Difference between revisions of "Part:BBa K5276001"
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Each recognition site consists of two incompletely symmetrical sequences on the left and right and a 2bp linker sequence between them. We named the left and right sequences of attB (attP) as B (P) type arms. | Each recognition site consists of two incompletely symmetrical sequences on the left and right and a 2bp linker sequence between them. We named the left and right sequences of attB (attP) as B (P) type arms. | ||
</p> | </p> | ||
| − | < img src="https://static.igem.wiki/teams/5276/wetlab/qzt/mechanism-of-recombinase.png" | + | <img src="https://static.igem.wiki/teams/5276/wetlab/qzt/mechanism-of-recombinase.png" alt="mechanism-of-recombinase" style="zoom:23%;" /> |
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In the free state, recombinase exists as a dimer (a), and the N-terminal domains (NTD) of the two integrases bind to each other; when attB & attP are present, the C-terminal domain (CTD) of the integrase will bind to the two arms of the binding site respectively (b); after CTD binding, the conformation changes, so that the dimers bound to attB & attP respectively form a tetramer (c); the tetramer conformation activates NTD, causing DNA being cut from the 2bp linker region in the middle of the binding site (e); after cutting, the two integrases on one side will rotate 180° relative to each other (f); and finally form a new attL & attR binding site (g). Therefore, as shown in Figure (g), the newly formed attL and attR are both composed of a B arm and a P arm. Since the two arms connecting the dimer are different, tetramers cannot form without RDF; thus, attL and attR cannot be reconstituted. If RDF is present, similar recombination will occur between the attL and attR sites, transforming back into attB and attP. | In the free state, recombinase exists as a dimer (a), and the N-terminal domains (NTD) of the two integrases bind to each other; when attB & attP are present, the C-terminal domain (CTD) of the integrase will bind to the two arms of the binding site respectively (b); after CTD binding, the conformation changes, so that the dimers bound to attB & attP respectively form a tetramer (c); the tetramer conformation activates NTD, causing DNA being cut from the 2bp linker region in the middle of the binding site (e); after cutting, the two integrases on one side will rotate 180° relative to each other (f); and finally form a new attL & attR binding site (g). Therefore, as shown in Figure (g), the newly formed attL and attR are both composed of a B arm and a P arm. Since the two arms connecting the dimer are different, tetramers cannot form without RDF; thus, attL and attR cannot be reconstituted. If RDF is present, similar recombination will occur between the attL and attR sites, transforming back into attB and attP. | ||
Revision as of 13:59, 26 September 2024
A118 recombinase
Abstract
A118 is a serine recombinase that mediates a reversible site-specific recombination system first discovered in bacteriophages. This site-specific recombination system is used by bacteriophages to integrate their genes into the host genomic DNA for proliferation and to transfer genes from the host genome to daughter phages by expressing recombination directionality factor (RDF) before the host is lysed and released. Specifically, A118 can mediate the excision or inversion of genes between a pair of specific recognition sites (attB and attP) and simultaneously convert this pair of sites into another pair of recognition sites (attL and attR). This site-specific recombination is reversible, and when the auxiliary protein RDF is present, the serine recombinase can reduce attL and attR to attB and attP.
It is worth noting that a recombinase can have multiple pairs of mutually orthogonal attB and attP recombination sites. For example, A118B-AA (BBa_K5276017) and A118P-AA (BBa_K5276018) are a pair of recombination sites of A118, while A118B-GG (BBa_K5276019) and A118P-GG (BBa_K5276020) are another pair of recombination sites orthogonal to that.
Molecular mechanism
Each recognition site consists of two incompletely symmetrical sequences on the left and right and a 2bp linker sequence between them. We named the left and right sequences of attB (attP) as B (P) type arms.
In the free state, recombinase exists as a dimer (a), and the N-terminal domains (NTD) of the two integrases bind to each other; when attB & attP are present, the C-terminal domain (CTD) of the integrase will bind to the two arms of the binding site respectively (b); after CTD binding, the conformation changes, so that the dimers bound to attB & attP respectively form a tetramer (c); the tetramer conformation activates NTD, causing DNA being cut from the 2bp linker region in the middle of the binding site (e); after cutting, the two integrases on one side will rotate 180° relative to each other (f); and finally form a new attL & attR binding site (g). Therefore, as shown in Figure (g), the newly formed attL and attR are both composed of a B arm and a P arm. Since the two arms connecting the dimer are different, tetramers cannot form without RDF; thus, attL and attR cannot be reconstituted. If RDF is present, similar recombination will occur between the attL and attR sites, transforming back into attB and attP.
The recognition site of the recombinase is directional. If a pair of attB and attP sites are in the same direction, the sequence between them will be removed; if the attB and attP sites are in opposite directions, the sequence between them will be flipped.
Directed Evolution
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal SpeI site found at 325
- 12INCOMPATIBLE WITH RFC[12]Illegal SpeI site found at 325
- 21COMPATIBLE WITH RFC[21]
- 23INCOMPATIBLE WITH RFC[23]Illegal SpeI site found at 325
- 25INCOMPATIBLE WITH RFC[25]Illegal SpeI site found at 325
- 1000INCOMPATIBLE WITH RFC[1000]Illegal SapI.rc site found at 721