Replication across template T/U by human DNA polymerase-iota.

Area of Science:

• Biochemistry
• Molecular Biology
• Enzymology

Background:

• Human DNA polymerase-iota (Poliota) exhibits preferential nucleotide incorporation opposite template purines.
• Poliota demonstrates significantly lower efficiency and fidelity when incorporating nucleotides opposite template pyrimidines, particularly thymine (T).

Purpose of the Study:

• To elucidate the structural basis for Poliota's low fidelity opposite template T/U.
• To understand the mechanism of preferential incorporation of dGTP over dATP opposite template T/U.

Main Methods:

• Analysis of Poliota active site accommodation of template T/U in binary and ternary complexes.
• Investigation of the conformational states of template T/U in the presence and absence of incoming nucleotides.
• Identification of specific molecular interactions governing nucleotide selection.

Main Results:

• Template T/U adopts both syn and anti conformations in binary complexes but predominantly the anti conformation in ternary complexes with dATP or dGTP.
• dATP and dGTP exhibit distinct insertion mechanisms opposite template T/U.
• A key hydrogen bond between the N2 amino group of dGTP and Gln59 of Poliota dictates the preferential incorporation of dGTP over dATP.

Conclusions:

• The conformational flexibility of template T/U and specific active site interactions are critical for Poliota's fidelity.
• A Gln59-dGTP hydrogen bond explains the observed bias towards incorporating incorrect guanine over adenine opposite template T/U, highlighting a novel mechanism in DNA repair fidelity.