7n2m
Crystal structure of DNA polymerase alpha catalytic core in complex with dCTP and template/primer having T-C mismatch at the post-insertion siteCrystal structure of DNA polymerase alpha catalytic core in complex with dCTP and template/primer having T-C mismatch at the post-insertion site
Structural highlights
FunctionDPOLA_HUMAN Plays an essential role in the initiation of DNA replication. During the S phase of the cell cycle, the DNA polymerase alpha complex (composed of a catalytic subunit POLA1/p180, a regulatory subunit POLA2/p70 and two primase subunits PRIM1/p49 and PRIM2/p58) is recruited to DNA at the replicative forks via direct interactions with MCM10 and WDHD1. The primase subunit of the polymerase alpha complex initiates DNA synthesis by oligomerising short RNA primers on both leading and lagging strands. These primers are initially extended by the polymerase alpha catalytic subunit and subsequently transferred to polymerase delta and polymerase epsilon for processive synthesis on the lagging and leading strand, respectively. The reason this transfer occurs is because the polymerase alpha has limited processivity and lacks intrinsic 3' exonuclease activity for proofreading error, and therefore is not well suited for replicating long complexes.[1] Publication Abstract from PubMedHuman DNA polymerase alpha (Polalpha) does not possess proofreading ability and plays an important role in genome replication and mutagenesis. Polalpha 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 Polalpha interaction with a mismatched template:primer. The structure of the human Polalpha 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 A 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 A and 0.7 A, respectively, and made one hydrogen bond. The binding studies conducted at a physiological salt concentration revealed that Polalpha 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, Polalpha showed a more than 10-fold higher selectivity for a correct duplex versus a mismatched one. According to pre-steady-state kinetic studies, human Polalpha 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. Structural and functional insight into mismatch extension by human DNA polymerase alpha.,Baranovskiy AG, Babayeva ND, Lisova AE, Morstadt LM, Tahirov TH Proc Natl Acad Sci U S A. 2022 Apr 26;119(17):e2111744119. doi: , 10.1073/pnas.2111744119. Epub 2022 Apr 25. PMID:35467978[2] From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine. See AlsoReferences
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