Mutagenic bypass of abasic DNA lesions during DNA and RNA synthesis in human cells
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Abstract
DNA molecules are constantly damaged by exogenous agents and endogenous cellular processes. The balance between repair and tolerance of the resulting DNA lesions is essential for the maintenance of genome integrity. DNA damage tolerance mechanisms allow cells to continue replication and transcription in the presence of damage. Unavoidably, lesion bypass by RNA or DNA polymerases comes at the expense of fidelity. During translesion DNA synthesis (TLS), specialised DNA polymerases enable replication opposite and beyond DNA lesions. However, their elevated rates of nucleotide misincorporation turn TLS into the major source of spontaneous mutations in human cells. Erroneous bypass of DNA lesions may also occur during transcription, promoting the generation of mutant transcripts in a process termed transcriptional mutagenesis (TM). The repeated translation of the mutated mRNAs can lead to the production and accumulation of abnormal proteins with pathogenic properties.
Abasic DNA lesions are extremely frequent in the genome and bear a huge miscoding potential due to their inherent lack of genetic information. Despite the abundance of this type of damage, current knowledge about its mutagenic properties is far from complete and is generally based on biochemical data obtained in cell-free systems as well as genetic evidence in prokaryotes and lower eukaryotes.
This work describes an innovative system for the direct detection of mutagenic bypass events via reporter reactivation assays in human cells. We design and generate a set of mutated EGFP reporters that encode for a non-fluorescent protein. The erroneous bypass of a specific lesion placed in the mutated nucleotide leads to the synthesis of a fluorescent EGFP protein that serves to quantify the miscoding capacity of the lesion. This approach is further applied in the characterisation of the erroneous bypass of abasic DNA lesions during DNA and RNA synthesis. Once the lesion is classified as mutagenic, analysis of the transcripts via RNA sequencing allows the determination of its mutation profile.
By placing repair-resistant abasic site analog tetrahydrofuran in the transcribed strand of these newly designed reporters, regain of EGFP fluorescence indicated that transcriptional mutagenesis occurs at the site of damage. The percentage of cells showing green fluorescence was a direct indication of TM events and it varied from 11% to 79% depending on the DNA repair capacity of the cell. Subsequent RNA sequencing analysis revealed that RNA pol II exclusively incorporates adenine at the lesion site. By using this novel TM assay as a tool to investigate repair, NER involvement in the repair of synthetic abasic lesions was shown for the first time in human cells.
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To detect mutations introduced during DNA synthesis opposite AP sites, this lesion was incorporated at the mutated position in the non-transcribed strand of the reporter and opposite to a gap. After transfection into several human cells, regain of EGFP fluorescence indicated that bypass of natural abasic sites, as well as their synthetic counterpart tetrahydrofuran, occurred mostly through adenine incorporation. Surprisingly, under the same conditions, natural abasic sites exhibited a different mutation profile as compared to tetrahydrofuran. While results showed that DNA polymerases mostly inserted adenine followed by guanine, cytosine, and thymine opposite natural AP sites, the incorporation of guanine was nearly absent opposite synthetic abasic lesions. This illustrates the importance of analyzing all chemical forms of a single adduct, which might lead to different mutagenic outcomes during DNA or RNA bypass events.
The miscoding property of abasic DNA lesions presents a challenge for the fidelity of replicational or transcriptional bypass processes. Most of these lesions arise from depurination reactions and cytosine deamination. Therefore, adenine leading the mutation pattern induced by these lesions implies that most AP sites are likely to be mutagenic. Thus, abasic lesions become a key source of mutagenesis in human cells, which entails important biological consequences. On one hand, it is likely that AP sites derived mutagenesis works as an important source of genetic diversity during evolution. On the other hand, mutations arising from the bypass of these lesions may play a critical role in cancer development and aggravation of degenerative diseases. In future studies, we can extend the established TM and TLS assays to investigate mutagenic bypass occurring at different DNA modifications. In addition, establishing the mutation pattern of additional DNA lesions can help to track the sources of mutations encountered in a broad range of human cellular backgrounds.