Structural and functional insight into mismatch extension by human DNA polymerase α

Andrey G. Baranovskiy, Nigar D. Babayeva, Alisa E. Lisova, Lucia M. Morstadt, Tahir H. Tahirov

Research output: Contribution to journalArticlepeer-review

3 Scopus citations

Abstract

Human DNA polymerase α (Polα) does not possess proofreading ability and plays an important role in genome replication and mutagenesis. Polα extends the RNA primers generated by primase and provides a springboard for loading other replication factors. Here we provide the structural and functional analysis of the human Polα interaction with a mismatched template:primer. The structure of the human Polα catalytic domain in the complex with an incoming deoxycytidine triphosphate (dCTP) and the template: primer containing a T-C mismatch at the growing primer terminus was solved at a 2.9 Å resolution. It revealed the absence of significant distortions in the active site and in the conformation of the substrates, except the primer 30-end. The T-C mismatch acquired a planar geometry where both nucleotides moved toward each other by 0.4 Å and 0.7 Å, respectively, and made one hydrogen bond. The binding studies conducted at a physiological salt concentration revealed that Polα has a low affinity to DNA and is not able to discriminate against a mispaired template:primer in the absence of deoxynucleotide triphosphate (dNTP). Strikingly, in the presence of cognate dNTP, Polα showed a more than 10-fold higher selectivity for a correct duplex versus a mismatched one. According to pre-steady-state kinetic studies, human Polα extends the T-C mismatch with a 249-fold lower efficiency due to reduction of the polymerization rate constant by 38-fold and reduced affinity to the incoming nucleotide by 6.6-fold. Thus, a mismatch at the postinsertion site affects all factors important for primer extension: affinity to both substrates and the rate of DNA polymerization.

Original languageEnglish (US)
Article number2111744119
JournalProceedings of the National Academy of Sciences of the United States of America
Volume119
Issue number17
DOIs
StatePublished - Apr 26 2022

Keywords

  • DNA polymerase α
  • DNA replication
  • crystal structure
  • kinetic studies
  • mismatch

ASJC Scopus subject areas

  • General

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