342:
the relative locations and orientations of the sites that are to be recombined, but also by the innate specificity of the site-specific system in question. Excisions and inversions occur if the recombination takes place between two sites that are found on the same molecule (intramolecular recombination), and if the sites are in the same (direct repeat) or in an opposite orientation (inverted repeat), respectively. Insertions, on the other hand, take place if the recombination occurs on sites that are situated on two different DNA molecules (intermolecular recombination), provided that at least one of these molecules is circular. Most site-specific systems are highly specialised, catalysing only one of these different types of reaction, and have evolved to ignore the sites that are in the "wrong" orientation.
338:
the "subunit rotation model" (Fig. 2). Independent of the model, DNA duplexes are situated outside of the protein complex, and large movement of the protein is needed to achieve the strand exchange. In this case the recombination sites are slightly asymmetric, which allows the enzyme to tell apart the left and right ends of the site. When generating products, left ends are always joined to the right ends of their partner sites, and vice versa. This causes different recombination hybrid sites to be reconstituted in the recombination products. Joining of left ends to left or right to right is avoided due to the asymmetric "overlap" sequence between the staggered points of top and bottom strand exchange, which is in stark contrast to the mechanism employed by tyrosine recombinases.
318:
54:(SSRs) perform rearrangements of DNA segments by recognizing and binding to short, specific DNA sequences (sites), at which they cleave the DNA backbone, exchange the two DNA helices involved, and rejoin the DNA strands. In some cases the presence of a recombinase enzyme and the recombination sites is sufficient for the reaction to proceed; in other systems a number of accessory proteins and/or accessory sites are required. Many different
328:
110:
132:
337:
It is still not entirely clear how the strand exchange occurs after the DNA has been cleaved. However, it has been shown that the strands are exchanged while covalently linked to the protein, with a resulting net rotation of 180Β°. The most quoted (but not the only) model accounting for these facts is
271:
residue conserves the energy that was expended in cleaving the DNA. Energy stored in this bond is subsequently used for the rejoining of the DNA to the corresponding deoxyribose hydroxyl group on the other DNA molecule. The entire reaction therefore proceeds without the need for external energy-rich
223:
Although the individual members of the two recombinase families can perform reactions with the same practical outcomes, the families are unrelated to each other, having different protein structures and reaction mechanisms. Unlike tyrosine recombinases, serine recombinases are highly modular, as was
92:
in length and consist of two motifs with a partial inverted-repeat symmetry, to which the recombinase binds, and which flank a central crossover sequence at which the recombination takes place. The pairs of sites between which the recombination occurs are usually identical, but there are exceptions
341:
The reaction catalysed by Cre-recombinase, for instance, may lead to excision of the DNA segment flanked by the two sites (Fig. 3A), but may also lead to integration or inversion of the orientation of the flanked DNA segment (Fig. 3B). What the outcome of the reaction will be is dictated mainly by
118:
Top: Traditional view including strand-exchange followed by branch-migration (proofreading). The mechanism occurs in the framework of a synaptic complex (1) including both DNA sites in parallel orientation. While branch-migration explains the specific homology requirements and the reversibility of
144:
Here, the synaptic complex arises from the association of pre-formed recombinase dimers with the respective target sites (CTD/NTD, C-/N-terminal domain). Like for Tyr-recombinases, each site contains two arms, each accommodating one protomer. As both arms are structured slightly differently, the
126:
This synaptic complex (1) arises from the association of two individual recombinase subunits ("protomers"; gray ovals) with the respective target site. Its formation depends on inter-protomer contacts and DNA bending, which in turn define the subunits (green) with an active role during the first
140:
Contrary to Tyr-recombinases, the four participating DNA strands are cut in synchrony at points staggered by only 2 bp (leaving little room for proofreading). Subunit-rotation (180Β°) permits the exchange of strands while covalently linked to the protein partner. The intermediate exposure of
122:
Bottom: Current view. Two simultaneous strand-swaps, each depending on the complementarity of three successive bases at (or close to) the edges of the 8-bp spacer (dashed lines indicate base-pairing). Didactic complications arise from the fact that, in this model, the synaptic complex must
163:
family. The names stem from the conserved nucleophilic amino acid residue present in each class of recombinase which is used to attack the DNA and which becomes covalently linked to it during strand exchange. The earliest identified members of the serine recombinase family were known as
232:
Recombination between two DNA sites begins by the recognition and binding of these sites β one site on each of two separate double-stranded DNA molecules, or at least two distant segments of the same molecule β by the recombinase enzyme. This is followed by
145:
subunits become conformationally tuned and thereby prepared for their respective role in the recombination cycle. Contrary to members of the Tyr-class the recombination pathway converts two different substrate sites (attP and attB) to site-hybrids
291:, cleave one DNA strand at a time at points that are staggered by 6β8bp, linking the 3' end of the strand to the hydroxyl group of the tyrosine nucleophile (Fig. 1). Strand exchange then proceeds via a crossed strand intermediate analogous to the
224:
first hinted by biochemical studies and later shown by crystallographic structures. Knowledge of these protein structures could prove useful when attempting to re-engineer recombinase proteins as tools for genetic manipulation.
401:
Bode, J; Schlake, T; asadasasada Iber, M; Schuebeler, D; Seibler, J; Snezhkov, E; Nikolaev, L (2000). "The transgeneticist's toolbox: novel methods for the targeted modification of eukaryotic genomes".
306:, but also new additions like ΟC31-, Bxb1-, and R4 integrases, cut all four DNA strands simultaneously at points that are staggered by 2 bp (Fig. 2). During cleavage, a proteinβDNA bond is formed via a
127:
crossover reaction. Both representations illustrate only one half of the respective pathway. These parts are separated by a
Holliday junction/isomerization step before the product (3) can be released.
298:
The mechanism and control of serine recombinases is much less well understood. This group of enzymes was only discovered in the mid-1990s and is still relatively small. The now classical members
237:, i.e. bringing the sites together to form the synaptic complex. It is within this synaptic complex that the strand exchange takes place, as the DNA is cleaved and rejoined by controlled
790:
Li, W.; Kamtekar, S; Xiong, Y; Sarkis, GJ; Grindley, ND; Steitz, TA (2005). "Structure of a
Synaptic Gamma Delta Resolvase Tetramer Covalently Linked to Two Cleaved DNAs".
149:. This explains the irreversible nature of this particular recombination pathway, which can only be overcome by auxiliary "recombination directionality factors" (RDFs).
283:
Although the basic chemical reaction is the same for both tyrosine and serine recombinases, there are some differences between them. Tyrosine recombinases, such as
1022:
Reed, R.R.; Grindley, N.D. (1981). "Transposon-mediated site-specific recombination in vitro: DNA cleavage and protein-DNA linkage at the recombination site".
530:
241:
reactions. During strand exchange, each double-stranded DNA molecule is cut at a fixed point within the crossover region of the recognition site, releasing a
314:
is replaced by a phosphoserine bond between a 5' phosphate at the cleavage site and the hydroxyl group of the conserved serine residue (S10 in resolvase).
1100:
Stark, M.W.; Sherratt, DJ; Boocock, MR (1989). "Site-specific recombination by Tn 3 resolvase: topological changes in the forward and reverse reactions".
376:
155:
Based on amino acid sequence homologies and mechanistic relatedness, most site-specific recombinases are grouped into one of two families: the
59:
608:
381:
210:
160:
156:
146:
119:
the process in a straightforward manner, it cannot be reconciled with the motions recombinase subunits have to undergo in three dimensions.
55:
481:
1065:
Reed, R.R.; Moser, C.D. (1984). "Resolvase-mediated recombination intermediates contain a serine residue covalently linked to DNA".
62:(RMCE), an advanced approach for the targeted introduction of transcription units into predetermined genomic loci, rely on SSRs.
684:"Cleavage of the site-specific recombination protein gamma delta resolvase: the smaller of two fragments binds DNA specifically"
577:
Nash, H. A. (1996). Site-specific recombination: integration, excision, resolution, and inversion of defined DNA segments.
1235:
1245:
1230:
1240:
561:
743:"Crystal structure of the site-specific recombinase gamma delta resolvase complexed with a 34 bp cleavage site"
371:
190:
273:
78:
1143:
Stark, W.M.; Boocock, M.R. (1994). "The
Linkage Change of a Knotting Reaction Catalysed by Tn3 Resolvase".
195:
65:
Site-specific recombination systems are highly specific, fast, and efficient, even when faced with complex
1225:
848:"Review: Site-specific recombinases: from tag-and-target- to tag-and-exchange-based genomic modifications"
277:
141:
double-strand breaks bears risks of triggering illegitimate recombination and thereby secondary reactions.
366:
361:
356:
288:
185:
35:
632:
Landy, A. (1989). "Dynamic, Structural, and
Regulatory Aspects of lambda Site-Specific Recombination".
911:
799:
695:
69:
genomes. They are employed naturally in a variety of cellular processes, including bacterial genome
311:
307:
257:
238:
169:
82:
1125:
1047:
895:
Van Duyne, G.D. (2002). "A structural view of tyrosine recombinase site-specific recombination".
877:
823:
772:
427:
299:
1201:
1160:
1117:
1082:
1039:
1004:
969:
869:
815:
764:
723:
649:
614:
604:
553:
504:
462:
419:
292:
43:
667:
Stark, W.M.; Boocock, M.R. (1995). "Topological selectivity in site-specific recombination".
1191:
1152:
1109:
1074:
1031:
996:
987:
Stark, W.M.; Boocock, M.R.; Sherratt, DJ (1992). "Catalysis by site-specific recombinases".
959:
926:
859:
807:
754:
713:
703:
641:
596:
545:
496:
482:"Site-directed genome modification: derivatives of BAL-modifying enzymes as targeting tools"
454:
411:
317:
176:
integrase (using attP/B recognition sites), differs from the now well-known enzymes such as
17:
512:
351:
284:
177:
70:
803:
699:
645:
245:
1196:
1179:
1178:
Sarkis, G.J; Murley, LL; Leschziner, AE; Boocock, MR; Stark, WM; Grindley, ND (2001).
718:
683:
600:
1219:
1113:
1035:
1000:
964:
947:
759:
742:
303:
249:
206:
42:
strand exchange takes place between segments possessing at least a certain degree of
1129:
1051:
948:"The integrase family of recombinases: organization and function of the active site"
827:
776:
431:
881:
261:
173:
94:
74:
500:
136:
Fig. 2. Ser-Recombinases: The (essentially irreversible) subunit-rotation pathway.
480:
Coates, C.J.; Kaminski, JM; Summers, JB; Segal, DJ; Miller, AD; Kolb, AF (2005).
1078:
682:
Abdel-Meguid, S.S.; Grindley, N.D.; Templeton, N.S.; Steitz, T.A. (April 1984).
327:
242:
51:
458:
931:
202:
165:
89:
81:. For the same reasons, they present a potential basis for the development of
66:
811:
253:
1205:
1156:
973:
873:
819:
708:
618:
549:
508:
466:
423:
1164:
1121:
1086:
1043:
1008:
768:
727:
653:
557:
445:
Kolb, A.F. (2002). "Genome
Engineering Using Site-Specific Recombinases".
864:
847:
268:
234:
181:
415:
264:
47:
326:
316:
130:
109:
108:
593:
Site-Specific DNA Recombinases as
Instruments for Genomic Surgery
322:
Fig. 3A. Reversible insertion and excision by the Cre-lox system.
131:
579:
Escherichia coli and
Salmonella: cellular and molecular biology
39:
123:
accommodate both substrates in an anti-parallel orientation.
531:"Inducible Gene Targeting in Mice Using the Cre/loxSystem"
172:, while the founding member of the tyrosine recombinases,
1180:"A model for the gamma-delta resolvase synaptic complex"
114:
Fig. 1. Tyr-Recombinases: Details of the crossover step.
295:
in which only one pair of strands has been exchanged.
194:). Famous serine recombinases include enzymes such as
595:. Advances in Genetics. Vol. 55. pp. 1β23.
88:
Recombination sites are typically between 30 and 200
248:, while the recombinase enzyme forms a transient
8:
671:. Oxford University Press. pp. 101β129.
912:"A mechanism for gene conversion in fungi"
1195:
963:
930:
863:
758:
717:
707:
332:Fig. 3B. Inversion by the Cre-lox system.
841:
839:
837:
32:conservative site-specific recombination
393:
377:Recombinase-mediated cassette exchange
60:recombinase-mediated cassette exchange
7:
382:Site-specific recombinase technology
646:10.1146/annurev.bi.58.070189.004405
260:between the hydroxyl group of the
25:
946:Grainge, I.; Jayaram, M. (1999).
591:Akopian, A.; Stark, W.M. (2005).
157:tyrosine (Tyr) recombinase family
1067:Cold Spring Harb Symp Quant Biol
965:10.1046/j.1365-2958.1999.01493.x
741:Yang, W.; Steitz, T.A. (1995).
209:(from the Tn3 transposon), and
56:genome modification strategies
1:
1197:10.1016/S1097-2765(01)00334-3
899:. ASM Press. pp. 93β117.
634:Annual Review of Biochemistry
601:10.1016/S0065-2660(05)55001-6
501:10.1016/j.tibtech.2005.06.009
1145:Journal of Molecular Biology
1114:10.1016/0092-8674(89)90111-6
1036:10.1016/0092-8674(81)90179-3
1001:10.1016/0168-9525(92)90327-Z
846:Turan, S.; Bode, J. (2011).
760:10.1016/0092-8674(95)90307-0
688:Proc. Natl. Acad. Sci. U.S.A
95:attP and attB of Ξ» integrase
1079:10.1101/sqb.1984.049.01.028
28:Site-specific recombination
18:Site specific recombination
1262:
459:10.1089/153623002753632066
101:Classification: tyrosine-
932:10.1017/S0016672300001233
447:Cloning & Stem Cells
372:Homologous recombination
191:Saccharomyces cerevisiae
161:serine (Ser) recombinase
812:10.1126/science.1112064
669:Mobile Genetic Elements
489:Trends in Biotechnology
79:mobile genetic elements
50:known as site-specific
1157:10.1006/jmbi.1994.1348
952:Molecular Microbiology
709:10.1073/pnas.81.7.2001
550:10.1006/meth.1998.0593
334:
324:
152:
128:
73:, differentiation and
910:Holliday, R. (1964).
367:Genetic recombination
362:FLP-FRT recombination
357:Cre-Lox recombination
330:
320:
310:reaction, in which a
196:gamma-delta resolvase
134:
112:
36:genetic recombination
865:10.1096/fj.11-186940
105:serine- recombinases
1236:Genetics techniques
804:2005Sci...309.1210L
700:1984PNAS...81.2001A
581:, 2, pp. 2363β2376.
416:10.1515/BC.2000.103
312:phosphodiester bond
308:transesterification
258:phosphodiester bond
239:transesterification
83:genetic engineering
1246:Molecular genetics
1231:Cellular processes
989:Trends in Genetics
529:Sauer, B. (1998).
335:
325:
252:to a DNA backbone
153:
129:
77:, and movement of
1241:Molecular biology
919:Genetics Research
858:(12): 4088β4107.
610:978-0-12-017655-7
410:(9β10): 801β813.
293:Holliday junction
44:sequence homology
16:(Redirected from
1253:
1210:
1209:
1199:
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934:
916:
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901:
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886:
885:
867:
843:
832:
831:
798:(5738): 1210β5.
787:
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732:
731:
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679:
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664:
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588:
582:
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560:. Archived from
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520:
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511:. Archived from
486:
477:
471:
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442:
436:
435:
398:
188:(from the yeast
30:, also known as
21:
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1016:
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147:(attL and attR)
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34:, is a type of
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22:
15:
12:
11:
5:
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1238:
1233:
1228:
1218:
1217:
1212:
1211:
1184:Molecular Cell
1170:
1135:
1092:
1057:
1014:
979:
938:
925:(2): 282β304.
902:
887:
833:
782:
753:(2): 193β207.
733:
674:
659:
624:
609:
583:
570:
567:on 2011-06-11.
521:
518:on 2006-08-29.
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246:hydroxyl group
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170:DNA invertases
106:
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58:, among these
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14:
13:
10:
9:
6:
4:
3:
2:
1258:
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1234:
1232:
1229:
1227:
1226:Biotechnology
1224:
1223:
1221:
1207:
1203:
1198:
1193:
1190:(3): 623β31.
1189:
1185:
1181:
1174:
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1166:
1162:
1158:
1154:
1150:
1146:
1139:
1136:
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1123:
1119:
1115:
1111:
1108:(4): 779β90.
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1103:
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1041:
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1033:
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1025:
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1006:
1002:
998:
995:(12): 432β9.
994:
990:
983:
980:
975:
971:
966:
961:
958:(3): 449β56.
957:
953:
949:
942:
939:
933:
928:
924:
920:
913:
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903:
898:
897:Mobile DNA II
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694:(7): 2001β5.
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647:
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640:(1): 913β41.
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598:
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584:
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547:
544:(4): 381β92.
543:
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510:
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502:
498:
495:(8): 407β19.
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304:Tn3 resolvase
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250:covalent bond
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214:C31 integrase
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207:Tn3 resolvase
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29:
19:
1187:
1183:
1173:
1151:(1): 25β36.
1148:
1144:
1138:
1105:
1101:
1095:
1070:
1066:
1060:
1030:(3): 721β8.
1027:
1023:
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592:
586:
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573:
562:the original
541:
537:
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513:the original
492:
488:
475:
453:(1): 65β80.
450:
446:
440:
407:
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340:
336:
331:
321:
297:
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262:nucleophilic
231:
222:
220:C31 phage).
217:
211:
199:
198:(from the Tn
189:
174:lambda phage
154:
150:
135:
113:
102:
87:
75:pathogenesis
64:
52:recombinases
31:
27:
26:
300:gamma-delta
243:deoxyribose
90:nucleotides
71:replication
1220:Categories
404:Biol. Chem
388:References
216:(from the
203:transposon
180:(from the
166:resolvases
67:eukaryotic
1073:: 245β9.
274:cofactors
254:phosphate
228:Mechanism
38:in which
1206:11583624
1130:46508016
1052:28410571
974:10577069
874:21891781
828:84409916
820:15994378
777:15849525
619:16291210
509:15993503
467:12006158
432:36479502
424:11076013
346:See also
276:such as
269:tyrosine
235:synapsis
182:P1 phage
1165:8196046
1122:2548736
1087:6099239
1044:6269756
1009:1337225
882:7075677
852:FASEB J
800:Bibcode
792:Science
769:7628011
728:6326096
696:Bibcode
654:2528323
558:9608509
538:Methods
256:. This
85:tools.
48:Enzymes
1204:
1163:
1128:
1120:
1085:
1050:
1042:
1007:
972:
880:
872:
826:
818:
775:
767:
726:
719:345424
716:
652:
617:
607:
556:
507:
465:
430:
422:
265:serine
184:) and
93:(e.g.
1126:S2CID
1048:S2CID
915:(PDF)
878:S2CID
824:S2CID
773:S2CID
565:(PDF)
534:(PDF)
516:(PDF)
485:(PDF)
428:S2CID
1202:PMID
1161:PMID
1118:PMID
1102:Cell
1083:PMID
1040:PMID
1024:Cell
1005:PMID
970:PMID
870:PMID
816:PMID
765:PMID
747:Cell
724:PMID
650:PMID
615:PMID
605:ISBN
554:PMID
505:PMID
463:PMID
420:PMID
302:and
200:1000
1192:doi
1153:doi
1149:239
1110:doi
1075:doi
1032:doi
997:doi
960:doi
927:doi
860:doi
808:doi
796:309
755:doi
714:PMC
704:doi
642:doi
597:doi
546:doi
497:doi
455:doi
412:doi
408:381
289:FLP
287:or
285:Cre
278:ATP
267:or
205:),
186:FLP
178:Cre
168:or
159:or
103:vs.
97:).
40:DNA
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1071:49
1069:.
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1028:25
1026:.
1003:.
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968:.
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954:.
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854:.
850:.
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771:.
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