Summary Note on site-specific recombinase system
Classification
Keywords for classification, which can be overlapped
- By Structure
- Tyrosine recombinase
- Serine recombinase
- Large Serine recombinase
- By Function
- Integrase
- Excisionase
- Invertase
- Resolvase
- Transposase
- other stuffs
Methods
- Discovering new site-specific recombinase
- Characterizing protein domains
- DNA binding domain
- Catalytic domain
- Finding the target-site
- Minimal specific sequence for targeting
- Looking into recombination mechanism
- DNA topologies
- Protein-DNA interaction
- Chronology of events
- Chemistry at active site
- Evaluating Efficiency
- Recombination yield
- Specificity
Site Specific recombinases
(see more experiment detail)
Name: P22, Host Salmonella tryphimurium
- Action: tyrosine integrase
- Structure: length:: 387 aa
- Target sequence:
attB:: has two inverted repeated sequence flanking an overlap region.
inverted repeated sequence (core-type).
The overlap region shares sequence with attP.
Crossing over occurs at the overlap region. overlap-region::TTCGTAA
attP has arm-type sites flanking core-type site.
IHF is required.
Name: HP1, Host: Haemophilus influenzae
- Action: tyrosine integrase
- Structure:
length:: 337 aa
crystal structure of C-terminal catalytic domain is known
attB:: has two inverted repeated sequence flanking an overlap region.
inverted repeated sequence (core-type).
The overlap region shares sequence with attP.
Crossing over occurs at the overlap region. overlap-region::TTTTAAA
attP has arm-type sites flanking core-type site.
Name: L5, Host: Mycobacterium smegmatis
Name: R4, Host: Streptomyces
- Action: tyrosine integrase
- Structure:
length:: 469 aa
crystal structure is unknown.
- Target sequence: attB:: has two inverted repeated sequence (core-type) flanking an overlap region .
attP:: has core-type sites and overlap region, without an arm-type site. Minimal attB and attP site
is <56 bps.
Excisionase has not yet been found.
Integration works in mammalian cell.
Name: TP901, Host: Lactococcus lactis
- Action: tyrosine integrase
- Structure:
length:: 485 aa.
crystal structure is unknown.
- Target sequence: attB:: has two inverted repeated sequence (core-type) flanking an overlap region.
attP:: has core-type sites and overlap region, without an arm-type site.
Minimal attB and attP site is <56 bps
- Mechanism:
- Excision: Excisionase has already been indentified.
- Usage:
Integration works in E.coli, mammalian cell and in vitro.
- Classification: tyrosine recombinase
- structure
- Length
- Amino-Acid-Sequence
- Domains
- six tyrosine kinase conserved residues: R212, K235, H308, R311, Y342, H308 (nucleophilic tyrosine and catalytic pentad)
- Target sequence: two identical simple sequence < 50 bp
- Mechanism:
- Engineering
- Mutation
- Mutation could change specificity to HK022's site
- Chimeric
- Directed-Evolution
- Exision:
Name: FLP, Host: Saccharomyces cerevisiae
- Classification: tyrosine recombinase
- structure
- Length
- Amino-Acid-Sequence
- Domains
- six tyrosine kinase conserved residues: R191, K223, H305, R308, W330, Y343 (nucleophilic tyrosine and catalytic pentad)
- Target sequence: two identical simple sequence < 50 bp
- Mechanism:
- Engineering
- Mutation
- Mutation could change specificity to HK022's site
- Chimeric
- Directed-Evolution
- Exision:
Name: XerC, Host: Escherichia coli
Name: Gamma-Delta, Host: Escherichia coli
- Classification: serine resolvase
- structure:
- Length: 183 aa
- Amino-Acid-Sequence
- Domains
- Catalytic site has R8, S10, D67, R68
- Li et al 2005 science
- Target sequence: two identical simple sequence < 50 bp
- Mechanism:
- two dimer per site (see diagram from the ref)
- Engineering
- Mutation
- Mutation could change specificity to HK022's site
- Chimeric
- Directed-Evolution
- Exision:
Name: Tn3, Host: Klebsiella pneumoniae
Action: resolvase
Name: gin (Phage Mu), Host: Escherichia coli
Action: invertase
questions/notes
- play with bacteriophage and microbial genome database, look for recombinase genes
- look at Tn4451 mechanism for boosting up integration, Lyras and Rood 2000
Database
Reference
- Groth AC & Calos MP (2004), Phage Integrases: Biology and Applications. J.Mol.Biol. 335:667-678
- Sclimenti CR, Thyagarajan B & Calos MP, Directed evolution of a recombinase for
improved genomic integration at a native human sequence. Nucl Acids Res, 29 5044-5051
- Grainge I & Sherratt DJ (2007), Site-Specific recombination. Molecular Genetics of Recombination, Springer
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