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Phosphoryl transfer in Flp recombination: a template for strand transfer mechanisms
vlEWS Rl?.II GENETIC RECOMBINATIONis the production of new DNA molecules from two parental DNA molecules and involves physical rearrangement of their informational content. There are several instances in which recombination occurs; for example, during meiosis and mitosis of eukaryotic cells, during a mixed infection of plasmids carrying different genetic markers and during integration of extrachromosomal elements into the host genome. Recombination may be general, occurring between two DNA substrates that share extensive homolo~, or sitespecific, being limited to two rela~i':ely short, specific DNA targets. Genetic exchanges can also take place between partners that share little or no homology as, for instance, in DNA transposition. In addition, recombination may occur in RNA molecules. For example, removal of introns by splicing is also a recombination reaction. Recombination in proteins via peptidyl transfer has also come to light, but will not be discussed here. The fundamental chemistry of recombination is phosphoryi transfer. Fhis reaction involves the translocation of a phosphodiester within a nucleic acid chain or between two nucleic acid chains (Fig. la). During each strand. transfer reaction, the transferred phosphodiester changes one of its neighboring nucleoside partners. Conservative recombination (for example, site-specific DNA recombination) is achieved by phosphoryl transfer alone, whereas nonconservative recombination (for example, DNA transposition) requires DNA synthesis in addition. Phosphoryl transfers are not only fundamental to DNA and RNA recombinations, but also to reactions catalysed by topoisomerases, DNA and RNA polymerases, nucleotidyl transferases, kinases, ligases and nucleases. In these cases the basic chemical reaction is a nucleophilic attack on the reactive phosphate (Fig. lb). Since the number of options available to these reactions is obviously limited, many of the biological catalysts that carry them out must exhibit common mechanistic features, This review aims to highlight these similarities ,sing site-specific DNA recombination as a model.
M. Jayaramis at the Departmentof Microbiology,Universityof Texasat Austin, Austin,TX78712, USA.
78
TIBS 19 - FEBRUARY 1 9 9 4
Phosphoryl transfer in Flp recombination: a template for strand transfer mechanisms MakkuniJayaram The basic chemistry involved in DNA recombination, RNA splicing and DNA transposition is a phosphoryl transfer reaction. This review is an attempt to provoke a unified thinking on the reaction mechanisms in these nucleic acid transactions. Some of the recent results with the FIp site-specific recombinase that reveal how the chemical reactivity for recombination is derived from cooperative protein-subunit interactions on the DNA substrate are discussed. At least some of the features of FIp reaction are likely to have global implications in other DNA and RNA strand-transfer systems.
Site-specific DNA recombination Conservative site-specific DNA recombination between two doublestranded DNA substrates can be thought of as the sum of four singlestrand recombination events. The reaction is completed in four times two (or eight) transesterification steps (Fig. 2). The absence of the 2'-hydroxyl in DNA (in contrast with RNA recombination, discussed later in this review) precludes it from providing the active nucleophile for the first transesterification (strand-cleavage) step. Instead, a nucleophile derived from the recombinase enzyme breaks the phosphodiester, forming a covalent link between the DNA and the enzyme. The cleavage reveals a sugar hydroxyl that provides the active nucleop!dle for the second transesterification step (strand exchange). This general mechanism is followed by recombinases belonging to the Int and resolvase/invertase familiesL The lnt family is headed by the ir,tegrase protein of phage L, which is the recombinase responsible for phage integration into and excision from the Escherichia coil genome. The resolvase/mvertase family includes prokaryotic recombinases that resolve co-integrate intermediates formed during DNA transposition. Recombination between the duplicated copies of the transposon within the co-integrate intermediate leads to the formation of simple integrants. This family also
includes recombinases that mediate inversion of DNA segments flanked by target recombination sites. The prototype member of the resolvase/invertase family is the resolvase protein of the transposon 78. The Int and resolvase/ invertase family recombinases execute a common chemistry of recombination by utilizing rather different reactive groupsL In the Int family reactions, strand cleavage is accomplished by the active-site tyrosine; the linkage between protein and DNA is through the 3'-phosphate; and the strand exchange is initiated by the 5'-hydroxyl of the nicked partner DNA (Fig. 2). However, in the resolvase/invertase reactions, the active residue in cleavage is the activesite serine instead of tyrosine; the recombinase-DNA linkage is through the 5'-phosphate; and the 3'-hydroxyl of the nicked DNA provides the active species for strand exchange. The recombinase enzyme makes a total of four breaks and four joints and leaves no loose ends to be tied up later. During strand cleavage, the nucleophile (the Z- or 3'-hydroxyl of DNA) as well as the target diester (the phosphodiester between DNA and the active-site tyrosine or serine) required for strand union are prod~ced on both partner substrates. Thus the chemistry of strand cutting, unlike the chemistry of transposition (see below), is conducive to reciprocal strand exchange. The int family reaction proceeds via pairwise © 1994,ElsevierScienceLtd 0968--0004/94/$07.00
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FEBRUARY1994
single-strand exchanges, whereas the invertase/resolvase family reaction occurs by double-strand breakage and reunion.
(a) P
~
P
~
P,w~^.,w~ "vvvg,
P
",
Transposition DNA transposition, by phage Mu l'~r example, differs from conservative DNA recombination in that the product of the reaction contains an increased number of phosphodiesters, reflecting the duplication of a short target site and of the transposon itself. The chemistry of transpositional recombination must accommodate this feature of the reaction. Unlike strand breakage in the lnt or resolvase/invertase reactions, cleavage of Mu ends is hydrolytic (Fig. 3), producing a 5'-phosphate and a 3'hydroxyl on each donor DNA strand. The 3'-hydroxyls then carry out the nucleophilic attack on two phosphodiesters placed five base-pairs ~ p ) apart on each strand of the target DNA. Thus, two recombinant strands are formed. However, production of each recombinant strand leaves behind a 5'-phosphate and a 3'-hydros! in the donor and the target, respectively2Gig. 3). In a reaction designed not to conserve the number of phosphodiesters, reciprocal strand exchange must be avoided. To this end the transposase has evolved to produce, during donor cleavage, the active nucleophile for the subsequent step (the 3'-hydroxyl of DNA), b u t n o potential target diester (protein-DNA linkage). The strand-transfer step of transposition (attack by the 3'-hydroxyl of the donor on the target) is chemically equivalent to the strand-joining step of site-specific recombination in its phosphodiester conservation. Here, one DNA phosphodiester is broken to form another (a recombinant one). Tying up the loose DNA ends left behind by the transposase requires DNA replication initiated at the 3'hydroxyl within the target DNA and ligation to the 5'-phosphate within the donor. The mechanism of retroviral integration bears remarkable resemblance to that of Mu strand transfer 3.
Recombination half-sites: mechanism of strand cleavage and exchange Half-site substrates for recombination are simplified recombination su~ strates, originally designed for analysis of the ~. lnt reaction4. They have subsequently been successfully adapted for a system that uses FIp, the sitespecific recombinase from Saccharomyces cerevisiae 5-7. During one round of
(b) 5 ' R 1 0 - - P - - OR23'
I
O X.....
P ..... /\ R10 O
O OR2
5 ' R 1 0 - - PX + HO - - R23'
R10 . . . . .
/ \P . . . . . O
X
OR~
5'R1-- OH + XP - - OR23'
Rgure1 The chemistry of recombination. (a) Two distinct blocks of genetic information (indicated by the thick and thin straight lines) on a single nucleic acid chain or on two separate chains are joined by phosphoryl transfer. (b) The phosphate at the recombination site is subject to nucleophilic attack. The DNA chains flanking this phosphate at the 5'- and 3'sides are denoted by Rz and R2, respectively. Depending on the line of entry of the nucleophile (X), strand breakage results in the production of a 3'- or 5'-phosphate linked to the nucleophile. The corresponding complementary product is a 5'- or a 3'-hydroxyl. The nucleophile may be derived from water (OH-) as in DNA transposition, from the protein catalyst as in site-specific recombination, from an exogenous nucleoside (3'-OH of guanosine) as in self-splicing, or from within the RNA chain (2'-OH) as in group II and messenger RNA splicing isee Refs 1, 3 and 19). The 3'- and 5'-hydroxyls produced by strand cleavage can be used as nucleophiles in the strand.joining reactions. recombination, a half-site substrate undergoes exactly half the chemistry that a full-site undergoes. A half-site harbors one recombinase-binding element, one cleavage site and one strand exchange point Gig. 4). The cleavage strand contains a short segment (two or three nucleotides) of the strandexchange region (spacer), while the other strand contains a full comp lement of the spacer with a 5'-hydroxyl terminus. This end mimics the normal product of the recombinase cleavage reaction (see Fig. 2). Cleavage of the substrate results in the covalent attachment of the Flp active-site tyrosine (Tyr343)s to the 3'-phosphate and loss of the short spacer from the reaction center by diffusion, causing strand breakage to be virtually irreversible. This allows the 5'-hydro~l to initiate the strand-transfer reaction by attacking the phosphodiester between DNA and Tyr343 of Flp Gig. 4). A combination of half-site substrates and Rp mutants blocked at the strand-cleavage or the strand-exchange step ('step
arrest mutants')9j° has revealed interesting new features of the Flp active site and of the recombination mechanism. Reactions between hybrid halfsite-recombinase complexes formed by Flp and 'step-arrest' Flp mutants 11 demonstrate that an Flp monomer bound to a half-site does not cleave that substrate, but cleaves the partner substrate, bound by a second Flp monomer (trans DNA cleavage). Note that the recombination reaction conserves the total number of phosphodiester bonds and therefore must have a AG° of zero or close to zero. Hence, if DNA cleavage occurs in cis, the forward reaction (recombination) must rely on some conformational change that ensures strand joining in trans. The trans cleavage mechanism may, in principle, combine strand breakage and conformational switching into a single step, thus leading the reaction towards the recombinant path. In a chemical sense, there is no obvious advantage of one cleavage mechanism over the other. However, trans cleavage, in the context of the
79
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(b) ;lesolvase /
Int family
invertase family p
p -,~
P
P :%==. . . . . - -. . . .
p--
Cleavage
Cleavage
OH?' P
~i ........
'm
~HO
- .............
...................
~
" 1
1 Rotation/ligation
~
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partial active-site configuration of the Flp monomer (see below), may be a safety mechanism that postpones strand cleavage until the recombination complex is fully assembled.
r--OH" OH P'---'-'-'~ '" Target
~.
Px
~--
I Strand transfer I
,
Px
~-"P HO ~---~
~
HO~ P~
Px
Figure 3 DNA transposition by phage Mu. The red lines represent Mu DNA. The DNA bordered by the target phosphodiesters, Px, is shown in blue, Attack by the waterderived nucleophile OH- results in DNA breakage at the ends of Mu DNA and exposure of the 3'-hydroxyl. This nucleophile then attacks the target DNA phosphodiester to form the strand-transfer product. This intermediate of Lransposition contains the 3'-hydroxyls for initiation of DNA polymerization and the 5'-phosphates for ligation. The finished product is known as the co-integrate.
80
........ :
--
Exchange
' "~,..--P HO
o
OH P
~
P
~
Figure 2 Strand-cleavage and strand-exchange steps of conservative site-specific DNA recombination. The red and blue lines represent the DNA strands flanking the strand-exchange region (green lines). (a) In the Int family recombinases, strand cleavage is accomplished by the recombinase nucleophile (the active-site tyrosine) causing covalent linkage between protein and DNA. Cleavage exposes the DNA nucleophile (5'-OH) for the exchange reaction. The reaction is initiated by one round of reciprocal singlestrand exchanges. Branch migration of the resulting Holliday junction and a second round of single-strand exchanges complete recombination. (b) In the resolvase/invertase family, the nucleophiles in the cleavage and exchange steps ale the active-site serine and 3'OH, respectively. Recombination is accomplished by concerted double-strand breakage, relative rotation of the cleaved recombination .complex and strand joining.
............. p
In contrast with FIp-medlated DNA scission, cleavage by ?8 resolvase occurs in cis ~2, and is double stranded (Fig. 2b). A resolvase dimer occupying a full resolvase-binding (res) site cu:t:s the same DNA molecule. A 180 rotation of one half of the cleaved recombination complex relative to the other aligns the substrates for strand joining in the recombinant mode, Such a rotary mechanism to fun:.tionally exchange recombinase molecules between substrates is not possible for lnt- or FIp-induced recombination as the reaction occurs in two steps of single-strand exchanges, Notice, however, that in the resolvase context, the term cis refers to the full site bound by a protein dimer, whereas it refers to the half-site bound by a protein monomer in the Flp context l~.r'. Therefore, within the full site, cleavage of the two half sites in trans by resolvase cannot be ruled out. It is not possible to tell wIlether the exchangesite phosphodiester is cleaved by the resolvase monomer a,djacent to it (cis) or by the one bound across the strandexchange regiou from it (trans).
Conservation of reaction mechanism within the Int family? The trans mode of DNA cleavage by Flp is probably the direct consequence of the way in which the active site is assembled. Each Flp monomer harbors a part of the active site, and the functional active site is assembled from amino acid residues contributed by
more than one monomer (the shared active site). A plausible scheme for the organization of the full active site is shown in Fig. 5. The formation of an active site at the interface of two protein subunits is similar to some of the allosteric enzymes, a classic example of which is aspartate transcarbamoylase ~5. The model predicts the catalytic eorisequences of pairing a wild-type Flp monomer with an RHR triad/Tyr343 double mutant of FIp, and of pairing a single, double or triple Flp mutant within the RHR triad with a Tyr343 mutant (Fig. 5). These predictions have been verified 16. We can now envisage a scheme that neatly unifies the strand cleavage and joining steps of recombination (Fig. 6). A monomer of FIp bound to the substrate renders the exchange-site phosphate susceptible to nucleophilic attack (activation in cis). The specificity of this activation must derive directly from the specificity of FIp-DNA contacts. The partner Flp molecule then delivers Tyr343 to execute strand cleavage (nucleophilic attack in trans). The strand-transfer reaction, which is chemically analogous to the strand-cleavage reaction, also conforms to the cis activation/trans nucleophilic attack paradigm. Here, the target diester (the 3'-phosphate linked to Tyr343 as a result of strand cleavage) is activated by the same Flp monomer that mediated cis activation during the cleavage step. The 5'-DNA hydroxyl is then
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®
delivered in trans, as was the tyrosine hydroxyl during cleavage. Indeed, a substrate that mimics the + HO'ITr Y cleavage intermediate (a , { ' ~ o , p < \ half site with its exchangeAAAGATCTOH ~-~.~.,)~,~ AAAGATCTOH - site phosphate linked to ....~ . ~ AA A G Tyr343 or a short Flp peptide via Tyr343 and conFigure 4 taining a 5'-spacer hydroxyl The half-site strand transfer. The Rp recombinase-binding element is represented by the horizontal pair on the second strand, analof arrows. The nucleotide sequence of the strand-exchange region (spacer) is indicated on both ogous to the cleaved interstrands. The phosphate at the exchange site is denoted by P. Strand cleavage results in the covalent mediate in Fig. 5) underattachment of recombinase to DNA. The short (5'HOTTT3') top-strand spacer, being unstably hydrogen goes strand transfer in the bonded to the bottom strand, diffuses away from the reaction center. Phosphoryl transfer initiated by the 5'-spacer hydroxyl of the bottom strand completes recombination. Consistent with the trans cleavpresence of the Tyr343 age model (see Rg. 5 and text for details), the dmgram implies that cleavage of the scissile phosphomutant of Flp, FIp(Y343F)]7. diester is not executed by the FIp monomer occupying the binding element adjacent to it. Furthermore, FIp(Y343F), which is completely inactive in full-site recombination, executes a small amount of strand-transfer (a) (b) (c) in a half site Is. A simple explanation for this unexpected reaction is that the 5'hydroxyl of the half site can, albeit inefficie~t!y, attack the phosphodiester directly, reducing the normal two-step (breakage followed by reunion) strandtransfer reaction to a single-step (concerted break-union) reaction. The analogy to RNA self-splicing (see below) and to Mu strand transfer is obvious. R~re 5 Is the partial active site~tram nucleoThe 'shared active site' of Rp. The RHR triad and Y refer to Arg191, His305 and Arg308, philic attack model unique to Flp, or and Tyr343, respectively, of Rp and correspond to the invariant tetrad residues within the perhaps the site-specific recombinases integrase family13,14. A plausible scheme for the functional association of two FIp of the yeast family (a subfamily within monomers is shown. (a) Two wild-type monomers can assemble two active sites (RHR-Y). the Int family), or is it applicable to the (b) An RHR-mutant and a Y-mutant can yield one functional active site. (©) No active site is entire Int family? There is no direct eviformed between a wild.type Rp and an (RHR)-Y double mutant. dence yet for a shared active site or for trans DNA cleavage In the ~. integrase reaction. However, no experiment protein assembly for initiation of nor- in this view of ;ecomEination, it is the unequivocally rules out such a mech- mal full-site recombination can avoid assembly of the synaptic complex that anism. There Is no compelling reason to wasteful strand-cutting and self-ligation. triggers the strand cleavage/exchange process. believe that all Int family proteins will share a common mode of subunit interGeneral Implications action, or follow the same rule of amino Enzymes that catalyse phosphoryl acid sharing in active-site assembly. transfers can be looked upon as an However, within the reaction complex array of biological solutions to similar consisting of two DNA substrates and chemical problems tested and in'lfour recombinase monomers, the proved over the course of evolution. active-site configuration may be simi'ar across the Int family. An analogy could be made with a jigsaw puzzle in which Rgure 6 the same final picture can be derived Strand cleavage and strand joining follow /Y from sets of differently shaped pieces. a cis activation/trans nucleophilic attack The folding pattern of each recombii P HO model. The specific contact between an nase would determine the shape of the Rp monomer and its target ONA activates a single phosphodiester within the individual piece. But tetrameric combiDNA chain (cis activation). The active-site nations of each of these pieces are nucleophile (Tyr343) is then delivered in likely to generate catalytic sites of trans by a second Rp monomer to effect identical or highly similar configurstrand breakage, The bound Rp then actiation. For recombinase proteins that vates in cis the phosphodiester formed exist in solution and bind substrate in between DNA and t'Fosine. The 5'-hydroxyl the monomeric form, the partial active from the nicked partner DNA carries out the trans nucleophilic attack to bring site is, in principle, a desirable attriabout strand joining. bute. The requirement of a tetrameric
1® i\
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The finite chemical repertoire available to biological catalysts limits the number of such solutions. Not surprisingly, many features of the chemistry of DNA recombination can also be gleaned in RNA recombination. In group i and group 11 RNA self-splicing and nuclear mRNA splicing (reactions within a singlestranded substrate), recombination is accomplished by two consecutive transesterification reactions ~9. The nucleophile that attacks the phosphate in the first transesterification step is provided by an exogenous uucleoside (3'-hydroxyl of guanosine in group ! splicing) or by the RNA itself (2'-hydroxyl in group II and mRNA splicing). This reaction produces a 3'-hydroxyl by chain breakage, thus yielding the nucleophile for the second transesterification step that completes the splicing event. The splicing reaction has strong chemical similarity to strand breakage and strand transfer during DNA transposition (see Fig. 3). The biochemicaily appealing suggestion that RNA editing in certain parasitic protozoa might be mechanistically analo;,ous to self-splicing has been made 2°.
The catalytic properties of protein and RNA enzymes are the result of their precise secondary and tertiary structures. The normal double-heiical arrangement of DNA, on the other hand, se,.,~rely restricts folding possibilities that can generate specific binding pockets. The absence of the 2'-hydroxyl further limits its chemical reactivity. However, if binding specificity can be achieved with single-stranded DNA2=, breakage and formation of bonds may indeed follow. DNA catalysis, therefore, is certainly not heresy
Acknowledgements I thank my colleagues, past and present, who contributed to studies on the Rp recombinase. I am grateful to Belinda Gonzales (Data Processing Department, U. T. Austin) for preparation o| figures. My laboratory is supported by a grant from the NIH. Partial support was provided by the Council of Tobacco Research-USA.
.References 1Craig, N.R.(1988;Annu. Re~ Genet. 22,
77-105
2 Mizuuchi, K. and Adzuma, K. (1991) Ce//66, 129-140 3 Engelman,A. et aL (1992) Ce1167, 1211-1221
4 Nunes-Duby,S. E. et al. (1989) Cell 59, 197-206 5 Amin, A. et a/. (1991) Mol. Cell Biol. 11, 4497-4508 6 Serre, M. C. etal. (1992) J. Mol. Biol. 225, 621-642 7 Qian, X-H. etal. (1992) J. BioL Chem. 267, 7794-7805 8 Evans, B. R. et aL (1990) J. Biol. Chem. 265, 18504--18510 9 Parsons, R. L. et al. (1988) Mol. Ceil. Biol. 8, 3303-3310 10 Parsons, R. L. etal. (1990) J. Biol. Chem. 265, 4527-4533 11 Chert,J. W. etal. (1992) Cell 69. 1-20 12 Droge, P. et al. (1990) Proc, Natl Acad. ScL USA 87, 5336-5340 13 Argos, P. etal. (1986) EMBO J. 5, 433-440 14 Abremski, K. and Hoess, R. H. (1992) Prot. Eng. 5, 87-91 I 5 Wente, S. R. and Schachman, H. K. (1987) Proc. Natl Acad. Sci. USA 84, 31-35 16 Chen,J. W. et al. (1993) J. Biol. Chem. 268, 14417-14425 17 Pan, H. and Sadowski, P. D. (1992) J. Biol. Chem. 267, 12397-12399 18 Serre, M. C. etal. (1993) J. Biol. Chem. 268, 455-463 19 Cech, T. R. (1990) Annu. Rev. Biochem. 59, 543-568 20 Cech,T. R. (1992) Cell, 64, 667--669 2I ElUngton,A. D. and Szostak,J. W. (1992) Nature. 355, 850-852
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M. Jayaramis at the Departmentof Microbiology,Universityof Texasat Austin, Austin,TX78712, USA.
78
TIBS 19 - FEBRUARY 1 9 9 4
Phosphoryl transfer in Flp recombination: a template for strand transfer mechanisms MakkuniJayaram The basic chemistry involved in DNA recombination, RNA splicing and DNA transposition is a phosphoryl transfer reaction. This review is an attempt to provoke a unified thinking on the reaction mechanisms in these nucleic acid transactions. Some of the recent results with the FIp site-specific recombinase that reveal how the chemical reactivity for recombination is derived from cooperative protein-subunit interactions on the DNA substrate are discussed. At least some of the features of FIp reaction are likely to have global implications in other DNA and RNA strand-transfer systems.
Site-specific DNA recombination Conservative site-specific DNA recombination between two doublestranded DNA substrates can be thought of as the sum of four singlestrand recombination events. The reaction is completed in four times two (or eight) transesterification steps (Fig. 2). The absence of the 2'-hydroxyl in DNA (in contrast with RNA recombination, discussed later in this review) precludes it from providing the active nucleophile for the first transesterification (strand-cleavage) step. Instead, a nucleophile derived from the recombinase enzyme breaks the phosphodiester, forming a covalent link between the DNA and the enzyme. The cleavage reveals a sugar hydroxyl that provides the active nucleop!dle for the second transesterification step (strand exchange). This general mechanism is followed by recombinases belonging to the Int and resolvase/invertase familiesL The lnt family is headed by the ir,tegrase protein of phage L, which is the recombinase responsible for phage integration into and excision from the Escherichia coil genome. The resolvase/mvertase family includes prokaryotic recombinases that resolve co-integrate intermediates formed during DNA transposition. Recombination between the duplicated copies of the transposon within the co-integrate intermediate leads to the formation of simple integrants. This family also
includes recombinases that mediate inversion of DNA segments flanked by target recombination sites. The prototype member of the resolvase/invertase family is the resolvase protein of the transposon 78. The Int and resolvase/ invertase family recombinases execute a common chemistry of recombination by utilizing rather different reactive groupsL In the Int family reactions, strand cleavage is accomplished by the active-site tyrosine; the linkage between protein and DNA is through the 3'-phosphate; and the strand exchange is initiated by the 5'-hydroxyl of the nicked partner DNA (Fig. 2). However, in the resolvase/invertase reactions, the active residue in cleavage is the activesite serine instead of tyrosine; the recombinase-DNA linkage is through the 5'-phosphate; and the 3'-hydroxyl of the nicked DNA provides the active species for strand exchange. The recombinase enzyme makes a total of four breaks and four joints and leaves no loose ends to be tied up later. During strand cleavage, the nucleophile (the Z- or 3'-hydroxyl of DNA) as well as the target diester (the phosphodiester between DNA and the active-site tyrosine or serine) required for strand union are prod~ced on both partner substrates. Thus the chemistry of strand cutting, unlike the chemistry of transposition (see below), is conducive to reciprocal strand exchange. The int family reaction proceeds via pairwise © 1994,ElsevierScienceLtd 0968--0004/94/$07.00
TIBS 1 9 -
REVIEWS
FEBRUARY1994
single-strand exchanges, whereas the invertase/resolvase family reaction occurs by double-strand breakage and reunion.
(a) P
~
P
~
P,w~^.,w~ "vvvg,
P
",
Transposition DNA transposition, by phage Mu l'~r example, differs from conservative DNA recombination in that the product of the reaction contains an increased number of phosphodiesters, reflecting the duplication of a short target site and of the transposon itself. The chemistry of transpositional recombination must accommodate this feature of the reaction. Unlike strand breakage in the lnt or resolvase/invertase reactions, cleavage of Mu ends is hydrolytic (Fig. 3), producing a 5'-phosphate and a 3'hydroxyl on each donor DNA strand. The 3'-hydroxyls then carry out the nucleophilic attack on two phosphodiesters placed five base-pairs ~ p ) apart on each strand of the target DNA. Thus, two recombinant strands are formed. However, production of each recombinant strand leaves behind a 5'-phosphate and a 3'-hydros! in the donor and the target, respectively2Gig. 3). In a reaction designed not to conserve the number of phosphodiesters, reciprocal strand exchange must be avoided. To this end the transposase has evolved to produce, during donor cleavage, the active nucleophile for the subsequent step (the 3'-hydroxyl of DNA), b u t n o potential target diester (protein-DNA linkage). The strand-transfer step of transposition (attack by the 3'-hydroxyl of the donor on the target) is chemically equivalent to the strand-joining step of site-specific recombination in its phosphodiester conservation. Here, one DNA phosphodiester is broken to form another (a recombinant one). Tying up the loose DNA ends left behind by the transposase requires DNA replication initiated at the 3'hydroxyl within the target DNA and ligation to the 5'-phosphate within the donor. The mechanism of retroviral integration bears remarkable resemblance to that of Mu strand transfer 3.
Recombination half-sites: mechanism of strand cleavage and exchange Half-site substrates for recombination are simplified recombination su~ strates, originally designed for analysis of the ~. lnt reaction4. They have subsequently been successfully adapted for a system that uses FIp, the sitespecific recombinase from Saccharomyces cerevisiae 5-7. During one round of
(b) 5 ' R 1 0 - - P - - OR23'
I
O X.....
P ..... /\ R10 O
O OR2
5 ' R 1 0 - - PX + HO - - R23'
R10 . . . . .
/ \P . . . . . O
X
OR~
5'R1-- OH + XP - - OR23'
Rgure1 The chemistry of recombination. (a) Two distinct blocks of genetic information (indicated by the thick and thin straight lines) on a single nucleic acid chain or on two separate chains are joined by phosphoryl transfer. (b) The phosphate at the recombination site is subject to nucleophilic attack. The DNA chains flanking this phosphate at the 5'- and 3'sides are denoted by Rz and R2, respectively. Depending on the line of entry of the nucleophile (X), strand breakage results in the production of a 3'- or 5'-phosphate linked to the nucleophile. The corresponding complementary product is a 5'- or a 3'-hydroxyl. The nucleophile may be derived from water (OH-) as in DNA transposition, from the protein catalyst as in site-specific recombination, from an exogenous nucleoside (3'-OH of guanosine) as in self-splicing, or from within the RNA chain (2'-OH) as in group II and messenger RNA splicing isee Refs 1, 3 and 19). The 3'- and 5'-hydroxyls produced by strand cleavage can be used as nucleophiles in the strand.joining reactions. recombination, a half-site substrate undergoes exactly half the chemistry that a full-site undergoes. A half-site harbors one recombinase-binding element, one cleavage site and one strand exchange point Gig. 4). The cleavage strand contains a short segment (two or three nucleotides) of the strandexchange region (spacer), while the other strand contains a full comp lement of the spacer with a 5'-hydroxyl terminus. This end mimics the normal product of the recombinase cleavage reaction (see Fig. 2). Cleavage of the substrate results in the covalent attachment of the Flp active-site tyrosine (Tyr343)s to the 3'-phosphate and loss of the short spacer from the reaction center by diffusion, causing strand breakage to be virtually irreversible. This allows the 5'-hydro~l to initiate the strand-transfer reaction by attacking the phosphodiester between DNA and Tyr343 of Flp Gig. 4). A combination of half-site substrates and Rp mutants blocked at the strand-cleavage or the strand-exchange step ('step
arrest mutants')9j° has revealed interesting new features of the Flp active site and of the recombination mechanism. Reactions between hybrid halfsite-recombinase complexes formed by Flp and 'step-arrest' Flp mutants 11 demonstrate that an Flp monomer bound to a half-site does not cleave that substrate, but cleaves the partner substrate, bound by a second Flp monomer (trans DNA cleavage). Note that the recombination reaction conserves the total number of phosphodiester bonds and therefore must have a AG° of zero or close to zero. Hence, if DNA cleavage occurs in cis, the forward reaction (recombination) must rely on some conformational change that ensures strand joining in trans. The trans cleavage mechanism may, in principle, combine strand breakage and conformational switching into a single step, thus leading the reaction towards the recombinant path. In a chemical sense, there is no obvious advantage of one cleavage mechanism over the other. However, trans cleavage, in the context of the
79
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(b) ;lesolvase /
Int family
invertase family p
p -,~
P
P :%==. . . . . - -. . . .
p--
Cleavage
Cleavage
OH?' P
~i ........
'm
~HO
- .............
...................
~
" 1
1 Rotation/ligation
~
p ..............
partial active-site configuration of the Flp monomer (see below), may be a safety mechanism that postpones strand cleavage until the recombination complex is fully assembled.
r--OH" OH P'---'-'-'~ '" Target
~.
Px
~--
I Strand transfer I
,
Px
~-"P HO ~---~
~
HO~ P~
Px
Figure 3 DNA transposition by phage Mu. The red lines represent Mu DNA. The DNA bordered by the target phosphodiesters, Px, is shown in blue, Attack by the waterderived nucleophile OH- results in DNA breakage at the ends of Mu DNA and exposure of the 3'-hydroxyl. This nucleophile then attacks the target DNA phosphodiester to form the strand-transfer product. This intermediate of Lransposition contains the 3'-hydroxyls for initiation of DNA polymerization and the 5'-phosphates for ligation. The finished product is known as the co-integrate.
80
........ :
--
Exchange
' "~,..--P HO
o
OH P
~
P
~
Figure 2 Strand-cleavage and strand-exchange steps of conservative site-specific DNA recombination. The red and blue lines represent the DNA strands flanking the strand-exchange region (green lines). (a) In the Int family recombinases, strand cleavage is accomplished by the recombinase nucleophile (the active-site tyrosine) causing covalent linkage between protein and DNA. Cleavage exposes the DNA nucleophile (5'-OH) for the exchange reaction. The reaction is initiated by one round of reciprocal singlestrand exchanges. Branch migration of the resulting Holliday junction and a second round of single-strand exchanges complete recombination. (b) In the resolvase/invertase family, the nucleophiles in the cleavage and exchange steps ale the active-site serine and 3'OH, respectively. Recombination is accomplished by concerted double-strand breakage, relative rotation of the cleaved recombination .complex and strand joining.
............. p
In contrast with FIp-medlated DNA scission, cleavage by ?8 resolvase occurs in cis ~2, and is double stranded (Fig. 2b). A resolvase dimer occupying a full resolvase-binding (res) site cu:t:s the same DNA molecule. A 180 rotation of one half of the cleaved recombination complex relative to the other aligns the substrates for strand joining in the recombinant mode, Such a rotary mechanism to fun:.tionally exchange recombinase molecules between substrates is not possible for lnt- or FIp-induced recombination as the reaction occurs in two steps of single-strand exchanges, Notice, however, that in the resolvase context, the term cis refers to the full site bound by a protein dimer, whereas it refers to the half-site bound by a protein monomer in the Flp context l~.r'. Therefore, within the full site, cleavage of the two half sites in trans by resolvase cannot be ruled out. It is not possible to tell wIlether the exchangesite phosphodiester is cleaved by the resolvase monomer a,djacent to it (cis) or by the one bound across the strandexchange regiou from it (trans).
Conservation of reaction mechanism within the Int family? The trans mode of DNA cleavage by Flp is probably the direct consequence of the way in which the active site is assembled. Each Flp monomer harbors a part of the active site, and the functional active site is assembled from amino acid residues contributed by
more than one monomer (the shared active site). A plausible scheme for the organization of the full active site is shown in Fig. 5. The formation of an active site at the interface of two protein subunits is similar to some of the allosteric enzymes, a classic example of which is aspartate transcarbamoylase ~5. The model predicts the catalytic eorisequences of pairing a wild-type Flp monomer with an RHR triad/Tyr343 double mutant of FIp, and of pairing a single, double or triple Flp mutant within the RHR triad with a Tyr343 mutant (Fig. 5). These predictions have been verified 16. We can now envisage a scheme that neatly unifies the strand cleavage and joining steps of recombination (Fig. 6). A monomer of FIp bound to the substrate renders the exchange-site phosphate susceptible to nucleophilic attack (activation in cis). The specificity of this activation must derive directly from the specificity of FIp-DNA contacts. The partner Flp molecule then delivers Tyr343 to execute strand cleavage (nucleophilic attack in trans). The strand-transfer reaction, which is chemically analogous to the strand-cleavage reaction, also conforms to the cis activation/trans nucleophilic attack paradigm. Here, the target diester (the 3'-phosphate linked to Tyr343 as a result of strand cleavage) is activated by the same Flp monomer that mediated cis activation during the cleavage step. The 5'-DNA hydroxyl is then
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delivered in trans, as was the tyrosine hydroxyl during cleavage. Indeed, a substrate that mimics the + HO'ITr Y cleavage intermediate (a , { ' ~ o , p < \ half site with its exchangeAAAGATCTOH ~-~.~.,)~,~ AAAGATCTOH - site phosphate linked to ....~ . ~ AA A G Tyr343 or a short Flp peptide via Tyr343 and conFigure 4 taining a 5'-spacer hydroxyl The half-site strand transfer. The Rp recombinase-binding element is represented by the horizontal pair on the second strand, analof arrows. The nucleotide sequence of the strand-exchange region (spacer) is indicated on both ogous to the cleaved interstrands. The phosphate at the exchange site is denoted by P. Strand cleavage results in the covalent mediate in Fig. 5) underattachment of recombinase to DNA. The short (5'HOTTT3') top-strand spacer, being unstably hydrogen goes strand transfer in the bonded to the bottom strand, diffuses away from the reaction center. Phosphoryl transfer initiated by the 5'-spacer hydroxyl of the bottom strand completes recombination. Consistent with the trans cleavpresence of the Tyr343 age model (see Rg. 5 and text for details), the dmgram implies that cleavage of the scissile phosphomutant of Flp, FIp(Y343F)]7. diester is not executed by the FIp monomer occupying the binding element adjacent to it. Furthermore, FIp(Y343F), which is completely inactive in full-site recombination, executes a small amount of strand-transfer (a) (b) (c) in a half site Is. A simple explanation for this unexpected reaction is that the 5'hydroxyl of the half site can, albeit inefficie~t!y, attack the phosphodiester directly, reducing the normal two-step (breakage followed by reunion) strandtransfer reaction to a single-step (concerted break-union) reaction. The analogy to RNA self-splicing (see below) and to Mu strand transfer is obvious. R~re 5 Is the partial active site~tram nucleoThe 'shared active site' of Rp. The RHR triad and Y refer to Arg191, His305 and Arg308, philic attack model unique to Flp, or and Tyr343, respectively, of Rp and correspond to the invariant tetrad residues within the perhaps the site-specific recombinases integrase family13,14. A plausible scheme for the functional association of two FIp of the yeast family (a subfamily within monomers is shown. (a) Two wild-type monomers can assemble two active sites (RHR-Y). the Int family), or is it applicable to the (b) An RHR-mutant and a Y-mutant can yield one functional active site. (©) No active site is entire Int family? There is no direct eviformed between a wild.type Rp and an (RHR)-Y double mutant. dence yet for a shared active site or for trans DNA cleavage In the ~. integrase reaction. However, no experiment protein assembly for initiation of nor- in this view of ;ecomEination, it is the unequivocally rules out such a mech- mal full-site recombination can avoid assembly of the synaptic complex that anism. There Is no compelling reason to wasteful strand-cutting and self-ligation. triggers the strand cleavage/exchange process. believe that all Int family proteins will share a common mode of subunit interGeneral Implications action, or follow the same rule of amino Enzymes that catalyse phosphoryl acid sharing in active-site assembly. transfers can be looked upon as an However, within the reaction complex array of biological solutions to similar consisting of two DNA substrates and chemical problems tested and in'lfour recombinase monomers, the proved over the course of evolution. active-site configuration may be simi'ar across the Int family. An analogy could be made with a jigsaw puzzle in which Rgure 6 the same final picture can be derived Strand cleavage and strand joining follow /Y from sets of differently shaped pieces. a cis activation/trans nucleophilic attack The folding pattern of each recombii P HO model. The specific contact between an nase would determine the shape of the Rp monomer and its target ONA activates a single phosphodiester within the individual piece. But tetrameric combiDNA chain (cis activation). The active-site nations of each of these pieces are nucleophile (Tyr343) is then delivered in likely to generate catalytic sites of trans by a second Rp monomer to effect identical or highly similar configurstrand breakage, The bound Rp then actiation. For recombinase proteins that vates in cis the phosphodiester formed exist in solution and bind substrate in between DNA and t'Fosine. The 5'-hydroxyl the monomeric form, the partial active from the nicked partner DNA carries out the trans nucleophilic attack to bring site is, in principle, a desirable attriabout strand joining. bute. The requirement of a tetrameric
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The finite chemical repertoire available to biological catalysts limits the number of such solutions. Not surprisingly, many features of the chemistry of DNA recombination can also be gleaned in RNA recombination. In group i and group 11 RNA self-splicing and nuclear mRNA splicing (reactions within a singlestranded substrate), recombination is accomplished by two consecutive transesterification reactions ~9. The nucleophile that attacks the phosphate in the first transesterification step is provided by an exogenous uucleoside (3'-hydroxyl of guanosine in group ! splicing) or by the RNA itself (2'-hydroxyl in group II and mRNA splicing). This reaction produces a 3'-hydroxyl by chain breakage, thus yielding the nucleophile for the second transesterification step that completes the splicing event. The splicing reaction has strong chemical similarity to strand breakage and strand transfer during DNA transposition (see Fig. 3). The biochemicaily appealing suggestion that RNA editing in certain parasitic protozoa might be mechanistically analo;,ous to self-splicing has been made 2°.
The catalytic properties of protein and RNA enzymes are the result of their precise secondary and tertiary structures. The normal double-heiical arrangement of DNA, on the other hand, se,.,~rely restricts folding possibilities that can generate specific binding pockets. The absence of the 2'-hydroxyl further limits its chemical reactivity. However, if binding specificity can be achieved with single-stranded DNA2=, breakage and formation of bonds may indeed follow. DNA catalysis, therefore, is certainly not heresy
Acknowledgements I thank my colleagues, past and present, who contributed to studies on the Rp recombinase. I am grateful to Belinda Gonzales (Data Processing Department, U. T. Austin) for preparation o| figures. My laboratory is supported by a grant from the NIH. Partial support was provided by the Council of Tobacco Research-USA.
.References 1Craig, N.R.(1988;Annu. Re~ Genet. 22,
77-105
2 Mizuuchi, K. and Adzuma, K. (1991) Ce//66, 129-140 3 Engelman,A. et aL (1992) Ce1167, 1211-1221
4 Nunes-Duby,S. E. et al. (1989) Cell 59, 197-206 5 Amin, A. et a/. (1991) Mol. Cell Biol. 11, 4497-4508 6 Serre, M. C. etal. (1992) J. Mol. Biol. 225, 621-642 7 Qian, X-H. etal. (1992) J. BioL Chem. 267, 7794-7805 8 Evans, B. R. et aL (1990) J. Biol. Chem. 265, 18504--18510 9 Parsons, R. L. et al. (1988) Mol. Ceil. Biol. 8, 3303-3310 10 Parsons, R. L. etal. (1990) J. Biol. Chem. 265, 4527-4533 11 Chert,J. W. etal. (1992) Cell 69. 1-20 12 Droge, P. et al. (1990) Proc, Natl Acad. ScL USA 87, 5336-5340 13 Argos, P. etal. (1986) EMBO J. 5, 433-440 14 Abremski, K. and Hoess, R. H. (1992) Prot. Eng. 5, 87-91 I 5 Wente, S. R. and Schachman, H. K. (1987) Proc. Natl Acad. Sci. USA 84, 31-35 16 Chen,J. W. et al. (1993) J. Biol. Chem. 268, 14417-14425 17 Pan, H. and Sadowski, P. D. (1992) J. Biol. Chem. 267, 12397-12399 18 Serre, M. C. etal. (1993) J. Biol. Chem. 268, 455-463 19 Cech, T. R. (1990) Annu. Rev. Biochem. 59, 543-568 20 Cech,T. R. (1992) Cell, 64, 667--669 2I ElUngton,A. D. and Szostak,J. W. (1992) Nature. 355, 850-852
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