Methods to Measure DNA Repair in Cells

There have been many methods developed for the measurement of DNA repair in a “test tube.” While these methods are powerful to reveal DNA repair capacity, it is limited by the fact that the complexities of the cell are not considered. In the first DNA repair blog in this three-part series, we explored the epigenetic role of DNA damage in G-quadruplex structures. In this second blog, we’ll take a look at recent efforts to develop methods that can measure DNA repair in a cellular context. See Part 3 of the series to learn more about RNA editing affects repair of DNA damage by the NEIL enzyme.

The DNA repair enzyme MUTYH is unique in that is responsible for removing the undamaged adenine base misplaced across from the highly common oxidative DNA damage 8-oxo-7,8-dihydroguanine, or OG. The syn conformer of OG is able to mimic thymine, and this allows for DNA polymerases often place adenine across from OG. If an OG:A mispair is left unrepaired, DNA replication can lead to a G:C to T:A transversion mutation. MUTYH acts as the last stand of defense before the transversion mutation is solidified.

Defects in MUTYH has been demonstrated to lead to a form of colorectal cancer known as MUTYH-associated polyposis (MAP). Over 300-MAP related mutations have been reported in the MUTYH gene, including many that are associated with different types of cancers, but several mutations are variants of unknown significance.2 This highlights the need to fully understand functional consequences of different variants so patients can properly be diagnosed and understand cancer risk. For many years, MUTYH has been studied in a “test tube” context to reveal the repair capabilities of the enzyme as well as cancer-associated variants. However, until recently, there were no methods to examine the enzyme’s ability to repair OG:A mispairs in a cellular context. Here, three different methods to examine cellular repair of  OG:A by MUTYH are presented.

Measuring Bacterial MutY Repair Using a Bacterial Cellular Repair Assay

One cellular reporter assay has been developed to examine bacterial MutY.3 In this assay, a plasmid can be digested with two restriction enzymes followed by insertion and ligation of duplex DNA containing the OG:A mispair (Figure 1). The plasmid can be transformed into bacterial cells containing MutY or lacking MutY (MutY+ and MutY- cells). Giving time for the enzyme to repair the OG:A mispair and for the plasmid to be replicated, the plasmids are then isolated from the bacteria. The plasmid inherently contains one BmtI restriction enzyme site already. If MutY did not repair the OG:A mispair, yielding a T:A base pair after replication, the plasmid can be digested with BmtI to yield one linear piece of DNA. However, if MutY does repair the OG:A mispair leading back to a G:C base pair after replication, this introduces a second BmtI restriction enzyme site into the plasmid. When digested with BmtI, the plasmid will now be cut into two different pieces. Sequencing can also be used to analyze the extent of T:A or G:C content (Figure 1). In this assay, OG:A-containing plasmids are fully repaired to the correct G:C base pairs in the presence of MutY. In the absence of MutY, there is a higher amount of T:A base pairs that are observed, indicating lower repair of OG. The background repair of the plasmid in MutY- cells is due to the dual-coding properties of OG that result in the ability of the lesion to also pair with C during replication. 

Measuring Human MUTYH Repair Capacity in Mammalian Cell Lines

Another reporter assay has been developed to examine the repair capabilities of mouse Mutyh as well as human MUTYH.4 In this assay, the OG:A mispair is placed into the coding region of the green fluorescent protein (GFP) gene such that repair of the adenine back to cytosine allows for the expression of full-length GFP, which fluoresces green (Figure 2). If A across from OG is not repaired by Mutyh/MUTYH, the template strand yields a stop codon that leads to a truncated GFP product that does not fluoresce. To insert the OG:A mispair into the plasmid, it is first digested from double-stranded DNA to single-stranded DNA by a phage. The OG-containing oligonucleotide is then annealed to the plasmid and the plasmid is extended back to double-stranded DNA by a DNA polymerase. The gap can then be sealed with a DNA ligase and ATP. The plasmid also contains the dsRed gene to be used as a transfection control. After transfection of the GFP/dsRed plasmid containing the OG:A mispair into mammalian cell lines and repair by Mutyh/MUTYH, flow cytometry can be used to quantify extent of repair. This is done by counting cells expressing both dsRed and GFP (indicating repair) and comparing it to cells only expressing dsRed (indicating no repair).

Measuring the Ability of MUTYH to Suppress G:C to T:A Transversion Mutations

Another cellular based assay known as the rifampicin resistance assay can be used to demonstrate the ability of MUTYH or cancer-associated variants to suppress G:C to T:A transversion mutations.5 This assay relies on the accumulation of mutations within the gene encoding for an RNA polymerase. If MUTYH repairs OG:A within the gene, the RNA polymerase is successfully generated within the cells. The cells are then treated with the antibiotic rifampicin which inhibits the RNA polymerase, which stops transcription and leads to cell death (Figure 3). This results in a low number of bacterial colonies on the plate. If, however, the MUTYH variant cannot successfully repair the OG:A mispairs that accumulate within the RNA polymerase gene, a mutant RNA polymerase is generated within the cell that is resistant to the antibiotic rifampicin. This allows for increased cell survival leading to a higher number of colonies on the plate. The number of rifampicin resistant colonies relative to the total number of cells in an untreated sample is reported as the mutation frequency. In conclusion, cells that have functional MUTYH will have a low mutation frequency, while cells that do not have functional MUTYH will have a high mutation frequency.

Overall, three assays have been presented which allows for researchers to assess MUTYH repair capacity in a cellular context. These assays allow for researchers to understand consequences of inheriting deficient versions of MUTYH which could help diagnose cancer risk.

References

David, S. S.; O’Shea, V. L.; Kundu, S. Base-Excision Repair of Oxidative DNA Damage. Nature 2007, 447 (7147), 941–950. https://doi.org/10.1038/nature05978.

Manlove, A. H.; McKibbin, P. L.; Doyle, E. L.; Majumdar, C.; Hamm, M. L.; David, S. S. Structure–Activity Relationships Reveal Key Features of 8-Oxoguanine: A Mismatch Detection by the MutY Glycosylase. ACS Chem. Biol. 2017, No. Figure 2, acschembio.7b00389. https://doi.org/10.1021/acschembio.7b00389.

Nuñez, N. N.; Khuu, C.; Babu, C. S.; Bertolani, S. J.; Rajavel, A. N.; Spear, J. E.; Armas, J. A.; Wright, J. D.; Siegel, J. B.; Lim, C.; et al. The Zinc Linchpin Motif in the DNA Repair Glycosylase MUTYH: Identifying the Zn2+ Ligands and Roles in Damage Recognition and Repair. J. Am. Chem. Soc. 2018, 140 (41), 13260–13271. https://doi.org/10.1021/jacs.8b06923.

Raetz, A. G.; Xie, Y.; Kundu, S.; Brinkmeyer, M. K.; Chang, C.; David, S. S. Cancer-Associated Variants and a Common Polymorphism of MUTYH Exhibit Reduced Repair of Oxidative DNA Damage Using a GFP-Based Assay in Mammalian Cells. Carcinogenesis 2012, 33 (11), 2301–2309. https://doi.org/10.1093/carcin/bgs270.

Raetz, A. G.; David, S. S. When You’re Strange: Unusual Features of the MUTYH Glycosylase and Implications in Cancer. DNA Repair (Amst). 2019, 80 (May), 16–25. https://doi.org/10.1016/j.dnarep.2019.05.005.