Recombinant Desulfotalea psychrophila Formamidopyrimidine-DNA glycosylase (mutM)

What is the role of Formamidopyrimidine-DNA glycosylase (MutM) in bacterial DNA repair?

Formamidopyrimidine DNA glycosylase (Fpg/MutM) is a functionally conserved repair enzyme that initiates the 8-oxoG repair pathway in eubacteria. It plays a critical role in removing oxidative damages in DNA, particularly 7,8-dihydro-8-oxoguanine (8-oxoG), which occurs due to reactive oxygen species produced during cellular metabolism or from environmental factors. Failure to replace 8-oxoG with the correct base can result in mutations, highlighting MutM's importance in maintaining genomic stability . In psychrophilic organisms like Desulfotalea psychrophila, this enzyme likely functions at temperatures below 0°C, adapting to permanently cold marine environments where this bacterium thrives .

How does Desulfotalea psychrophila MutM differ from mesophilic bacterial MutM proteins?

While the search results don't provide specific information on D. psychrophila MutM, we can infer that as a psychrophilic protein, it likely exhibits structural adaptations that enable activity at low temperatures compared to mesophilic homologs. D. psychrophila encodes nine putative cold shock proteins and nine potentially cold shock-inducible proteins , suggesting its proteins, including MutM, may have evolved specific amino acid compositions and conformational flexibility to function at temperatures as low as -1.8°C where the organism can grow . The genome analysis of D. psychrophila strain LSv54 revealed many striking differences compared to other sulfate reducers , which may extend to its DNA repair machinery including MutM.

What are the substrate specificities of bacterial MutM proteins?

Based on the research with Mycobacterium smegmatis, MutM proteins predominantly recognize and excise 8-oxoG lesions from DNA. Biochemical analysis of mycobacterial Fpg showed that it possesses substrate specificities similar to MutM in E. coli . MutM typically functions in conjunction with MutY (which excises misincorporated A opposite 8-oxoG) as part of a coordinated repair system. This complementary action helps prevent mutations from becoming permanent during DNA replication . In the case of D. psychrophila, its MutM would presumably function similarly but at lower temperatures.

What are the optimal conditions for expressing recombinant D. psychrophila MutM?

Given D. psychrophila's psychrophilic nature, recombinant expression of its MutM likely requires specialized conditions. For optimal expression, consider the following parameters based on the organism's characteristics:

How can one purify active recombinant D. psychrophila MutM?

For purification of active D. psychrophila MutM, a cold-adapted purification strategy is essential:

• Maintain all purification steps at 2-8°C to preserve enzymatic activity

• Include reducing agents (2-5 mM DTT or β-mercaptoethanol) to protect potential catalytic cysteine residues

• Consider using a combination of ion exchange chromatography followed by affinity chromatography

• If using His-tagged constructs, employ IMAC purification with imidazole gradients at cold temperatures

• Perform dialysis steps gradually at 4°C to remove imidazole while minimizing thermal stress on the protein
  The purification protocol should account for the potential cold-lability of the enzyme, as psychrophilic enzymes often demonstrate reduced stability at temperatures above their physiological range .

What assays are suitable for measuring D. psychrophila MutM activity?

Several established assays can be adapted for measuring D. psychrophila MutM activity:

• Oligonucleotide cleavage assay: Using synthetic DNA substrates containing 8-oxoG lesions labeled with fluorescent reporters to detect enzymatic activity at different temperatures (particularly sub-zero temperatures)

• Gel-shift assays: To measure binding affinity to damaged DNA substrates

• Base excision assays: Using HPLC or LC-MS methods to quantify the release of modified bases from DNA substrates

• Circular dichroism spectroscopy: To monitor structural changes at different temperatures and correlate with activity
  When conducting these assays, it's crucial to perform temperature-dependent studies (from -5°C to 25°C) to determine the temperature optimum and range for the psychrophilic enzyme .

How does D. psychrophila MutM activity correlate with survival at sub-freezing temperatures?

D. psychrophila has been demonstrated to survive and proliferate, at least temporarily, at temperatures down to -5°C and -10°C . The activity of DNA repair enzymes like MutM likely plays a crucial role in this cold adaptation. At subfreezing temperatures, DNA damage from reactive oxygen species can still occur, potentially at higher relative rates due to increased dissolved oxygen concentrations in cold waters. MutM activity under these extreme conditions may represent an essential adaptation that allows D. psychrophila to maintain genomic integrity despite the challenging environment.
Researchers investigating this correlation should consider designing experiments that measure MutM activity in native cell extracts at various subfreezing temperatures and correlate these findings with mutation rates and cell viability under oxidative stress conditions .

What role might D. psychrophila MutM play in astrobiology and the search for extraterrestrial life?

D. psychrophila serves as an important model organism for understanding potential life in extraterrestrial environments, particularly Mars and icy moons with similar conditions to Earth's polar regions . The functionality of critical DNA repair enzymes like MutM at subfreezing temperatures provides valuable insights into the minimum requirements for DNA maintenance in extraterrestrial environments.
Research in this area should focus on:

• Testing D. psychrophila MutM activity in simulated Martian regolith containing various sulfate minerals

• Examining how radiation levels typical of Martian or icy moon surfaces affect MutM function

• Investigating whether D. psychrophila MutM contains structural adaptations that might be universal requirements for cold-adapted DNA repair enzymes across different planetary environments
  These investigations could provide valuable biomarkers for astrobiology missions searching for life in cold, sulfate-rich environments throughout the solar system .

How does oxidative stress response in D. psychrophila compare to other extremophiles regarding MutM function?

Unlike hyperthermophilic organisms like Archaeoglobus fulgidus (another sulfate reducer), D. psychrophila has evolved to handle oxidative stress at cold temperatures . This likely requires unique adaptations in its DNA repair machinery, including MutM. A comparative analysis of mutation patterns between D. psychrophila and other extremophiles would be valuable.
For example, in Mycobacterium smegmatis, Fpg (MutM) deficiency leads to enhanced mutator phenotype and susceptibility to hydrogen peroxide, with a predominance of A to G (or T to C) mutations, unlike E. coli where C to A mutations predominate . D. psychrophila may show yet another distinct mutation spectrum under oxidative stress due to its psychrophilic adaptations and specific genomic G+C content.

How did the MutM repair pathway evolve in cold-adapted bacteria?

The evolution of MutM in cold-adapted bacteria like D. psychrophila likely involved selecting for variants that maintain catalytic efficiency at low temperatures while potentially sacrificing thermal stability. D. psychrophila's genome contains more than 30 two-component regulatory systems, which suggest sophisticated adaptation mechanisms to environmental stresses . The evolutionary history of MutM in this organism may reveal important insights about adaptation to extreme cold.
A phylogenetic analysis comparing MutM sequences across psychrophilic, mesophilic, and thermophilic bacteria could identify specific amino acid substitutions associated with cold adaptation. These findings could contribute to understanding the evolutionary constraints on DNA repair systems in extreme environments.

What does genome analysis reveal about the base excision repair network in D. psychrophila?

The genome sequence of D. psychrophila strain LSv54 consists of a 3,523,383 bp circular chromosome with 3,118 predicted genes and two plasmids . Although specific analysis of the base excision repair network isn't detailed in the available search results, we can infer that as a psychrophilic organism living in permanently cold marine sediments, D. psychrophila likely possesses a robust DNA repair system including MutM.
A comprehensive analysis of the D. psychrophila genome for all base excision repair (BER) components would help identify whether this organism employs unique strategies for handling DNA damage at low temperatures. Such analysis should include identification of all glycosylases, AP endonucleases, DNA polymerases, and ligases involved in the BER pathway, with special attention to cold-specific adaptations .

What are common challenges when working with recombinant psychrophilic enzymes like D. psychrophila MutM?

Researchers working with recombinant psychrophilic enzymes frequently encounter these challenges:

• Thermal instability: Psychrophilic enzymes often denature at moderate temperatures (sometimes even room temperature)

• Expression difficulties: Low yields when expressed in mesophilic hosts due to protein misfolding

• Activity measurement: Standard enzyme assays may require modification for low-temperature conditions

• Storage considerations: Rapid activity loss during freeze-thaw cycles or extended storage
  To address these challenges, researchers should consider using specialized expression systems for cold-adapted proteins, performing all experimental procedures at reduced temperatures, and developing appropriate buffer conditions that enhance enzyme stability without compromising activity .

How can temperature-activity profiles be accurately determined for D. psychrophila MutM?

Accurate determination of temperature-activity profiles for psychrophilic enzymes requires specialized approaches:

• Use temperature-controlled reaction chambers capable of precise regulation from -10°C to 40°C

• Include cryoprotectants such as glycerol in reaction buffers to prevent freezing at sub-zero temperatures

• Apply longer incubation times at lower temperatures to compensate for reduced reaction rates

• Include appropriate controls with mesophilic MutM homologs to benchmark relative activities
  The experimental design should account for both the absolute activity at each temperature and the stability of the enzyme over time at those temperatures. This dual approach provides important insights into the enzyme's adaptation to cold environments and its potential utility in various research applications .

What are promising applications for recombinant D. psychrophila MutM in genomic research?

Recombinant D. psychrophila MutM offers several promising applications in genomic research:

• Development of cold-active enzymes for low-temperature DNA manipulation and sequencing

• Creation of specialized DNA repair cocktails for ancient DNA research where sample heating is detrimental

• Investigation of structural determinants that confer cold activity without sacrificing catalytic efficiency

• Study of DNA damage recognition mechanisms under extreme environmental conditions
  The psychrophilic nature of this enzyme may provide unique advantages for specific biotechnological applications requiring low-temperature processing of nucleic acids .

How might D. psychrophila MutM contribute to understanding mutation patterns in permanently cold environments?

D. psychrophila MutM likely plays a key role in determining mutation patterns in permanently cold environments. As seen in Mycobacterium smegmatis, MutM deficiency can lead to specific mutation signatures—A to G (or T to C) mutations, with a shift toward C to G (or G to C) mutations under oxidative stress .
Future research should examine whether similar or different mutation signatures occur in D. psychrophila under various stress conditions, especially at temperatures approaching the lower limit of the organism's growth range. Such studies would provide insights into how DNA repair mechanisms shape bacterial evolution in extreme environments and potentially reveal previously unknown mechanisms of genomic stability maintenance in psychrophiles .