EG10651 andreavz

From BioE80 Boot
Jump to: navigation, search

Author Information

Andrea Villarreal

Basic Information

  • ID: EG10651
  • Name: deoxyribonuclease IV [Escherichia coli], endonuclease IV, rfo

Deoxy.png

  • Organism: E. Coli, Escherichia coli K-12 substr. MG1655
  • UniProt ID: P0A6C1 (END4_ECOLI) [1]
  • Description:
  • Function:
Endonuclease IV plays a role in DNA repair. AP endonuclease, specifically, catalyzes the incision of DNA exclusively at AP sites, and therefore prepares DNA for subsequent excision, repair synthesis and DNA ligation.[2] It cleaves phosphodiester bonds at apurinic or apyrimidinic sites (AP sites) to produce new 5'-ends that are base-free deoxyribose 5-phosphate residues. It preferentially attacks modified AP sites created by bleomycin and neocarzinostatin. [3] Endonucleases are enzymes that cleave the phosphodiester bond within a polynucleotide chain. E. coli cells contain two AP endonucleases: endonuclease IV (endoIV) and exonuclease III (exoIII).
"Damaged bases in DNA occur due to alkylating agents, ionizing radiation or oxidants such as superoxide radicals, hydrogen peroxide and hydroxyl radicals. "The Base excision repair (BER) pathway is the predominant DNA damage repair pathway for the processing of small base lesions, derived from oxidation and alkylation damages." [4] Base excision repair (BER) works to repair damaged DNA by multiple steps: removal of the damaged base by a cleavage of the glycosidic bond by a glycosylase enzyme generating an apyyrimidinic or apurinic (AP) site; removal of the AP site by an AP endonuclease; resynthesis of the excised strand by DNA polymerase using the intact strand as template and ligation by DNA ligase. Nfo is one of two (the other being XthA) 5' AP endonucleases in Escherichia coli. Nfo has been purified and shown to catalyze the formation of single-stand breaks in double stranded AP DNA [Levin88, Cunningham86]. Nfo has been shown to function in a manner similar to Exonuclease III (XthA) in removing phosphoglycoaldehyde, phosphate, deoxyribose-5-phosphate, 4-hydroxy-2-pentenal residues and urea residues [Doetsch90]. Unlike XthA, Nfo does not have a 3' exonuclease activity." [5]
  • Reaction: a single stranded DNA + n H2O → n a 5'-phosphooligonucleotide
  • Location: The location where these protein works is the cytosol, which is the aqueous component of the cytoplasm in a cell within which various organelles and particles are suspended.
  • Pathways: This protein belongs primarily to the Base Excision Repair (BER) pathway.


  • DNA Length: 858 base pairs.
  • DNA sequence:

ATG AAA TAC ATT GGA GCG CAC GTT AGT GCT GCT GGC GGT CTG GCA AAT GCC GCA ATT CGC GCC GCC GAA ATC GAC GCA ACC GCG TTT GCC TTG TTC ACC AAA AAC CAA CGT CAG TGG CGT GCC GCA CCG CTC ACG ACG CAA ACC ATC GAT GAA TTC AAA GCC GCC TGT GAA AAA TAT CAC TAC ACA TCG GCG CAA ATT CTT CCC CAC GAC AGT TAT CTG ATT AAC CTC GGA CAT CCG GTC ACT GAA GCT CTG GAA AAA TCG CGC GAT GCC TTT ATA GAT GAA ATG CAG CGT TGC GAA CAG CTG GGG CTT TCT TTG CTC AAC TTC CAC CCT GGC AGC CAT CTG ATG CAG ATT TCA GAA GAG GAT TGC CTT GCG CGT ATT GCC GAA TCC ATC AAC ATT GCG CTG GAT AAA ACT CAA GGT GTG ACA GCG GTG ATA GAA AAC ACC GCC GGT CAG GGC AGT AAC TTA GGG TTT AAA TTC GAA CAT CTC GCG GCG ATT ATC GAC GGC GTG GAA GAT AAA TCC CGC GTC GGC GTC TGC ATT GAT ACC TGC CAT GCT TTC GCT GCC GGG TAT GAT TTG CGT ACT CCA GCC GAA TGC GAG AAA ACA TTC GCG GAT TTT GCC CGT ACT GTC GGC TTT AAG TAT CTG CGC GGG ATG CAC CTT AAC GAT GCG AAA AGC ACC TTT GGC AGC CGC GTT GAC CGC CAT CAT AGC CTC GGT GAA GGC AAT ATC GGT CAT GAT GCG TTC CGC TGG ATC ATG CAG GAC GAC CGT TTC GAC GGC ATT CCG CTG ATC CTC GAA ACC ATC AAC CCG GAT ATC TGG GCA GAA GAG ATC GCC TGG CTG AAA GCG CAA CAA ACT GAA AAA GCG GTA GCC TGA

  • Amino Acid length: 285 amino acids.
  • Amino Acid sequence:

MKYIGAHVSA AGGLANAAIR AAEIDATAFA LFTKNQRQWR AAPLTTQTID EFKAACEKYH YTSAQILPHD SYLINLGHPV TEALEKSRDA FIDEMQRCEQ LGLSLLNFHP GSHLMQISEE DCLARIAESI NIALDKTQGV TAVIENTAGQ GSNLGFKFEH LAAIIDGVED KSRVGVCIDT CHAFAAGYDL RTPAECEKTF ADFARTVGFK YLRGMHLNDA KSTFGSRVDR HHSLGEGNIG HDAFRWIMQD DRFDGIPLIL ETINPDIWAE EIAWLKAQQT EKAVA

Function and Homologs

  • Product: 5'-phosphooligonucleotide end-products.
  • Closest homologous proteins: The top (max three) homologous proteins to this protein, as identified by BLAST searches.
    • endonuclease IV (Shigella dysenteriae Sd197) [6]
      • Max score: 593
      • Query Cover: 100%
      • E-Value: 0.0
      • Ident: 100%
    • deoxyribonuclease IV (Shigella sonnei) [7]
      • Max score: 590
      • Query Cover: 100%
      • E-Value: 0.0
      • Ident: 99%
    • apurinic endonuclease (APN1) (Escherichia sp. KTE11) [8]
      • Max score: 589
      • Query Cover: 100%
      • E-Value: 0.0
      • Ident: 99%

Eg10651.png

The relation between this protein and MMSYN1_0109 that is the equivalent in the JCVI-Syn3.0 organism is one of functionality. The two proteins, which carry the same name, carry out the same functionality in DNA repair. This is why the two proteins were chosen as equivalent in the two organisms.

Expression

  • Expression Level: Medium. 281 Proteins/Cell
  • Expression Level Hypothesis: The Base Excision Repair pathway is a key repair mechanism in the E. Coli organism. In it, the endonuclease IV protein always contributes to the success of the repair of the DNA damages that occur in the organism. This being said, it is not the most abundant protein that is part of the pathway. It follows that the expression level of the protein in this organism be medium.
  • Expression Level References and Description: The information for this expression level was found through the database for the Escherichia coli (strain K12) organism's genes. The name of the protein is Endonuclease IV (nfo). In 2015 Schmidt et al reported a "quantitative proteome" of E. coli, which measures the levels of all expressed proteins under different growth conditions [9] In E.coli the major AP endonuclease is ExoIII (Ec-ExoIII) representing 90% of the cellular AP endonuclease activity, while EndoIV (Ec-EndoIV) accounts for 10% of the total activity. [10] This means to say that this protein is not as needed as its counterpart when repairing DNA. The medium expression level makes sense because of this.
  • Expression Time: Late
  • Expression Level Hypothesis: The Base Excision Repair (BER) pathway, the one the Endonuclease IV protein is a part of, is a repair mechanism in the organism that repairs spontaneous DNA damages that do not significantly distort the DNA helix structure. Because this pathway focuses on smaller DNA lesions that are the effect of time and decay, it follows that this protein (and this pathway) should be expressed late on in the organism's life as it is not too pressing to take care of these repairs as part of the organism's lifestyle.
  • Expression Time References and Description: "Damaged bases in DNA occur due to alkylating agents, ionizing radiation or oxidants such as superoxide radicals, hydrogen peroxide and hydroxyl radicals." [11] Base excision repair (BER) is an evolutionarily conserved pathway, which could be considered the "workhorse" repair mechanism of the cell. In particular, BER corrects most forms of spontaneous hydrolytic decay products in DNA, as well as everyday oxidative and alkylative modifications to bases or the sugar phosphate backbone. [12] "Base excision repair (BER) corrects small base lesions that do not significantly distort the DNA helix structure. Such damage typically results from deamination, oxidation, or methylation. Much of the damage is the result of spontaneous decay of DNA (Lindahl 1993), although similar damage may also be caused by environmental chemicals, radiation, or treatment with cytostatic drugs". [13]

Gene Context

The AP endonuclease module is part of the Base Excision Repair (BER) mechanism/pathway that repairs damaged DNA throughout the organism's lifetime. BER is important for removing damaged bases that could otherwise cause mutations by mispairing or lead to breaks in DNA during replication. The other components in this module are: DNA glycosylases, AP endonuclease, End processing enzymes, DNA polymerases, Flap endonucleases. This section will focus on the AP endonuclease as a functional module for the protein being studied. Ko03410.png

  • Other Components: There are four types of AP endonucleases, classified according to their function and place of incision: Class I AP endonucleases, Class II AP endonucleases, Class III AP endonucleases, Class IV AP endonucleases. The protein being studied in this wiki specifically corresponds to this last class of endonuclease, which is the only type found in E. Coli. Nfo is one of two (the other being XthA) 5' AP endonucleases in Escherichia coli. Because of this, there are no other components in this pathway.
  • Possible Dependencies: DNA Glycosylases. "BER is normally defined as DNA repair initiated by lesion-specific DNA glycosylases and completed by either of the two sub-pathways: short-patch BER where only one nucleotide is replaced and long-patch BER where 2-13 nucleotides are replaced. Each sub-pathway of BER relies on the formation of protein complexes that assemble at the site of the DNA lesion and facilitate repair in a coordinated fashion." [14]
  • Process: DNA repair, removal of the AP site by an AP endonuclease
    • Inputs: A single stranded DNA, n H2O
    • Outputs: n a 5'-phosphooligonucleotide

Construct

  • Synthesis Score: The synthesis score of your construct: 1, 2,3
  • Predicted Translation Rate: Prediction of construct translation rate from the RBS calculator
  • Design Notes and Details: For example, had to use a rare codon to fix folding energy;
  • GenBank File: A link to the GenBank file. file