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Updated: Nov 1, 2025
Visualizing and Quantifying Endonuclease-Based Site-Specific DNA Damage
Published on: August 21, 2021
Mechanism of genome instability mediated by human DNA polymerase mu misincorporation
Miao Guo1,2, Yina Wang1,2, Yuyue Tang1,2
1Institute of Biophysics, College of Life Sciences, Zhejiang University, Hangzhou, Zhejiang, China.
Abstract:
Pol μ is capable of performing gap-filling repair synthesis in the nonhomologous end joining (NHEJ) pathway. Together with DNA ligase, misincorporation of dGTP opposite the templating T by Pol μ results in a promutagenic T:G mispair, leading to genomic instability. Here, crystal structures and kinetics of Pol μ substituting dGTP for dATP on gapped DNA substrates containing templating T were determined and compared. Pol μ is highly mutagenic on a 2-nt gapped DNA substrate, with T:dGTP base pairing at the 3' end of the gap. Two residues (Lys438 and Gln441) interact with T:dGTP and fine tune the active site microenvironments. The in-crystal misincorporation reaction of Pol μ revealed an unexpected second dGTP in the active site, suggesting its potential mutagenic role among human X family polymerases in NHEJ.
Insights
Pol μ polymerase causes genomic instability by misincorporating nucleotides during DNA repair. Structural and kinetic studies reveal its mutagenic potential in the nonhomologous end joining pathway.
Area of Science:
- Biochemistry
- Molecular Biology
- Genetics
Background:
- DNA polymerase mu (Pol μ) participates in DNA repair via the nonhomologous end joining (NHEJ) pathway.
- Misincorporation of nucleotides by Pol μ can lead to DNA mispairs and genomic instability.
Purpose of the Study:
- To investigate the mechanism of Pol μ-mediated nucleotide misincorporation.
- To compare the enzymatic activity and structural basis of Pol μ with dGTP versus dATP incorporation.
Main Methods:
- X-ray crystallography to determine Pol μ structures with gapped DNA substrates.
- Kinetic assays to measure enzyme activity and misincorporation rates.
- Site-directed mutagenesis to analyze key active site residues.
Main Results:
- Pol μ exhibits high mutagenicity on a 2-nt gapped DNA substrate, specifically misincorporating dGTP opposite a templating T.
- Crystal structures reveal interactions between Pol μ residues Lys438 and Gln441 with the T:dGTP mispair.
- An unexpected second dGTP molecule was observed in the Pol μ active site during in-crystal reactions.
Conclusions:
- Pol μ's active site residues fine-tune its mutagenic potential during NHEJ.
- The presence of a second dGTP suggests a novel mechanism for Pol μ-induced mutagenesis in human X family polymerases.
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