Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis
Induction and repair of DNA double strand breaks: The increasing spectrum of non-homologous end joining pathways
Introduction
The elucidation of DNA structure and the central dogma of molecular biology placed the DNA molecule as the carrier of genetic information at the center of all life processes. To be a reliable carrier of genetic information, the DNA molecule must be faithfully replicated and distributed to cell progeny. Moreover, the DNA must be protected against accidental modifications induced by various physical and chemical environmental agents, from intracellular oxidative stress, as well as from damage induced as by-product of errors during scheduled biological processes, such as DNA replication, V(D)J recombination, etc. If allowed to persist, such lesions will alter the DNA sequence and will cause mutations or other genomic rearrangements. This in turn will ultimately lead to genomic instability and cancer.
Despite the impressively high number of lesions forming in cellular DNA from the above sources, only an extremely small fraction ultimately persists or has adverse biological consequences. This is because cells have evolved highly efficient repair mechanisms to remove DNA lesions and thus to maintain genomic integrity [1], [2], [3]. DNA repair mechanisms, in their vast majority, have evolved on the premise that DNA lesions mainly affect only one of the DNA strands. Thus, after removal of the lesion, the faithful restoration of the damaged DNA strand is easily possible using information available on the complementary, non-damaged strand. This conceptual foundation of many DNA repair pathways is undermined when two or more lesions are induced directly opposite, or in close apposition, to each other, affecting thus locally both DNA strands. Under such circumstances, an intact complementary strand is lacking for the restoration of the genetic information and it becomes necessary to develop more complex mechanisms for the faithful restoration of the DNA molecule, or, alternatively, to accept solutions that may compromise genomic integrity.
Multiple lesions affecting both DNA strands, located within about one helical turn of the DNA molecule, have been termed by John Ward locally multiply damage sites (LMDS or simply MDS) [4], but are frequently also referred to as complex or clustered DNA damage [5]. The processes leading to the induction and the mechanisms involved in the repair of complex DNA damage are currently the subject of intensive research [6], [7].
The formation of complex DNA damage requires the practically simultaneous generation of multiple lesions randomly distributed on both DNA strands. Purely statistical considerations easily demonstrate that such induction characteristics are highly unlikely as a result of accidental chemical modifications (base hydrolysis, oxidation, deamination, or sugar oxidation) occurring in the DNA under normal conditions of cell growth and maintenance. They are also highly unlikely after exposure of cells to chemicals modifying the DNA – for example by oxidation, methylation, acetylation, etc., – at least at treatment conditions compatible with the cell survival.
Induction of complex DNA damage with relatively high efficiency is a unique characteristic of ionizing radiation (IR). IR damages molecules, such as the DNA, by imparting energy in forms capable of causing their ionization—hence the term ionizing radiation. In chemical terms, the electron loss associated with ionization is equivalent to oxidation. Energy is transferred to irradiated matter mainly by energetic electrons that are generated after exposure of cells to photons or by other types of directly ionizing particles. Ionizations are not evenly distributed in space. Rather, they are confined along the tracks of the primary ionizing particles, or of the secondarily produced electrons. Densely ionizing charged particles and all electrons near the ends of their tracks produce, as consequence of their physical properties, multiple ionizations confined in a relatively small volume. It is precisely this spatial accumulation of multiple ionizations (clustering) that causes the generation of complex DNA damage by simultaneously ionizing the DNA molecule at multiple neighboring locations (Fig. 1). Damage complexity can be further increased by attacks from OH radicals produced within the ionization cluster from water molecules surrounding the DNA (Fig. 1).
Complex DNA damage can be highly diverse. It may comprise random combinations in different numbers of strand breaks, various forms of base damage and sugar damage to generate a highly complex spectrum of compound lesions that present a problem not only to the cellular repair systems, but also to anyone who attempts its systematic classification.
Section snippets
The DNA double-strand break: a simple form of a complex lesion
Among the different forms of complex DNA damage, the DNA double-strand break, DSB, has received particular attention during the last 30 years. In the simplest scenario, a DSB is formed when two single-strand breaks (SSBs) are induced in close proximity on opposite DNA strands to divide in two a linear DNA molecule, or to linearize a circular DNA molecule (Fig. 1). However, more complex arrangements of lesions at a DSB can also be envisioned. This can include the presence of additional SSBs and
Homologous recombination repair (HRR)
As discussed above, the characteristics of a DSB compromise the faithful restoration of the damaged DNA molecule using information available on a complementary strand. Not surprisingly, therefore, the only known repair pathway capable of faithfully restoring the DNA molecule utilizes information available on a homologous DNA segment—hence the term homologous recombination repair. As source of the homologous sequence serves a DNA segment on the same DNA molecule, on a homologous chromosome, or
Evidence for backup pathways of NHEJ (B-NHEJ)
Early studies using SV40 DNA substrates tested in cultured monkey cells showed efficient DNA end joining in the absence of key D-NHEJ factors and provided evidence for alternative end-joining [83]. This interpretation was also in line with experiments showing extensive plasmid end joining in-vitro in reactions assembled with extracts of mutants deficient in either KU or LIG4 [84]. Analysis of junctions generated in these experiments showed that while similar basic concepts apply to end joining
The enzymatic make-up of B-NHEJ
The characterization of enzymatic activities contributing to B-NHEJ, the coordination of their functions, and their interactions with components of D-NHEJ and HRR has evolved as a prime area of current research in the field. Since DNA ligation is the last step of the end joining reaction and D-NHEJ is exclusively dependent on LigIV, it was reasonable to inquire which ligase is involved in B-NHEJ. Genetic and biochemical experiments point to the function of DNA ligase III (LigIII) in this
B-NHEJ and the formation of chromosomal translocations
The discontinuity of genetic material and its organization in distinct chromosomes is a prerequisite for the formation of chromosome aberrations. Chromosome aberrations are structural abnormalities in the chromosomes caused by large-scale rearrangements in the constituting DNA molecule of the same or different chromosomes [119]. Fusions, deletions or insertions of chromosomal arms, occurring as a result of chromosomal translocations, are directly implicated in cell killing, genomic instability
B-NHEJ and telomere maintenance
Given the promiscuity of NHEJ pathways, it is not surprising that eukaryotic cells rely on highly ordered DNA regions of repetitive elements, working together with protein complexes to protect their chromosome ends (telomeres) from illegitimate fusion and deterioration in addition to providing an efficient mechanism to complete DNA synthesis. The main enzymatic activities involved in telomere maintenance are: telomerase, which provides a reliable mechanism to accomplish replication at telomeric
B-NHEJ in the development of the immune system
The development of B- and T-cells requires successful V(D)J and class switch recombination [141]. While V(D)J recombination assembles immunoglobulin heavy and light chain exons in immature B-cells, CSR exchanges immunoglobulin constant region exons in peripheral B-lymphocytes. Both processes are initiated by the formation of DSBs produced by specific enzymatic activities at defined genomic locations.
Specific cleavage near the recombination signal sequences (RSS), within V, D and J regions is
Concluding remarks
Cells place particular importance in the processing of DNA ends generated by DSBs, telomere attrition or other scheduled cellular processes. This is documented by the highly sophisticated system that detects their presence and coordinates the numerous cellular responses including diverse repair pathways. Recent evidence suggests that in addition to HRR and the classical DNA-PK dependent NHEJ, cells engage alternative pathways of NHEJ operating as a backup. In this scheme, B-NHEJ features as a
Conflict of interest
None.
Acknowledgements
Work supported by grants from the DFG, BMBF (KVSF) and the EU (NOTE).
References (158)
- et al.
DNA-PKcs deficiency leads to persistence of oxidatively induced clustered DNA lesions in human tumor cells
Free Rad. Biol. Med.
(2010) - et al.
Analysis of DNA double-strand break repair pathways in mice
Mutat. Res./Fund. Mol. Mech. Mutagen.
(2007) - et al.
Quantifying clustered DNA damage induction and repair by gel electrophoresis, electronic imaging and number average length analysis
Mutat. Res./Fund. Mol. Mech. Mutagen.
(2003) - et al.
Dealing with DNA damage: relationships between checkpoint and repair pathways
Mutat. Res./Rev. Mutat. Res.
(2010) - et al.
Assembly of checkpoint and repair machineries at DNA damage sites
Trends Biochem. Sci.
(2010) - et al.
Rad54, the motor of homologous recombination
DNA Rep. (Amst.)
(2010) - et al.
DNA double-strand break repair: from mechanistic understanding to cancer treatment
DNA Rep. (Amst.)
(2007) - et al.
Homologous recombination-mediated double-strand break repair
DNA Rep. (Amst.)
(2004) - et al.
Processing of 3′-phosphoglycolate-terminated DNA double strand breaks by artemis nuclease
J. Biol. Chem.
(2007) - et al.
Tyrosyl-DNA phosphodiesterase and the repair of 3′-phosphoglycolate-terminated DNA double-strand breaks
DNA Rep. (Amst.)
(2009)