MUTM E.Coli

Formamidopyrimidine-DNA Glycosylase E.Coli Recombinant
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Cat. No.
BT22849
Source
E.coli.
Synonyms
Formamidopyrimidine-DNA glycosylase, FPG.
Appearance
Sterile Filtered colorless solution.
Purity
Greater than 90% as determined by SDS-PAGE.
Usage
THE BioTek's products are furnished for LABORATORY RESEARCH USE ONLY. The product may not be used as drugs, agricultural or pesticidal products, food additives or household chemicals.
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Description

Enzymatic Activities

MUTM operates through three interconnected functions to repair oxidative DNA damage:

  1. DNA Glycosylase Activity: Excises oxidized purines (e.g., 8-oxo-7,8-dihydroguanine, Fapy lesions) and some pyrimidine derivatives .

  2. AP Lyase Activity:

    • β-elimination: Cleaves the 3’-phosphodiester bond at abasic sites.

    • δ-elimination: Cleaves the 5’-phosphodiester bond, generating a 3’-phosphate terminus .

  3. Schiff Base Intermediate Formation: The N-terminal proline (Pro1) initiates catalysis via a covalent intermediate with damaged DNA .

Biological Role in Oxidative Damage Repair

MUTM collaborates with MutY and MutT in a tripartite defense system against 8-oxoguanine (8-oxoG)-induced mutations:

ProteinFunctionMutation Prevented
MutM (FPG)Removes 8-oxoG paired with cytosineG:C → T:A transversions
MutYExcises adenine mispaired with 8-oxoG or guanineA:T → C:G transversions
MutTHydrolyzes 8-oxo-dGTP in nucleotide pools, preventing incorporation into DNAA:T → C:G transversions

  • Synergistic Effects: Deletion of both mutM and mutY increases G:C → T:A transversions by 800-fold compared to wild-type E. coli .

  • Repair Efficiency: MUTM corrects ~10,000 lesions per cell daily under normal oxidative stress .

Research Findings and Applications

  • Mutation Spectrum Analysis:

    • Strains lacking mutM show no significant mutation rate increase alone but exhibit 10-fold higher G:C → T:A transversions when combined with mutY deletions .

    • MUTM repairs 8-oxoG lesions 50 times faster than endogenous DNA polymerases replicate past them .

  • Industrial Relevance:

    • Recombinant MUTM (e.g., ProSpec Bio ENZ-589) is utilized in vitro to study oxidative DNA damage mechanisms .

    • Insights from MUTM’s repair pathways inform synthetic biology approaches to enhance bacterial genome stability .

Interaction with Other Repair Systems

MUTM’s activity intersects with mismatch repair (MMR) and nucleotide excision repair:

  • MMR Synergy: While MMR primarily corrects replication errors, MUTM addresses oxidative lesions unaddressed by MutS/MutL systems .

  • Dam Methylation Sites: MUTM-independent mutations at Dam (5’-GATC-3’) sites highlight alternative repair bottlenecks .

Product Specs

Introduction
MUTM, a crucial base excision repair enzyme, plays a vital role in identifying and removing various oxidized purines from damaged DNA. Its non-dismissable nature highlights its importance in rapidly repairing substrate lesions on chromosomes. Moreover, MUTM effectively repairs a significant portion of lesions recognized by Endo III, indicating its substantial involvement in the overall repair of both purine and pyrimidine damage within living organisms.
Description
Recombinantly produced in E. coli, MUTM is a single polypeptide chain consisting of 289 amino acids (specifically, amino acids 1 to 269). It possesses a molecular mass of 32.4kDa. For purification purposes, a 20 amino acid His-tag is fused to the N-terminus of MUTM, followed by purification using proprietary chromatographic techniques.
Physical Appearance
A clear and sterile solution.
Formulation
The MUTM solution, provided at a concentration of 0.5mg/ml, is formulated with the following components: 20mM Tris-HCl buffer (pH 8.0), 100mM Nacl, 1mM DTT, and 20% glycerol.
Stability
For short-term storage (2-4 weeks), the MUTM vial should be stored at 4°C. For extended storage periods, freezing at -20°C is recommended. To further enhance long-term stability, adding a carrier protein (0.1% HSA or BSA) is advisable. Minimize repeated freeze-thaw cycles.
Purity
SDS-PAGE analysis demonstrates a purity exceeding 90%.
Synonyms
Formamidopyrimidine-DNA glycosylase, FPG.
Source
E.coli.
Amino Acid Sequence
MGSSHHHHHH SSGLVPRGSH MPELPEVETS RRGIEPHLVG ATILHAVVRN GRLRWPVSEE IYRLSDQPVL SVQRRAKYLL LELPEGWIII HLGMSGSLRI LPEELPPEKH DHVDLVMSNG KVLRYTDPRR FGAWLWTKEL EGHNVLTHLG PEPLSDDFNG EYLHQKCAKK KTAIKPWLMD NKLVVGVGNI YASESLFAAG IHPDRLASSL SLAECELLAR VIKAVLLRSI EQGGTTLKDF LQSDGKPGYF AQELQVYGRK GEPCRVCGTP IVATKHAQRA TFYCRQCQK

Product Science Overview

Classification and Structure

Fpg is classified as a bifunctional DNA glycosylase with both N-glycosylase and apurinic/apyrimidinic (AP) lyase activities . The enzyme contains a zinc-finger motif near its C-terminal, which is essential for its DNA-binding and catalytic activities .

Biological Properties

Fpg recognizes and excises oxidatively damaged purines, such as 8-oxo-7,8-dihydroguanine (8-oxoG) and 2,6-diamino-4-hydroxy-5-formamidopyrimidine (FapyG) . The N-glycosylase activity of Fpg cleaves the N-glycosidic bond, releasing the damaged base and creating an AP site. The AP lyase activity then cleaves the DNA backbone at the AP site via β- and δ-elimination, resulting in a single-nucleotide gap with 3’ and 5’ phosphate termini .

Expression Patterns and Tissue Distribution

In E. coli, the expression of Fpg is regulated by the soxRS and oxyR regulons, which are activated in response to oxidative stress . The recombinant form of Fpg is typically expressed in E. coli strains engineered to overproduce the enzyme for research and industrial applications .

Biological Functions and Modes of Action

The primary function of Fpg is to protect cells from the mutagenic effects of oxidative DNA damage. By excising damaged bases and initiating the BER pathway, Fpg helps maintain the integrity of the genetic material . This process is vital for preventing mutations that could lead to various diseases, including cancer.

Regulatory Mechanisms

The activity of Fpg is tightly regulated by cellular oxidative stress levels. The soxRS and oxyR regulons control the transcription of the fpg gene, ensuring that the enzyme is produced in response to increased oxidative damage . Additionally, post-translational modifications and interactions with other DNA repair proteins may further modulate Fpg activity.

Applications

Recombinant Fpg is widely used in research to study DNA repair mechanisms and to assess oxidative DNA damage in various experimental systems . Its ability to recognize and excise specific damaged bases makes it a valuable tool for understanding the molecular basis of DNA repair and the effects of oxidative stress on genomic stability.

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