Establishing Linkages Among DNA Damage, Mutagenesis, and Genetic Diseases

Abstract

DNA damage by chemicals, radiation, or oxidative stress leads to a mutational spectrum, which is complex because it is determined in part by lesion structure, the DNA sequence context of the lesion, lesion repair kinetics, and the type of cells in which the lesion is replicated. Accumulation of mutations may give rise to genetic diseases such as cancer and therefore understanding the process underlying mutagenesis is of immense importance to preserve human health. Chemical or physical agents that cause cancer often leave their mutational fingerprints, which can be used to back-calculate the molecular events that led to disease. To make a clear link between DNA lesion structure and the mutations a given lesion induces, the field of single-lesion mutagenesis was developed. In the last three decades this area of research has seen much growth in several directions, which we attempt to describe in this Perspective.

Figure 1.

Chemical damage to the genome and its role in genetic diseases. There are many ways that the genome can become damaged. Inflammation and endogenous metabolic processes generate a host of reactive oxygen (e.g., hydroxyl radical), nitrogen (e.g., nitric oxide), and halogen (e.g., HOCl) species that directly damage DNA. Some of these agents damage DNA indirectly by reacting with lipids in biological membranes, to yield electrophilic lipid-derived products, or by damaging the nucleotide pool to form pool-originated mutagens such as the deoxynucleoside triphosphate of 7,8-dihydro-8-oxoguanine (8-OxoG). DNA lesions, also referred to here as adducts, are depicted as L1, L2, etc. Ionizing and nonionizing radiations similarly form many lesions, as do a host of organic and inorganic agents that are either inherently able to attack DNA directly or do so via electrophiles produced by oxidations mediated by enzymes such as the cytochrome P450s. The collection of lesions formed can be repaired but, if repair fails, polymerases attempt to replicate them. In some cases, DNA synthesis is blocked, potentially leading to a lethal outcome. In some other cases, lesion bypass occurs but occurs at the expense of fidelity, leading to a collection of mutations. Mutations that activate protooncogenes, such a RAS, or inactivate tumor suppressor genes, such as TP53, are particularly dangerous as they may propel the cell along the pathway to malignant transformation.

Figure 2.

Technologies discussed in this paper. (A) Mutational spectra classically are produced by damaging the genome of a cell or vector and replicating the damaged piece of DNA in cells that have normal or disabled DNA repair status or normal or altered replicative status. Mutations are determined and plotted along the sequence of the piece of DNA as shown. (B) Often it is of value to test the hypothesis that a given mutation might have been caused by a specific DNA adduct. In that case, an oligodeoxynucleotide is synthesized with the adduct at a specific site. The oligonucleotide is spliced into the genome of a vector, usually a plasmid or viral genome, and replicated in living cells. Mutant progeny are sequenced. The type, amount and genetic requirements for mutagenesis are thereby determined, along with the potential genotoxicity of the lesion. Most of the lesions discussed in the text were evaluated by this site-specific mutagenesis strategy.

Figure 3.

Structures of dF and dQ, which lack Watson—Crick hydrogen-bonding ability but possess shapes similar to dT and dA, respectively.

Figure 4.

Structures of some of the DNA lesions discussed in this paper.

Figure 5.

(A) O⁶-mG, a minor lesion formed by methylating agents, is efficiently repaired by DNA alkyltransferases (depicted here as a Pacman symbol), but any unrepaired O⁶-mG will give rise to G:C→A:T mutations. The lollipop symbol designates a DNA methylating agent, which forms O⁶-mG. As shown, the repair protein transfers the methyl group to itself, rendering the repair protein inactive. If the adduct evades repair, it can be replicated in an error-free manner with C inserted as the opposing base (panel B, left) or in an error-prone manner with T inserted opposite the lesion (panel B, right).The pairing of O⁶-mG with T results, after the next round of replication, in a G:C→A:T mutation. The base pairing modes of O⁶-mG with C and T were determined from NMR and crystal structure studies (panel B).^,

Figure 6.

Simplified scheme showing a pattern of DNA damage from an agent that, either directly or upon metabolic activation, reacts with various positions in the genome. The blue bars represent the level of damage at the bases, and the red bars specify the sites where mutations occurred. The height of the red bars represents the mutational frequency at the specified base. The asterisks (at positions 1, 3, and 5) indicate hotspots of lesion formation in panel A. Repair of these lesions is also nonrandom, and lesions at certain sites (e.g., 3, 4, and 5) are repaired more efficiently than at other sites as shown in panel C. Mutagenic consequences are nonrandom as well, as, for example, mutations are detected only in positions 2, 4, 5, and 7, as shown in panel B when replication occurrs in the absence of repair. If replication takes place after DNA repair, a much lower level of mutations is detected (shown in panel D). In many cases, replication of the damaged DNA may take place partly before repair and partly after repair, and the relative proportions of these two competitive events depend on a variety of factors. It should also be noted that most mutagens form multiple types of lesions, which further diversifies the mutational spectrum, and both the efficiency of repair and mutagenesis may be different for the leading strand and the lagging strand of the DNA helix.

Figure 7.

Nucleotide incorporation patterns opposite an AP-site differs in different types of cells or organisms.

Figure 8.

UV and hydroxyl radical-induced tandem DNA damages.

Figure 9.

Types and frequencies of single-base substitutions induced by G[8,5-Me]T (G^T; left panel) and T[5-Me,8]G (T^G; right panel) detected in HEK 293T cells. The colors used in the bar graph represent T (red), A (green), G (blue), and C (yellow).

Figure 10.

Model for DNA damage tolerance by HDR. A replication-stalling DNA lesion (indicated by the red filled circle) can be bypassed by either TLS or HDR. The latter involves a template switching mechanism, which can be initiated by strand invasion followed by making a copy of the complementary strand from the sister chromatid. Template switching can also happen by regression of the stalled fork, synthesis of the DNA complementary to the damaged site, and reversion. Various other models of HDR have been proposed. The structures in the boxes depict the bypassed lesion, by whichever mechanism the cell uses.