Crystal structure, biochemical and cellular activities demonstrate separate functions of MTH1 and MTH2

Area of Science:

• Biochemistry
• Molecular Biology
• Cancer Research

Background:

• Cancer involves deregulated redox metabolism, causing oxidative damage to cellular components like deoxyribonucleoside triphosphates (dNTPs).
• Targeting dNTP pool sanitizing enzymes, particularly MTH1, is a promising anticancer strategy.
• NUDT15 (MTH2) is considered a human homologue of bacterial MutT with 8-oxo-dGTPase activity.

Purpose of the Study:

• To investigate the substrate specificity and biological relevance of NUDT15 as an 8-oxo-dGTPase.
• To compare the activity of NUDT15, NUDT17, and NUDT18 with MTH1 in sanitizing the dNTP pool.
• To elucidate the structural basis for substrate preference differences between NUDT15 and MTH1.

Main Methods:

• X-ray crystallography to determine the NUDT15 structure.
• Enzyme activity assays to assess substrate preferences.
• Depletion studies (e.g., siRNA or CRISPR) to evaluate the impact of NUDT15 on cancer cells.
• Profiling of NUDT17 and NUDT18 activities.

Main Results:

• The crystal structure of NUDT15 was determined, revealing key features influencing substrate binding.
• NUDT15 demonstrated a preference for nucleotide substrates other than 8-oxo-dGTP.
• Depletion of NUDT15 did not affect 8-oxo-dGTP incorporation into DNA or cancer cell survival.
• NUDT17 and NUDT18 exhibited significantly lower activity against oxidized nucleotides compared to MTH1.

Conclusions:

• NUDT15 is not a biologically significant 8-oxo-dGTPase in the context of cancer cell survival.
• MTH1 remains the most important enzyme for sanitizing the cellular dNTP pool against oxidative damage.
• Structural differences explain the distinct substrate specificities of NUDT15 and MTH1.