Due to the extreme specificity of genetic information, accurate DNA replication is critical to maintaining normal cell function. DNA polymerases are key proteins that catalyze DNA replication and proofread the DNA for any errors as new nucleotides are being incorporated. Without this proofreading ability, the DNA can accumulate with significant mutations. This leads to life-threatening diseases such as cancer. Therefore, the fidelity of DNA polymerases is important in DNA replication.
An example of what can occur when DNA polymerase’s proofreading capabilities are compromised is seen in individuals with mutations in the η (Pol η) gene. This polymerase is part of a family of polymerases involved in DNA translesion synthesis from UV-induced lesions. Individuals with this mutation develop hypersensitivity to UV-radiation. In some cases, an affected individual might develop skin cancer.
In recent work, Chang et. al investigated the structural components responsible for DNA polymerase fidelity. The key enzymatic reaction in DNA synthesis is the nucleotidyl transfer reaction, known to be dependent on metal ions. A variety of DNA polymerases have similar active sites containing two or three conserved acidic residues that coordinate with at least two metal ions. It was originally believed that two metal ions were sufficient for the catalysis of this reaction, but recent research has found that three metal ions are actually involved. This suggests the potential for a three-metal ion dependent process and the key structural determinants for fidelity remain unclear.
The authors made use of x-ray time-resolved crystallography to investigate the dynamic catalytic mechanism of DNA polymerase Pol η with atomic resolution. They used the metal ions Mg 2+ and Mn 2+ because each ion has been shown to impact DNA polymerase fidelity by affecting nucleotide misincorporation. In this study, Chang et al. captured the pre-, intra- and post-reaction states of Pol η misincorporating nucleotides during DNA synthesis. They discovered the accuracy of nucleotide incorporation was greatly impacted by the alignment of the primer 3’-OH
The accuracy of DNA synthesis was in part determined through visualizing and quantifying DNA separation on polyacrylamide urea gels using the Azure Sapphire Biomolecular Imager. The researchers observed the third metal ion binding site had to be in an ideal position in order for nucleotidyl transfer to occur.
There was a noted difference in Mn2+ and Mg2+ with misincorporation happening more often when Mn2+ was used resulting in more error-prone polymerase catalysis of DNA synthesis.
This study highlights the essential roles of the three separate metal ions in DNA synthesis, specifically Pol η . It supports the idea that the third metal ion is catalytic and drives nucleotidyl transfer through stabilization of the transition state.
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- Chang, C., Lee Luo, C. & Gao, Y. In crystallo observation of three metal ion promoted DNA polymerase misincorporation. Nat Commun 13, 2346 (2022). https://doi.org/10.1038/s41467-022-30005-3