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Published on: September 25, 2017
Replication of a carcinogenic nitropyrene DNA lesion by human Y-family DNA polymerase
Kevin N Kirouac1, Ashis K Basu, Hong Ling
1Department of Biochemistry, Medical Sciences Building 334, University of Western Ontario, London, ON N6A 5C1, Canada.
Abstract:
Nitrated polycyclic aromatic hydrocarbons are common environmental pollutants, of which many are mutagenic and carcinogenic. 1-Nitropyrene is the most abundant nitrated polycyclic aromatic hydrocarbon, which causes DNA damage and is carcinogenic in experimental animals. Error-prone translesion synthesis of 1-nitropyrene-derived DNA lesions generates mutations that likely play a role in the etiology of cancer. Here, we report two crystal structures of the human Y-family DNA polymerase iota complexed with the major 1-nitropyrene DNA lesion at the insertion stage, incorporating either dCTP or dATP nucleotide opposite the lesion. Polι maintains the adduct in its active site in two distinct conformations. dCTP forms a Watson-Crick base pair with the adducted guanine and excludes the pyrene ring from the helical DNA, which inhibits replication beyond the lesion. By contrast, the mismatched dATP stacks above the pyrene ring that is intercalated in the helix and achieves a productive conformation for misincorporation. The intra-helical bulky pyrene mimics a base pair in the active site and facilitates adenine misincorporation. By structure-based mutagenesis, we show that the restrictive active site of human polη prevents the intra-helical conformation and A-base misinsertions. This work provides one of the molecular mechanisms for G to T transversions, a signature mutation in human lung cancer.
Insights
Nitrated polycyclic aromatic hydrocarbons, like 1-nitropyrene, cause DNA damage. Human DNA polymerase iota
Area of Science:
- Molecular Biology
- Environmental Health
- Structural Biology
Background:
- Nitrated polycyclic aromatic hydrocarbons (NPAHs) are environmental pollutants.
- 1-Nitropyrene (1-NP) is a common NPAH, mutagenic and carcinogenic, causing DNA damage.
- Translesion synthesis of 1-NP DNA lesions contributes to cancer etiology.
Purpose of the Study:
- To elucidate the structural mechanisms of DNA polymerase iota (Polι) in replicating 1-NP DNA lesions.
- To understand how Polι incorporates nucleotides opposite the 1-NP adduct.
- To investigate the role of DNA polymerase eta (Polη) in preventing misincorporation.
Main Methods:
- X-ray crystallography to determine the structures of human Polι complexed with 1-NP DNA lesions.
- Site-directed mutagenesis to study the function of Polη.
Main Results:
- Two distinct conformations of Polι with the 1-NP lesion were observed during nucleotide insertion.
- Incorporation of dCTP opposite the lesion inhibited replication, excluding the pyrene ring from DNA.
- Mismatched dATP incorporation was facilitated by intercalation of the pyrene ring within the DNA helix.
- Human Polη's active site prevents intra-helical conformation and A-base misinsertion.
Conclusions:
- The study reveals molecular mechanisms for G to T transversions, a common mutation in lung cancer.
- Polι's ability to accommodate the 1-NP adduct in different conformations dictates nucleotide incorporation.
- Polη acts as a barrier against misincorporation of adenine opposite the 1-NP lesion.
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