DNA Damage: From Threat to Treatment

Abstract

DNA is the source of genetic information, and preserving its integrity is essential in order to sustain life. The genome is continuously threatened by different types of DNA lesions, such as abasic sites, mismatches, interstrand crosslinks, or single-stranded and double-stranded breaks. As a consequence, cells have evolved specialized DNA damage response (DDR) mechanisms to sustain genome integrity. By orchestrating multilayer signaling cascades specific for the type of lesion that occurred, the DDR ensures that genetic information is preserved overtime. In the last decades, DNA repair mechanisms have been thoroughly investigated to untangle these complex networks of pathways and processes. As a result, key factors have been identified that control and coordinate DDR circuits in time and space. In the first part of this review, we describe the critical processes encompassing DNA damage sensing and resolution. In the second part, we illustrate the consequences of partial or complete failure of the DNA repair machinery. Lastly, we will report examples in which this knowledge has been instrumental to develop novel therapies based on genome editing technologies, such as CRISPR-Cas.

Keywords: CRISPR-Cas; DNA damage; DNA damage response (DDR); HDR; NHEJ; cancer; cell-cycle; gene editing; genome integrity.

Figure 1

Schematic of the major DNA lesions experienced by cellular genomic DNA: The table below indicates the type of DNA lesion depicted in the figure above, its leading cause and the DNA repair pathway engaged for its resolution.

Figure 2

Resolution of a DNA double-stranded break (DSB): Following the recognition and marking of a DSB by the concerted action of key proteins such as ATM (ataxia-telangiectasia mutated), ATR (ataxia telangiectasia and Rad3-related) and DNA-PKcs (DNA-dependent protein kinase catalytic subunit), the repair can follow different pathways. On one hand, if the DNA ends are protected by the Ku70/Ku80 complex, the DSB is generally repaired via the non-homologous end-joining (NHEJ) pathway (depicted in A). In this case, an intermediate end-processing step might be necessary if the protected ends are not compatible. On the other hand, resection of the DNA ends by the MRN/CtIP (MRE11-RAD50-NBS1/C-terminal-binding protein interacting protein) complex results in NHEJ inhibition. Based on the length of homology DNA fragments revealed during end-resection, the DSB can be repaired either via homology-directed repair (HDR) (B) or microhomology-mediated end joining (MMEJ)/ single-strand annealing (SSA) (C). The key factors involved in the DNA repair pathways depicted are indicated within the figure.

Figure 3

Genome editing using designer nucleases (DN): (A) Targeting of the DN to the coding region of a gene promotes the formation of a DSB which is typically repaired via NHEJ. The subsequent formation of indel mutations (grey box) may generate a premature stop codon, eventually leading to gene inactivation. (B) DN can be targeted to regulatory elements (RE) in order to disrupt the binding site of an activating transcription factor (TF). As a result, downstream gene transcription might be reduced or abolished in a cell-specific fashion as described in Section 6.1. (C) Two DNs can be directed to sites flanking an exon containing a non-sense mutation for its deletion. The resulting gene might result in the generation of a truncated, albeit partially functional, protein. (D) Targeting of a DN in proximity of a genetic mutation and simultaneous delivery of a donor template harboring the correct genetic sequence might result in precise gene editing via harnessing of the homology directed repair pathway.