Recombinant Methanococcus maripaludis N-glycosylase/DNA lyase (MMP0304)
Description
Introduction to Recombinant Methanococcus maripaludis N-glycosylase/DNA lyase (MMP0304)
The compound Recombinant Methanococcus maripaludis N-glycosylase/DNA lyase (MMP0304) refers to an enzyme derived from the archaeon Methanococcus maripaludis. This enzyme exhibits both N-glycosylase and DNA lyase activities, indicating its involvement in DNA repair mechanisms. M. maripaludis is a methanogenic archaeon, well-studied for its genetic accessibility and rapid growth, making it a valuable model organism in laboratory settings .
2.1. Glycosylase Activity
N-glycosylases are enzymes that catalyze the hydrolysis of the N-glycosidic bond between a base and the deoxyribose sugar in DNA. This activity is crucial for removing damaged or modified bases, such as 8-oxoguanine (GO), from the DNA backbone . In Escherichia coli, the GO-system, which includes enzymes like MutM (also known as Fpg), functions as a DNA glycosylase/lyase to excise GO and its ring-opened form, formamidopyrimidine (Fapy), from base pairs with cytosine .
2.2. AP Lyase Activity
AP lyases catalyze the cleavage of the phosphodiester backbone at abasic sites (AP sites) in DNA. These sites occur when a base is removed, but the sugar-phosphate backbone remains intact. Some DNA glycosylases, like Tb-AGOG from Thermococcus barophilus Ch5, possess both glycosylase and AP lyase activities, acting bifunctionally in DNA repair . Studies on Tb-AGOG have identified specific residues crucial for AP lyase activity, such as E79, K163, Y174, and D229 .
3.1. Repair of Oxidative DNA Damage
Organisms like Pyrobaculum aerophilum, which thrive at high temperatures, require efficient DNA repair mechanisms to counteract the increased rates of spontaneous hydrolysis and oxidation . P. aerophilum expresses a GO-glycosylase/lyase that effectively removes aberrant bases from both single- and double-stranded DNA .
3.2. NrpR Regulation
Nitrogen assimilation in Methanococcus maripaludis is regulated by transcriptional repression involving a palindromic 'nitrogen operator' repressor binding sequence. The nitrogen repressor, NrpR, isolated from M. maripaludis, plays a key role in this regulation. Deletion of the nrpR gene results in the loss of nitrogen operator binding activity and the loss of repression of nif (nitrogen-fixation) and glnA (glutamine synthetase) gene expression in vivo .
4.1. Markerless Mutagenesis
Markerless mutagenesis techniques are employed to create precise genetic alterations without leaving foreign DNA sequences in the genome . This is particularly useful in M. maripaludis to study gene function. The method uses a negative selection strategy based on the M. maripaludis hpt gene, which encodes hypoxanthine phosphoribosyltransferase, conferring sensitivity to 8-azahypoxanthine .
4.2. CRISPR-Cas12a System
The CRISPR-Cas12a system has been developed for efficient genome editing in Methanococcus maripaludis . This system utilizes Cas12a from Lachnospiraceae bacterium ND2006 (LbCas12a) in combination with the homology-directed repair machinery present in M. maripaludis . It allows for gene deletions with high success rates, even given the hyperpolyploidy of M. maripaludis .
Product Specs
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Target Background
KEGG: mmp:MMP0304
STRING: 267377.MMP0304
Q&A
What is MMP0304 and what is its primary function in Methanococcus maripaludis?
MMP0304 is an N-glycosylase/DNA lyase enzyme found in the archaeon Methanococcus maripaludis S2. Based on functional annotations and sequence homology, it belongs to a family of DNA repair enzymes that participate in base excision repair (BER) pathways. The enzyme exhibits dual functionality:
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N-glycosylase activity: Recognizes and removes damaged DNA bases by cleaving the N-glycosidic bond
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DNA lyase activity: Catalyzes the cleavage of the DNA backbone at the resulting abasic site via β-elimination
MMP0304 appears in multiple functional modules (22, 33, 43, 152) within the M. maripaludis gene network , suggesting it plays multifaceted roles in DNA maintenance pathways within this archaeal species.
How does MMP0304 compare to other archaeal DNA repair enzymes?
MMP0304 shares functional similarities with other archaeal DNA repair enzymes but has some distinct characteristics:
Unlike some bacterial DNA glycosylases that catalyze β,δ-elimination (e.g., E. coli MutM), archaeal enzymes like MMP0304 typically generate 3′-α,β unsaturated aldehyde termini through β-elimination, similar to human OGG1 .
What are the optimal conditions for MMP0304 activity?
While specific conditions for MMP0304 have not been directly reported, optimal conditions can be inferred from studies on similar archaeal DNA repair enzymes and M. maripaludis growth characteristics:
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Temperature: Likely 30-40°C, as M. maripaludis grows optimally at 37°C
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pH: Neutral to slightly alkaline (pH 7.0-8.0)
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Buffer composition: Typically contains:
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20-50 mM Tris-HCl or HEPES
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50-100 mM NaCl or KCl
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1-5 mM DTT or β-mercaptoethanol
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1-10 mM EDTA
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5-10% glycerol for stability
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Unlike mesophilic enzymes, MMP0304 may exhibit higher thermostability reflective of its archaeal origin, though M. maripaludis is not a hyperthermophile like some other archaea.
What are effective strategies for recombinant expression of MMP0304?
Several expression systems have been successfully used for archaeal proteins, with the following considerations specifically relevant to MMP0304:
