves the recombinases, or are regulators of them, is still an open question. Double-strand DNA breaks are made, and probably the two inside ends are quickly ligated together in a precise fashion (signal joint). After processing of the outside ends (involving hairpin formation, trimming by exonucleases and addition by terminal transferase), the two coding ends are joined.
T1G DECEMBER1992 VOL. 8 NO. 12
~04
~]~NTRODUCTION
recombination mechanisms analysed to date involve a covalent intermediate between a hydroxyl residue on the recombination enzyme and phosphates in the DNA backbone; this bond conserves the high energy of the phosphodiester bond, so that subsequent ligation to 3' hydroxyls in the DNA can proceed without exogenous energy carriers such as ATP. For the invertases and resolvases, the step following strand transfer of DNA to protein is the exchange of the four partners. The four DNA ends and the proteins covalently attached to each of them form a (probably twofold) symmetric complex, held together by interactions between the proteins. One 180 ° rotation could exchange the position of the subunits in this complex. What is not yet understood in structural terms is how the free rotation can occur without dissociation of the complex. The phage ~, Int protein and the yeast FLP protein exchange strands in a different fashion: in these cases Holliday junctions are formed (four-branched DNA structures, which result because only one pair of DNA strands has been exchanged9). The last step in the reaction is the precise reverse of the first step: the phosphodiester bonds are transferred back from protein to DNA again. Since the cuts in the recombination sites are made in a staggered fashion, correct base pairing between the protruding singlestranded ends of the partners is necessary before all new phosphodiester bonds can be made. In certain cases, some phosphodiester bonds can be made without proper base pairing, for example the phage )~ Int protein can ligate the first strand in the absence of base pairing, and Tn3 resolvase has been reported to ligate substrates that have imperfect complementarity in their extended single-strand ends. Invertases show the interesting mechanistic phenomenon of 'selectivity', that is, they sense whether their two recombination sites are in the correct (in this case inverted) orientation, and will not act efficiently on direct repeats. The opposite preference is found for resolvases: these proteins are 30% identical in sequence to the inver-
tases, but will only act efficiently on direct rather than inverted repeats. The explanation for the difference lies in the role that accessory sites play in the establishment of a synaptic complex (see Stark et al.9). Those recombinases that do not depend on accessory sites for synapsis (e.g. yeast FLP, or phage P1 Cre) do not show selectivity.
v-J joining and related systems Many switching systems have not been studied in vitro in any detail, and consequently not much is known about the switching mechanism. In most of these cases all that has been done is 'book keeping' of sequences present before and after the switch. In addition, in some cases (e.g. that of V-J joining) artificial substrates were tested in vivo to determine substrate requirements, and to study how substrate structure determines product structure. The review by Gellert 1° demonstrates that this type of book keeping, in combination with careful genetic analysis, can lead to a good picture of several steps in the switching reaction. In principle, the recombination removes a stretch of DNA between the V and J segments. The intervening sequence is released as a circle; the signal joint in the circle is precise. The coding joint between the V and J segments is imprecise. Mutant analysis also points to a difference between the two types of joints: the scid mutation of the mouse does not affect the formation of the signal joint, but impairs the joining of the coding ends. The controlled imprecision at the coding joint is one of the most interesting aspects of this mechanism. The imprecision introduces an extra possibility for sequence variation, and thus enhancement of the repertoire of rearranged genes. One of the enzymes that is probably responsible for the imprecision has been identified: terminal deoxynucleotidyl transferase (TdT) was shown to be capable of adding nontemplated nucleotides at the junction. Forced activation of v - J joining in cell lines that do not express TdT results in coding joints without nontemplated sequences, and additional forced expression of TdT results in joints with these TI6 DECEMBER 1 9 9 2 VOL. 8 NO. 12
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sequences 11. A beginning has recently been made on the biochemical analysis of this remarkable switching system. An important advance is the demonstration of a double-strand DNA break that occurs in T cell receptor DNA only in cells in which switching occurs (in the thymus) 12. In DNA from wild-type animals only the broken signal ends could be found, and the corresponding coding ends were not detectable; interestingly, the coding ends could be observed in DNA isolated from scid mutants, and they have an unusual hairpin structure 13. It is possible that the scid gene product plays a role in the processing of these hairpins.
Other mechanisms Chromosome diminution in nematodes may be the first genetic switch described, but only recently has its mechanism been studied in some detail (see Tobler et al.l~). The germ-line configuration of the genome differs considerably from that of the somatic cells, and the process of removal of a considerable part of the genome is referred to as diminution. The nematode Parascaris removes 85% of its germ-line DNA (mainly repetitive sequences) in the soma. Ascaris removes 25%. Cuts are apparently made in a region-specific not a sitespecific manner and the broken ends are elongated by a telomerase. Ciliates (unicellular protozoa) show a similar phenomenon. One nucleus is kept apart for the transmission of DNA to the next generation; this DNA is not transcribed, and the nucleus is small (micronucleus). Two other nuclei (the macronuclei) contain many copies of what could be referred to as 'messenger DNA', each containing one gene. These nuclei do not contribute to the genome of subsequent generations of cells. The conversion from germ-line to somatic configuration can be quite spectacular, as illustrated by the alignment of the micronuclear and macronuclear versions of the actin I gene in the ciliate Oxytricha (see Prescottl5). Not only are the intervening sequences removed from the gene, but the order of the coding sequences after the switch is different from that before the
~'~NTRODUCTION switch. The information for unscrambling seems to reside at least partially in short stretches of homology at the recombination sites, but since these are sometimes only 3 bp long, more information be hidden somewhere to guide precise unscrambling.
must
Why they do it The advantages of DNA rearrangements as a molecular basis for" choice of cell program are obvious: they allow efficient use of the genetic material, control over the rate of switching between types, control of the repertoire, and, in many cases, reversibility. Therefore, one of the most striking aspects of DNA rearrangements is their apparent absence in most multicellular animals and plants (with the exception of the immune system). The discovery of the expression of the RAG genes (see Oettinger 16) in cells of the nervous system has led to speculations about possible roles for gene switching in the establishment of nerve cell function 17. Experimental support for this notion seemed to come from the detection of a V-J joining event in the brain of transgenic mice TM, but a recent interpretation of these experiments suggests that they may have been based on artifacts 19. At present, it seems fair to say that in all likelihood cell lineage in multicellular organisms is not determined by DNA sequence alterations. To venture a few speculations as to why that might be: (1) It may not be necessary. Several alternative modes of epigenetic inheritance may be sufficient for cell determination (phosphorylation of proteins, chromatin structure, methylation of DNA, etc.). (2) It may not be possible. Most of the switching systems that are described here involve a chanceand-selection mechanism, where many cells switch, and only the successful survivors make it. This is true for parasites that escape the immune system (illustrated by the African trypanosome, where only a few out of many millions of cells survive a fever peak, to cause the next). This is also the case for their opponents, maturing pre-B,or pre-T cells, where one successful switch activates the next step. The presence of abundant clones allows this
'wasteful' strategy. In a spatially ordered solid tissue, the option of continuing with only the productively switched cells from a random population would be more problematic. (3) DNA shuffling may be too risky. In animals, one incorrectly switched cell may be fatal for the whole organism. The only vertebrate tissue in which DNA rearrangements seem to play an integral role in cell determination, the immune system, shows this quite well: many leukemias result from incorrect programmed DNA rearrangements (v-J joining). An enhanced risk of cancer is the price leukocytes pay for the ability to change their genomes. The 'Why?' question is always dangerous in biology. The nematode Ascaris reshuffles its whole genome during development, whereas the related nematode Caenorhabditis elegans does not. The spirochaete B. hermsii can persist in the mammalian host by antigenic variation, but the highly related spirochaete B. burgdorferi seems to be doing pretty well without it (and is currently causing a major epidemic of Lyme disease in the western world). If one were not aware of Ascaris or B. hermsii, one might have wasted effort on futile arguments to explain w h y nematodes do not rearrange chromosomes, and Borrelia does not vary antigens. Likewise we should be cautious not to overinterpret the absence of DNA rearrangements in animals. We do not live in the best of all worlds, but in the world as we find it, and no laws of physics or chemistry would be broken if one day we were to find a new switching system that determines a step in animal development.
silencing of transcription (see Haber2). Studying structure still seems the best way to study function, perhaps because it is the least influenced by the bias and preconceived ideas of the researcher.
Acknowledgements I have kept literature references to a minimum by referring as much as possible to the reviews in this issue; my apologies to those whose work has not been cited directly. I thank Scan Colloms, Gloria Rudenko and Piet Borst for comments on the manuscript. Research in my lab is supported by a Pioneer grant from the Dutch Science Foundation NWO.
References 1 Borst, P. and Greaves, D.R. (1987) Science 235, 658-667 2 Haber, J.E. (1992) Trends Genet. 8, 446-452 3 Thompson, C.B. (1992) Trends Genet. 8, 416-422 4 Van der Ploeg, L.H.T., Gottesdiener, K. and Lee, M.G-S. (1992) Trends Genet. 8, 452-457 5 Turner, C.M. and BarryJ.D. (1989) Parasitology 99, 67-75 6 Plasterk, R.H.A., Simon, M.I. and Barbour, A.G. (1986) in Antigenic Variation in Infectious Diseases
7 8 9 10 11 12
13 14 15
Study structure!
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Although the systems described in the following articles are brought together because they share a function, most of the emphasis in each of the articles is on structure and mechanism. The continuing story of mating-type regulation in yeast is a beautiful illustration of h o w detailed analysis of a mechanism, employing biochemical as well as genetic techniques, has led to new insights into other areas: the regulation of gene expression in the cell cycle, or the role of chromatin structure in local
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18 19 20 21
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(Birkbeck, T.H. and Penn, C.W., eds), pp. 127-146, IRL Press Robertson, B.D. and Meyer, T.F. (1992) Trends Genet. 8, 422-427 van de Putte, P. and Goosen, N. (1992) Trends Genet. 8, 457-462 Stark, W.M., Boocock, M.R. and Sherratt, D.J. (1992) Trends Genet. 81 432-439 Gellert, M. (1992) Trends Genet. 8, 408-412 Kallenbach, S., Doyen, N., Fanton d'Andon, M. and Rougeon, F. (1992) Proc. Natl Acad. Sci. USA 89, 2799-2803 Roth, D.B. etal. (1992) Ce1169, 41-53 Roth, D.B. et al. Cell (in press) Tobler, H., Etter, A. and Mfiller, F. (1992) Trends Genet, 8, 427-432 Prescott, D.M. (1992) Trends Genet. 8, 439-445 Oettinger, M.A. (1992) Trends Genet. 8, 413-416 Chun, J.J. etaL (1991) Ce1164, 189--200 Matsuoka, M. et al. (1991) Science 254, 81-86 Abeliovich, A. et al. (1992) Science 257, 404-407 Mizuuchi, K. and Adzuma, K. (1991) Cell66, 129-140 Benjamin, H.W. and Kleckner, N. (1992) Proc. Natl Acad. Sci. USA 89, 4648-4652 Engelman, A., Mizuuchi, K. and Craigie, R. (1991) Ce1167, 1211-1221 Vink, C. et al. (1991) Nucleic Acids Res. 19, 6691-6698 Kaufman, P.D. and Rio, D.C. (1992) Cell 69, 27-39
