7k96
Human DNA polymerase beta ternary complex with templating cytosine and incoming deoxyguanosine diphosphate
Structural highlights
FunctionDPOLB_HUMAN Repair polymerase that plays a key role in base-excision repair. Has 5'-deoxyribose-5-phosphate lyase (dRP lyase) activity that removes the 5' sugar phosphate and also acts as a DNA polymerase that adds one nucleotide to the 3' end of the arising single-nucleotide gap. Conducts 'gap-filling' DNA synthesis in a stepwise distributive fashion rather than in a processive fashion as for other DNA polymerases.[1] [2] [3] [4] Publication Abstract from PubMedDNA polymerases play vital roles in the maintenance and replication of genomic DNA by synthesizing new nucleotide polymers using nucleoside triphosphates as substrates. Deoxynucleoside triphosphates (dNTPs) are the canonical substrates for DNA polymerases; however, some bacterial polymerases have been demonstrated to insert deoxynucleoside diphosphates (dNDPs), which lack a third phosphate group, the gamma-phosphate. Whether eukaryotic polymerases can efficiently incorporate dNDPs has not been investigated, and much about the chemical or structural role played by the gamma-phosphate of dNTPs remains unknown. Using the model mammalian polymerase (Pol) beta, we examine how Pol beta incorporates a substrate lacking a gamma-phosphate [deoxyguanosine diphosphate (dGDP)] utilizing kinetic and crystallographic approaches. Using single-turnover kinetics, we determined dGDP insertion across a templating dC by Pol beta to be drastically impaired when compared to dGTP insertion. We found the most significant impairment in the apparent insertion rate (k(pol)), which was reduced 32000-fold compared to that of dGTP insertion. X-ray crystal structures revealed similar enzyme-substrate contacts for both dGDP and dGTP. These findings suggest the insertion efficiency of dGDP is greatly decreased due to impairments in polymerase chemistry. This work is the first instance of a mammalian polymerase inserting a diphosphate nucleotide and provides insight into the nature of polymerase mechanisms by highlighting how these enzymes have evolved to use triphosphate nucleotide substrates. Mechanism of Deoxyguanosine Diphosphate Insertion by Human DNA Polymerase beta.,Varela FA, Freudenthal BD Biochemistry. 2021 Feb 9;60(5):373-380. doi: 10.1021/acs.biochem.0c00847. Epub , 2021 Jan 21. PMID:33475337[5] From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine. See AlsoReferences
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