Mutations: The dark side of DNA repair
DNA can be damaged in many ways—bases can, for example, be oxidized, alkylated or deaminated—so cells need a way to repair DNA damage. Sometimes it is possible to reverse the process that caused these ‘base lesions’, but cells tend to employ a more complex approach that is called the base excision repair (BER) pathway. This process involves three basic steps: first, enzymes called nucleases break the DNA strand that is damaged on either side of the lesion so that the section containing the lesion can be removed; second, DNA polymerases make a new stretch of DNA to replace the section that has been removed, using the undamaged strand as a template; third, DNA ligases join this new stretch to the existing DNA.
However, there is a ‘dark side’ to DNA repair because the process by which the replacement DNA is synthesized is inherently error-prone. Moreover, DNA is normally tightly packaged inside the nucleus to protect it from damage: however, the BER pathway involves regions of DNA being unpackaged, and this provides other enzymes and repair pathways with access to the DNA, which can lead to lesions and mutations. The overall result is that DNA repair can sometimes lead to more rather than less damage and, in some cases, this damage can ultimately kill the cell (Fu et al., 2012).
Large-scale DNA sequencing has revealed a complex pattern of lesions and mutations that includes mutational hotspots and mutations that are linked to specific sequences of bases. In some cases the mutations appear to be linked to enzymes belonging to the APOBEC family—which change the cytosine bases in DNA into uracil bases—being able to access the DNA (Roberts et al., 2013; Nik-Zainal et al., 2014). Finally, some seemingly random mutations occur with high frequency.
Now, in eLife, Jia Chen, Brendan Miller and Anthony Furano—who all are based at the National Institute of Diabetes and Digestive and Kidney Diseases—have used DNA sequencing to explore the accuracy of the BER pathway in mammalian cells (Chen et al., 2014). They used genetic techniques to deliberately introduce lesions associated with different sub-pathways of the BER pathway. These lesions included: a simple strand break between two neighbouring bases (which is known as a nick); replacing the cytosine base opposite a guanine base with an abasic site; and various types of base mismatches (such as thymine/guanine, uracil/guanine or hydroxymethyl-uracil/guanine instead of cytosine/guanine). The lesions were individually engineered into episomal DNA, and the episomes were allowed to undergo repair in mammalian cells and then isolated. After passage through E. coli, mutants were selected, and the DNA was subjected to sequence analysis.
As expected, most of the lesions were repaired by the BER pathway (Figure 1), but there were interesting differences in the repair of the different types of lesions. In some cases, for example, there were mutations in the template strand, rather than the strand containing the lesion, which suggests that various enzymes had been able to access the template strand during the repair process.
Moreover, mutations were observed in the bases on one side of the base lesion (distal to the lesion). These ‘flanking mutations’ were shown to depend on the initial steps of the BER pathway in most cases. A number of enzymes are known to be capable of exposing single-stranded DNA on either side of a nick, but it seems surprising that the molecular machinery responsible for the BER pathway allowed these enzymes to have access to the lesions. Previously it was thought that each enzyme in the BER pathway ‘handed over’ to the next enzyme in a highly coordinated process that left little scope for third parties to have access to the DNA (Prasad et al., 2010), but the presence of the flanking mutations suggests that the lesion recognition and hand-over process is not always able to keep third parties away from the lesion.
So what actually causes the flanking mutations? Chen, Miller and Furano suggest that the BER pathway is ‘hijacked’ by another DNA repair pathway called the mismatch repair system (which, as its name suggests, repairs base mismatches). They provided evidence for this by showing that depleting factors involved in the mismatch repair system results in a reduction in flanking mutations.
Chen et al. also noticed that many of the flanking mutations involved cytosine bases that were preceded by thymine bases, with the cytosine bases changing to uracil bases. This pattern suggested that certain APOBEC enzymes—those that have a preference for the TpC sequence in single-stranded DNA—were involved. To test this idea Chen et al. depleted the level of APOBEC enzymes and, as they predicted, there was a drop in the number of flanking mutations. Taken together, the results point to the idea that the mismatch repair machinery exposes single-stranded DNA that serves as a substrate for the APOBEC enzymes, which remove amine groups from the cytosine bases to leave uracil bases (Roberts et al., 2012, 2013). DNA synthesis to copy the single-stranded DNA then leads to mutations.
Overall, the results reported by Chen, Miller and Furano open up a whole new range of questions about the introduction of mutations during DNA repair.
References
-
Balancing repair and tolerance of DNA damage caused by alkylating agentsNature Reviews Cancer 12:104–120.https://doi.org/10.1038/nrc3185
-
Substrate channeling in mammalian base excision repair pathways: passing the batonJournal of Biological Chemistry 285:40479–40488.https://doi.org/10.1074/jbc.M110.155267
Article and author information
Author details
Publication history
- Version of Record published: May 20, 2014 (version 1)
Copyright
© 2014, Wilson
This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited.
Metrics
-
- 3,146
- views
-
- 104
- downloads
-
- 5
- citations
Views, downloads and citations are aggregated across all versions of this paper published by eLife.
Download links
Downloads (link to download the article as PDF)
Open citations (links to open the citations from this article in various online reference manager services)
Cite this article (links to download the citations from this article in formats compatible with various reference manager tools)
Further reading
-
- Biochemistry and Chemical Biology
- Microbiology and Infectious Disease
To date, all major modes of monoclonal antibody therapy targeting SARS-CoV-2 have lost significant efficacy against the latest circulating variants. As SARS-CoV-2 omicron sublineages account for over 90% of COVID-19 infections, evasion of immune responses generated by vaccination or exposure to previous variants poses a significant challenge. A compelling new therapeutic strategy against SARS-CoV-2 is that of single-domain antibodies, termed nanobodies, which address certain limitations of monoclonal antibodies. Here, we demonstrate that our high-affinity nanobody repertoire, generated against wild-type SARS-CoV-2 spike protein (Mast et al., 2021), remains effective against variants of concern, including omicron BA.4/BA.5; a subset is predicted to counter resistance in emerging XBB and BQ.1.1 sublineages. Furthermore, we reveal the synergistic potential of nanobody cocktails in neutralizing emerging variants. Our study highlights the power of nanobody technology as a versatile therapeutic and diagnostic tool to combat rapidly evolving infectious diseases such as SARS-CoV-2.
-
- Biochemistry and Chemical Biology
Phosphoinositide 3-kinase (PI3K) beta (PI3Kβ) is functionally unique in the ability to integrate signals derived from receptor tyrosine kinases (RTKs), G-protein coupled receptors, and Rho-family GTPases. The mechanism by which PI3Kβ prioritizes interactions with various membrane-tethered signaling inputs, however, remains unclear. Previous experiments did not determine whether interactions with membrane-tethered proteins primarily control PI3Kβ localization versus directly modulate lipid kinase activity. To address this gap in our knowledge, we established an assay to directly visualize how three distinct protein interactions regulate PI3Kβ when presented to the kinase in a biologically relevant configuration on supported lipid bilayers. Using single molecule Total Internal Reflection Fluorescence (TIRF) Microscopy, we determined the mechanism controlling PI3Kβ membrane localization, prioritization of signaling inputs, and lipid kinase activation. We find that auto-inhibited PI3Kβ prioritizes interactions with RTK-derived tyrosine phosphorylated (pY) peptides before engaging either GβGγ or Rac1(GTP). Although pY peptides strongly localize PI3Kβ to membranes, stimulation of lipid kinase activity is modest. In the presence of either pY/GβGγ or pY/Rac1(GTP), PI3Kβ activity is dramatically enhanced beyond what can be explained by simply increasing membrane localization. Instead, PI3Kβ is synergistically activated by pY/GβGγ and pY/Rac1 (GTP) through a mechanism consistent with allosteric regulation.