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

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dc.contributor.authorBaranovskiy, A.G.
dc.contributor.authorBabayeva, N.D.
dc.contributor.authorLisova, A.E.
dc.contributor.authorMorstadt, L.M.
dc.contributor.authorTahirov, T.H.
dc.date.issued2022
dc.description.abstractHuman 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 3′-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.
dc.description.statementofresponsibilityAndrey G. Baranovskiya, Nigar D. Babayevaa, Alisa E. Lisovaa, Lucia M. Morstadta, and Tahir H. Tahirova
dc.identifier.citationProceedings of the National Academy of Sciences of USA, 2022; 119(17):e2111744119-1-e2111744119-7
dc.identifier.doi10.1073/pnas.2111744119
dc.identifier.issn0027-8424
dc.identifier.issn1091-6490
dc.identifier.orcidLisova, A.E. [0000-0002-3647-6460]
dc.identifier.urihttps://hdl.handle.net/2440/146472
dc.language.isoen
dc.publisherThe National Academy of Sciences (NAS)
dc.rights© 2022 the Author(s). Published by PNAS. This article is distributed under Creative Commons Attribution-NonCommercial-NoDerivatives License 4.0 (CC BY-NC-ND).
dc.source.urihttps://doi.org/10.1073/pnas.2111744119
dc.subjectDNA polymerase α; DNA replication; crystal structure; mismatch; kinetic studies
dc.subject.meshHumans
dc.subject.meshDNA Polymerase I
dc.subject.meshDNA Primers
dc.subject.meshDNA Replication
dc.subject.meshCatalytic Domain
dc.subject.meshKinetics
dc.titleStructural and functional insight into mismatch extension by human DNA polymerase α.
dc.typeJournal article
pubs.publication-statusPublished

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