MMSYN1 0109 andreavz

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Author Information

Andrea Villarreal

Basic Information

  • ID: MMSYN1_0109
  • Name: apurinic endonuclease (APN1), deoxyribonuclease IV [Mycoplasma mycoides]

0109.png

  • Organism: JCVI-Syn3.0, Mycoplasma mycoides subsp. capri str. GM12
  • UniProt ID: F4MNZ7 (F4MNZ7_MYCML) [1]
  • Description:
  • Function:
This protein is a probable endonuclease IV (endonucleases are enzymes that cleave the phosphodiester bond within a polynucleotide chain), plays a role in DNA repair. "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." [2] AP endonuclease, specifically, catalyzes the incision of DNA exclusively at AP sites, and therefore prepares DNA for subsequent excision, repair synthesis and DNA ligation.[3]
"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." [5] This protein binds 3 Zn2+ ions and can also bind Mn2+ ions.
  • 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.
  • Pathway: The protein in question belongs to the Base Excision Repair (BER) pathway.
  • DNA Length: 867 base pairs.
  • DNA sequence:

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

  • Amino Acid length: 289 amino acids.
  • Amino Acid sequence:

MNKVLLGCHVSMNKQNNYLVGSVNEAISYKANTFMIFTGPPQSTLRTNTNHLYINQMHELMNSYKIDAKDLVVHAPYIINIANSVDQNKWKFAVDFLIQEIKRCEEIKIPTLVLHPGSHTTGNYKDSLNQIIKALDIVSNYQVNVKIALETMSGKGTEVCSKLEDFKYILDNVKNKDKVGVCLDTCHLHDAGYDLSKWDEFKEQMKQNFDLNKVLCIHLNDSKNMISSHKDRHANIGYGYVGFDTLVNVVFDKDFSNISKILETPYIDKKPPYKIEIEDLLNKTFTNRL

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.
    • deoxyribonuclease IV (Mycoplasma capricolum) [6]
      • Max score: 584
      • Query Cover: 100%
      • E-Value: 0.0
      • Ident: 98%
    • deoxyribonuclease IV (Mycoplasma leachii) [7]
      • Max score: 580
      • Query Cover: 100%
      • E-Value: 0.0
      • Ident: 97%
    • endonuclease (Mycoplasma capricolum) [8]
      • Max score: 575
      • Query Cover: 100%
      • E-Value: 0.0
      • Ident: 96%

Compare0109.png

Although the most probable answer for this piece of the page is deoxyribonuclease IV [Escherichia coli], this protein has an 89% query cover and a 30% ident. These numbers don't indicate a lot of similarities in the amino acid sequence, but the functionality between the E. Coli equivalent and this one for Mycoplasma Mycoides is extremely similar.

Expression

  • Expression Level: High. Proteins/Cell: 550
The expression level of this protein was searched for using two names the protein goes by:
  • Deoxyribonuclease IV: The name of the protein after blasting the amino acid sequence. The Mycoplasma genitalium organism has a protein with the same name with 550 Proteins/Cell.
  • Apurinic Endonuclease: The name in the JCVI database. The Mycoplasma genitalium organism has a protein with the same name with 435 Proteins/Cell.
These proteins all have high expression levels, which means the protein chosen to be most similar to the one studied in this wiki doesn't matter. However, for the sake of choosing, Deoxyribonuclease will be chosen based on its perfect match when blasting the sequence.
  • Expression Level Hypothesis: The Base Excision Repair pathway is a key repair mechanism in the Mycoplasma Mycoides organism. In it, the endonuclease IV protein always contributes to the success of the repair of the DNA damages that occur in the organism. While the BER pathway can divide into longer or shorter repairs, the protein is used in both sub-pathways to create a nick in the backbone of the AP site. It follows that the protein should be expressed in a high level for this organism.
  • Expression Level References and Description: The information for this expression level was found through the database for the Mycoplasma genitalium organism's genes. Accurate measurements of protein levels in Mycoplasma mycoides are difficult to come by, so this expression level is based on the Mycoplasma genitalium organism. The equivalent gene that encodes for pdhC does have a known expression level. Since these genes have the same function and produce the same protein, it is inferred that the expression level will be the same. [9]
  • 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." [10] 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. [11] "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". [12]

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 Mycoplasma Mycoides. 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." [13]
  • 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