User:Pakpoom Subsoontorn/Notebook/Genetically Encoded Memory/2008/10/10: Difference between revisions

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==questions/notes==
==questions/notes==
*ask Michele about site-specific recombinase database
*play with bacteriophage and microbial genome database, look for recombinase genes
*read Allan's paper on site-specificity of lambda integrase..esp 2002 paper
*look at Tn4451 mechanism for boosting up integration, Lyras and Rood 2000


==Database ==
==Database ==

Revision as of 20:17, 21 October 2008

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Summary Note on site-specific recombinase system

  • Insert content here...

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
    • Integron

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

Name: lambda, Host: Escherichia Coli

  • Classification: tyrosine, integrase
  • Structure:
    • Length 356 aa.
    • Amino-Acid-Sequence
    • Domains
      • crystal structure of C-terminal catalytic domain is known
      • N-terminal 1-64 aa binds to arm-type site.
      • C-terminal 65-169 aa binds to low affinity core-type site.
      • 170-356 aa is the minimal catalytic domain which include six tyrosine kinase conserved residues: R212, K235, H308, R311, Y342, H308 (nucleophilic tyrosine and catalytic pentad)
  • Target sequence: diagram (from Groth and Calos, 2004)
    • attB:: has two inverted repeated sequence flanking an overlap region. Inverted repeated sequence (core-type) is about 9-13 bps.
    • attP is more complicated, with arm-type sites flanking core-type site.
    • The overlap region (between attP and attB) is about 6-8 bps and shares sequence with attP. Here is where crossing over occur.
      • overlap-region:TTTATAC:
  • Mechanism: high affinity binding at arm-region of attP, low affinity binding at core-region. Integration

host factor (IHF, from HimA and hip genes) is required for sharp bending in DNA, allowing the integrase bound at arm-type site to also bind at the low affinity core-type sites. Break one strand at the time. Tyrosine phosphate binds to 3'end . 5'OH is free. Integration involve holiday junction

  • Engineering
    • Mutation
      • Mutation could change specificity to HK022's site
    • Chimeric
    • Directed-Evolution
  • Exision: Excision is mediated by viral Xis excisionase and enhanced by host FIS.
    • Unnatural-Host
    • the mutant works in mammalian cell (see more experiment detail)



Name: HK022, Host: Escherichia Coli

  • Action: tyrosine integrase
  • Structure: length:: 357 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::AGGTGAA attP has arm-type sites flanking core-type site.

  • Mechanism:

Integrases from lambda and HK022 recognize the same arm-type site. However, the integrases notice the difference in core-type site. IHF is required.




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.

  • Mechanism:

IHF is required.


Name: HP1, Host: Haemophilus influenzae

  • Action: tyrosine integrase
  • Structure:

length:: 337 aa crystal structure of C-terminal catalytic domain is known

  • 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::TTTTAAA attP has arm-type sites flanking core-type site.

  • Mechanism:



Name: L5, Host: Mycobacterium smegmatis



Name: PhiC31, Host: Streptomyces lividans

  • Action: tyrosine integrase
  • Structure:

length:: 613 aa crystal structure is unknown.

  • Target sequence: attB:: has two inverted repeated sequence (core-type) flanking an overlap region (TTG). Minimal attB is 34 bps while minimal attP is 39 bp for e.coli intramolecular integration.

attP:: has core-type sites and overlap region, without an arm-type site

  • Change specificity:

Mutation/directed evolution could change specificity to pseudo attP site (HpsA, 44% identity)

  • Mechanism:
  • Excision:

Excisionase has not yet been found.

  • Usage:

Integration works in E.coli, mammalian and in vitro


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.

  • Excision:

Excisionase has not yet been found.

  • Usage:

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.



Name: Cre (P1), Host: Escherichia coli

  • Classification: tyrosine recombinase
  • structure
    • Length
    • Amino-Acid-Sequence
    • Domains
      • six tyrosine kinase conserved residues: R173, K201, R292, H289, W305, Y324 (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:
    • Unnatural-Host




  • 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:
    • Unnatural-Host





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:
    • Unnatural-Host



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
    • res-I, res-II, res-III
  • Mechanism:
    • two dimer per site (see diagram from the ref)
  • Engineering
    • Mutation
      • Mutation could change specificity to HK022's site
    • Chimeric
    • Directed-Evolution
  • Exision:
    • Unnatural-Host






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

  1. Groth AC & Calos MP (2004), Phage Integrases: Biology and Applications. J.Mol.Biol. 335:667-678
  2. 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

  1. Grainge I & Sherratt DJ (2007), Site-Specific recombination. Molecular Genetics of Recombination, Springer


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