NEIL1 Human

What is the fundamental structure and function of human NEIL1?

NEIL1 is a DNA glycosylase that recognizes and excises oxidized DNA bases as part of the base excision repair (BER) pathway. Unlike its bacterial homolog, NEIL1 contains a unique "zincless finger" structural motif composed of two antiparallel β-strands that mimics the antiparallel β-hairpin zinc finger found in other Fpg/Nei family members but lacks zinc-binding residues . This structural feature appears critical for glycosylase activity, as mutations to conserved residues within this motif significantly reduce enzymatic function . The protein also contains an N-terminal domain harboring catalytic residues P2, E3, and K54, and DNA binding motifs including a helix-two-turns-helix (H2TH) region .

What are the primary DNA substrates recognized by NEIL1?

NEIL1 exhibits versatile substrate specificity, recognizing:

• Oxidized pyrimidines in DNA

• Hydantoin lesions

• Formamidopyrimidines

• Thymine residues oxidized at the methyl group

• Stereoisomers of thymine glycol

• 5-carboxylcytosine (5caC), an intermediate in DNA demethylation pathways

Methodological considerations: Substrate specificity varies with DNA structure. NEIL1 has been reported to be active on single-stranded DNA and DNA with bubble or forked structures, though some studies suggest higher efficiency on single-stranded substrates . When testing NEIL1 activity, researchers should employ both single- and double-stranded DNA substrates containing various oxidized bases.

How does NEIL1 deficiency affect physiological functions in animal models?

NEIL1-deficient mice exhibit multiple phenotypes:

• Impaired memory retention in water maze tests without abnormalities in motor performance, anxiety, or fear conditioning

• Increased brain damage and worsened functional outcomes in focal ischemia/reperfusion models of stroke

• Development of metabolic syndrome including severe obesity, dyslipidemia, fatty liver disease, and hyperinsulinemia

Research approach: Behavioral testing combined with histological assessment of brain damage following ischemic injury has established NEIL1's neuroprotective role . Incision capacity assays on substrates containing oxidized bases (such as 5-hydroxyuracil) can reveal differences in DNA repair efficiency between wild-type and NEIL1-deficient tissues .

What techniques are most effective for characterizing NEIL1 enzymatic activity?

Several complementary approaches are recommended:

Critical controls: Include catalytically inactive NEIL1 mutants (P2T or E3Q variants) to confirm specificity . The P2T variant can still bind DNA substrates, making it useful for distinguishing binding from catalytic activity .

How can researchers effectively study NEIL1-protein interactions?

Multiple analytical approaches are required:

• Far-western analysis: Use purified recombinant histidine-tagged full-length NEIL1 (NEIL1-FL) and truncated polypeptides (NEIL1-∆40, NEIL1-∆56, NEIL1-∆78, NEIL1-∆100) to map interaction domains

• Size exclusion chromatography (SEC): Evaluate complex formation under various solvent conditions. Note that interactions may be sensitive to salt concentration (e.g., 300 mM NaCl may not support complex formation between NEIL1 and TFAM)

• SEC-MALS-SAXS methods: Determine absolute molar mass and stoichiometry of binding. This approach has demonstrated that NEIL1 and TFAM form a ternary complex only in the presence of DNA, with a molar mass of approximately 91.2 kDa

• Hydrogen-deuterium exchange mass spectrometry (HDX-MS): Identify regions involved in binding interactions. HDX-MS analysis over a time course (30 seconds to 30 minutes) can reveal protection patterns indicating binding interfaces

What techniques best elucidate NEIL1's role in DNA demethylation pathways?

NEIL1's involvement in demethylation requires specialized approaches:

• Direct enzymatic assays: Test NEIL1's ability to excise 5caC from DNA in comparison to TDG. Current evidence shows NEIL1 exhibits detectable but comparatively weak glycosylase activity against 5caC-containing DNA

• Structural modeling: Based on crystal structures of NEIL1 with other substrates (like thymine glycol), researchers can construct models of Michalis complexes with 5caC-containing DNA. Models suggest the NEIL1 base pocket can accommodate 5caC, with potential salt bridge formation between R257 and the carboxylate group of 5caC

• Cooperative function assays: Investigate how NEIL1 stimulates TDG activity on 5fC and 5caC substrates. Importantly, this stimulation appears specific to demethylation intermediates and does not require NEIL1's glycosylase or lyase activities

• Reporter assays: Test wild-type cells versus TDG-deficient cells for their ability to rescue expression from methylated and TET-oxidized plasmids, with and without NEIL1 expression

How do NEIL1 variants affect DNA repair efficiency and disease susceptibility?

Methodological approach:

• Express recombinant wild-type and variant NEIL1 proteins

• Compare glycosylase/lyase activities on defined substrates

• Measure DNA binding affinity through EMSA

• Assess structural impacts through crystal structure analysis or modeling

• Correlate biochemical findings with phenotypic data from model systems

The significance of NEIL1 variants is highlighted by the finding that NEIL1 substrate specificity can be altered by ADAR1-catalyzed RNA editing, leading to enzymes with either lysine or arginine (K242 and R242) forming a side wall of the active site pocket . These alterations affect substrate recognition and processing efficiency.

What are the best experimental designs to investigate NEIL1's role in mitochondrial DNA maintenance?

Given NEIL1's interaction with mitochondrial transcription factor A (TFAM) , researchers should consider:

• Subcellular fractionation: Isolate mitochondria to assess NEIL1 localization and activity

• Enzymatic assays with mitochondrial lysates: Compare incision capacity on oxidized substrates between wild-type and NEIL1-deficient mitochondrial extracts. Studies have shown lower incision capacity in mitochondrial lysates from unstressed old NEIL1-deficient mice

• Interaction studies: Use far-western analysis and SEC-MALS-SAXS to characterize NEIL1-TFAM interactions. Current research indicates that NEIL1 interacts with TFAM via its N-terminal regions, including protein lacking the established protein interaction domain

• Environmental factors: Consider that interactions between NEIL1 and TFAM can be modulated by local environment such as salt concentrations and protein availability

How should researchers investigate NEIL1's neuroprotective functions?

Based on findings that NEIL1 deficiency results in greater brain damage following ischemic stroke , recommended approaches include:

• Focal ischemia/reperfusion models: Compare infarct volume and functional outcomes between wild-type and NEIL1-deficient mice

• Behavioral testing: Employ water maze tests to assess memory retention, complemented by tests of motor performance, anxiety, and fear conditioning

• Biochemical analyses: Measure oxidative DNA damage accumulation and repair capacity in different brain regions

• Mechanistic studies: Investigate how neuronal excitation affects NEIL1 activity, as glutamate receptor activation has been linked to both DNA damage and repair enhancement

What controls and validation steps are essential when studying NEIL1 enzymatic activity?

Essential controls include:

• Catalytically inactive NEIL1 mutants (P2T or E3Q variants)

• DNA binding assays to distinguish binding from catalysis

• Comparison with other glycosylases (particularly TDG) on the same substrates

• Activity assays under varying buffer conditions to establish optimal parameters

Validation approaches:

• HDX-MS to confirm protection patterns upon DNA binding. Key protected regions include residues 2-28, 79-93, 163-180, 183-198, 228-255, and 256-271

• Crystal structure validation to confirm that peptides showing protection in HDX-MS are indeed present within the interaction interface of the NEIL1-DNA complex

How can researchers accurately characterize complex formation between NEIL1 and other proteins?

When studying NEIL1's interactions with proteins like TFAM, several technical considerations emerge:

• Buffer conditions significantly impact complex detection: In SEC studies, NEIL1 and TFAM elute as separate peaks in solvent containing 300 mM NaCl, suggesting no complex formation under these conditions

• DNA presence may be required: SEC-MALS analysis reveals complex formation only in the presence of DNA, with a higher molar mass peak of 91.2 kDa corresponding to a potential ternary complex of TFAM-NEIL1-DNA

• Stoichiometry determination requires advanced techniques: The ternary complex may have a stoichiometric ratio of either 1:1:1 (theoretical MW 83.9 kDa) or 1:1:2 (theoretical MW 97.5 kDa)

• Transient interactions require specialized detection: Given the likely transient nature of these interactions, there may be only small fractions of complex present in equilibrated mixtures, necessitating sensitive detection methods beyond standard SEC