A sensor complements the steric gate when DNA polymerase ϵ discriminates ribonucleotides

Area of Science:

• Molecular Biology
• Biochemistry
• Structural Biology

Background:

• Cellular environments have high ribonucleotide (NTP) and low deoxyribonucleotide (dNTP) concentrations.
• DNA polymerases must discriminate against NTPs to prevent DNA synthesis errors.
• The steric gate model in B-family DNA polymerases proposes discrimination via a tyrosine clash with the ribonucleotide's 2'-hydroxyl.

Purpose of the Study:

• To elucidate the mechanism by which DNA polymerases sense and discriminate against NTPs.
• To investigate the role of the finger domain in ribonucleotide discrimination.
• To challenge and refine existing models of nucleotide discrimination in DNA polymerases.

Main Methods:

• X-ray crystallography of B-family DNA polymerase with UTP or CTP.
• Molecular dynamics simulations.
• Biochemical assays and yeast genetics.

Main Results:

• Identified a novel mechanism for NTP sensing in the finger domain of DNA polymerases.
• Demonstrated that steric hindrance, not a polar filter, is mediated by an amino acid residue in the finger domain.
• Confirmed that both the finger domain sensor and the palm domain steric gate are crucial for NTP discrimination.
• Found the identified sensor residue to be conserved across B-family polymerases.

Conclusions:

• DNA polymerase discrimination against NTPs involves both a steric gate in the palm domain and a steric hindrance sensor in the finger domain.
• The finger domain sensor mechanism is likely conserved among B-family DNA polymerases and potentially other polymerase types.
• This finding refines our understanding of DNA synthesis fidelity and error prevention mechanisms.