Difference between revisions of "Part:BBa K5477002"

 
 
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<partinfo>BBa_K5477002 short</partinfo>
 
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RAD27 encodes a 5' to 3' exonuclease and 5' flap endonuclease, which is essential for Okazaki fragment processing and maturation during DNA replication. It plays a role in long-patch base-excision repair, large loop repair (LLR), and ribonucleotide excision repair. RAD27 removes RNA flaps in RNA:DNA hybrid intermediates during lagging strand synthesis. It can also relocalize to the cytosol under hypoxic conditions. Based on the figure, pRAD27 functions as a weak constitutive promoter in yeast (1,2,3,4,6,7). Figure 3A shows the relative strengths of 19 constitutive promoters by measuring fluorescence from two reporters, mRuby2 and Venus. The plot highlights three key promoters: pTDH3 (strong), pRPL18B (medium), and pREV1 (weak) (5). The horizontal and vertical bars represent the range of fluorescence from four biological replicates, with the intersection indicating the median. The inset also includes results from testing a third reporter, mTurquoise2, demonstrating consistent promoter strength across different reporter proteins. From the data, pRAD27 appears to be positioned in the lower range of constitutive promoter strengths.
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RAD27 encodes a 5' to 3' exonuclease and 5' flap endonuclease, which is essential for Okazaki fragment processing and maturation during DNA replication. It plays a role in long-patch base-excision repair, large loop repair (LLR), and ribonucleotide excision repair. RAD27 removes RNA flaps in RNA:DNA hybrid intermediates during lagging strand synthesis. It can also relocalize to the cytosol under hypoxic conditions (1) (2) (3) (4) (6) (7).  
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https://static.igem.wiki/teams/5477/promoters.gif
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Figure 3A shows the relative strengths of 19 constitutive promoters by measuring fluorescence from two reporters, mRuby2 and Venus. The plot highlights three key promoters: pTDH3 (strong), pRPL18B (medium), and pREV1 (weak) (5). The horizontal and vertical bars represent the range of fluorescence from four biological replicates, with the intersection indicating the median. The inset also includes results from testing a third reporter, mTurquoise2, demonstrating consistent promoter strength across different reporter proteins. From the data, pRAD27 appears to be positioned in the lower range of constitutive promoter strengths. Based on the figure, pRAD27 functions as a weak constitutive promoter in yeast (5).
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pRAD27 was used to drive the expression of the AhR receptor module [https://parts.igem.org/Part:BBa_K5477024 | BBa_K5477024].
  
  
 
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===Usage and Biology===
 
===Usage and Biology===
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===References===
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1. Ayyagari R, Gomes XV, Gordenin DA, Burgers PM. Okazaki fragment maturation in yeast. I. Distribution of functions between FEN1 AND DNA2. J Biol Chem. 2003 Jan 17;278(3):1618-25. doi: 10.1074/jbc.M209801200. Epub 2002 Nov 6. PMID: 12424238.
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2. Dastidar RG, Hooda J, Shah A, Cao TM, Henke RM, Zhang L. The nuclear localization of SWI/SNF proteins is subjected to oxygen regulation. Cell Biosci. 2012 Aug 29;2(1):30. doi: 10.1186/2045-3701-2-30. PMID: 22932476; PMCID: PMC3489556.
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3. Kao HI, Henricksen LA, Liu Y, Bambara RA. Cleavage specificity of Saccharomyces cerevisiae flap endonuclease 1 suggests a double-flap structure as the cellular substrate. J Biol Chem. 2002 Apr 26;277(17):14379-89. doi: 10.1074/jbc.M110662200. Epub 2002 Feb 1. PMID: 11825897.
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4. Laverde EE, Polyzos AA, Tsegay PP, Shaver M, Hutcheson JD, Balakrishnan L, McMurray CT, Liu Y. Flap Endonuclease 1 Endonucleolytically Processes RNA to Resolve R-Loops through DNA Base Excision Repair. Genes (Basel). 2022 Dec 29;14(1):98. doi: 10.3390/genes14010098. PMID: 36672839; PMCID: PMC9859040.
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5. Lee, Michael & DeLoache, William & Cervantes, Bernardo & Dueber, John. (2015). A Highly Characterized Yeast Toolkit for Modular, Multipart Assembly. ACS synthetic biology. 4. 10.1021/sb500366v.
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6. Sommer D, Stith CM, Burgers PM, Lahue RS. Partial reconstitution of DNA large loop repair with purified proteins from Saccharomyces cerevisiae. Nucleic Acids Res. 2008 Aug;36(14):4699-707. doi: 10.1093/nar/gkn446. Epub 2008 Jul 15. PMID: 18628298; PMCID: PMC2504288.
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7. Sparks JL, Chon H, Cerritelli SM, Kunkel TA, Johansson E, Crouch RJ, Burgers PM. RNase H2-initiated ribonucleotide excision repair. Mol Cell. 2012 Sep 28;47(6):980-6. doi: 10.1016/j.molcel.2012.06.035. Epub 2012 Aug 2. PMID: 22864116; PMCID: PMC3470915.

Latest revision as of 20:09, 1 October 2024


pRAD27 - weak constitutive promoter in Saccharomyces cerevisiae

RAD27 encodes a 5' to 3' exonuclease and 5' flap endonuclease, which is essential for Okazaki fragment processing and maturation during DNA replication. It plays a role in long-patch base-excision repair, large loop repair (LLR), and ribonucleotide excision repair. RAD27 removes RNA flaps in RNA:DNA hybrid intermediates during lagging strand synthesis. It can also relocalize to the cytosol under hypoxic conditions (1) (2) (3) (4) (6) (7).


promoters.gif

Figure 3A shows the relative strengths of 19 constitutive promoters by measuring fluorescence from two reporters, mRuby2 and Venus. The plot highlights three key promoters: pTDH3 (strong), pRPL18B (medium), and pREV1 (weak) (5). The horizontal and vertical bars represent the range of fluorescence from four biological replicates, with the intersection indicating the median. The inset also includes results from testing a third reporter, mTurquoise2, demonstrating consistent promoter strength across different reporter proteins. From the data, pRAD27 appears to be positioned in the lower range of constitutive promoter strengths. Based on the figure, pRAD27 functions as a weak constitutive promoter in yeast (5).

pRAD27 was used to drive the expression of the AhR receptor module | BBa_K5477024.


Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal SpeI site found at 289
    Illegal SpeI site found at 426
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal SpeI site found at 289
    Illegal SpeI site found at 426
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 102
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal SpeI site found at 289
    Illegal SpeI site found at 426
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal SpeI site found at 289
    Illegal SpeI site found at 426
  • 1000
    COMPATIBLE WITH RFC[1000]



References

1. Ayyagari R, Gomes XV, Gordenin DA, Burgers PM. Okazaki fragment maturation in yeast. I. Distribution of functions between FEN1 AND DNA2. J Biol Chem. 2003 Jan 17;278(3):1618-25. doi: 10.1074/jbc.M209801200. Epub 2002 Nov 6. PMID: 12424238.

2. Dastidar RG, Hooda J, Shah A, Cao TM, Henke RM, Zhang L. The nuclear localization of SWI/SNF proteins is subjected to oxygen regulation. Cell Biosci. 2012 Aug 29;2(1):30. doi: 10.1186/2045-3701-2-30. PMID: 22932476; PMCID: PMC3489556.

3. Kao HI, Henricksen LA, Liu Y, Bambara RA. Cleavage specificity of Saccharomyces cerevisiae flap endonuclease 1 suggests a double-flap structure as the cellular substrate. J Biol Chem. 2002 Apr 26;277(17):14379-89. doi: 10.1074/jbc.M110662200. Epub 2002 Feb 1. PMID: 11825897.

4. Laverde EE, Polyzos AA, Tsegay PP, Shaver M, Hutcheson JD, Balakrishnan L, McMurray CT, Liu Y. Flap Endonuclease 1 Endonucleolytically Processes RNA to Resolve R-Loops through DNA Base Excision Repair. Genes (Basel). 2022 Dec 29;14(1):98. doi: 10.3390/genes14010098. PMID: 36672839; PMCID: PMC9859040.

5. Lee, Michael & DeLoache, William & Cervantes, Bernardo & Dueber, John. (2015). A Highly Characterized Yeast Toolkit for Modular, Multipart Assembly. ACS synthetic biology. 4. 10.1021/sb500366v.

6. Sommer D, Stith CM, Burgers PM, Lahue RS. Partial reconstitution of DNA large loop repair with purified proteins from Saccharomyces cerevisiae. Nucleic Acids Res. 2008 Aug;36(14):4699-707. doi: 10.1093/nar/gkn446. Epub 2008 Jul 15. PMID: 18628298; PMCID: PMC2504288.

7. Sparks JL, Chon H, Cerritelli SM, Kunkel TA, Johansson E, Crouch RJ, Burgers PM. RNase H2-initiated ribonucleotide excision repair. Mol Cell. 2012 Sep 28;47(6):980-6. doi: 10.1016/j.molcel.2012.06.035. Epub 2012 Aug 2. PMID: 22864116; PMCID: PMC3470915.