| Autor: |
Baranovskiy, Andrey G., Babayeva, Nigar D., Lisova, Alisa E., Morstadt, Lucia M., Tahirov, Tahir H. |
| Předmět: |
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| Zdroj: |
Proceedings of the National Academy of Sciences of the United States of America; 4/26/2022, Vol. 119 Issue 17, p1-7, 14p |
| Abstrakt: |
Human DNA polymerase a (Pola) does not possess proofreading ability and plays an important role in genome replication and mutagenesis. Pola 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 Pola interaction with a mismatched template:primer. The structure of the human Pola 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 Pola 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, Pola showed a more than 10-fold higher selectivity for a correct duplex versus a mismatched one. According to pre-steady-state kinetic studies, human Pola 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. [ABSTRACT FROM AUTHOR] |
| Databáze: |
Complementary Index |
| Externí odkaz: |
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