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

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***N-terminal 1-64 aa binds to arm-type site.
***N-terminal 1-64 aa binds to arm-type site.
***C-terminal 65-169 aa binds to low affinity core-type site.
***C-terminal 65-169 aa binds to low affinity core-type site.
***170-356 aa is the minimal catalytic domain which include catalytic tyrosine and RKHRK pentad
***170-356 aa is the minimal catalytic domain which include six tyrosine kinase conserved residues: R212, K235, H308, R311, Y342, H308
<ref>Deborah Solomon. [http://www.nytimes.com/2006/01/08/magazine/08wwln_q4.html?_r=1&n=Top/Reference/Times%20Topics/People/H/Hart,%20Gary&oref=slogin Over Life on the Hill]. ''[[New York Times]]'', January 8, 2006.</ref>
*Target sequence: [[Media:lambdaInt.gif|diagram (from Groth and Calos, 2004) ]]
*Target sequence: [[Media:lambdaInt.gif|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.  
**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.  
**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.  
**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
***overlap-region:TTTATAC:
*Change specificity:
*Mechanism: high affinity binding at arm-region of attP, low affinity binding at core-region. Integration
*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  
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.
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
*Engineering
**Mutation
**Mutation

Revision as of 19:40, 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

[1]

  • 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

  • Action: recombinase
  • Target sequence: two identical simple sequence < 50 bp



Name: FLP, Host: Saccharomyces cerevisiae

  • Action: invertase
  • Target sequence: two identical simple sequence < 50 bp



Name: XerC, Host: Escherichia coli


Name: Gamma-Delta, Host: Escherichia coli Action: resolvase

  • Structure:

length:: 183 aa some crystal structure with res target have been identified.

  • Target sequence: TATTATAAAT
  • Usage:

Integration works in mammalian cell.


Name: Tn3, Host: Klebsiella pneumoniae Action: resolvase


Name: gin (Phage Mu), Host: Escherichia coli Action: invertase

questions/notes

  • ask Michele about site-specific recombinase database
  • read Allan's paper on site-specificity of lambda integrase..esp 2002 paper

Database

Reference

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

335:667-678

  1. 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. ↑ Deborah Solomon. Over Life on the Hill. New York Times, January 8, 2006.
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