7N2M

Crystal structure of DNA polymerase alpha catalytic core in complex with dCTP and template/primer having T-C mismatch at the post-insertion site

Summary for 7N2M

• Entry DOI: 10.2210/pdb7n2m/pdb
• Related: 4qcl
• Descriptor: DNA polymerase alpha catalytic subunit, RNA (5'-R(*GP*CP*CP*UP*GP*GP*AP*GP*CP*GP*C)-3'), DNA (5'-D(*AP*T*AP*GP*TP*CP*GP*CP*TP*CP*CP*AP*GP*GP*C)-3'), ... (10 entities in total)
• Functional Keywords: dna replication, dna polymerase, t-c mismatch, rna primer, dna template, human protein, dna polymerase alpha, transferase-dna-rna complex, transferase/dna/rna
• Biological source: Homo sapiens (Human); More
• Total number of polymer chains: 3
• Total formula weight: 114817.49

Authors

Tahirov, T.H.,Baranovskiy, A.G.,Babayeva, N.D. (deposition date: 2021-05-29, release date: 2022-04-20, Last modification date: 2023-10-18)

Primary citation

Baranovskiy, A.G.,Babayeva, N.D.,Lisova, A.E.,Morstadt, L.M.,Tahirov, T.H.
Structural and functional insight into mismatch extension by human DNA polymerase alpha.
Proc.Natl.Acad.Sci.USA, 119:e2111744119-e2111744119, 2022

PubMed 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 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.

PubMed: 35467978
DOI: 10.1073/pnas.2111744119

Experimental method

X-RAY DIFFRACTION (2.9 Å)