Induction and repair of DNA double strand breaks: The increasing spectrum of non-homologous end joining pathways

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Abstract

A defining characteristic of damage induced in the DNA by ionizing radiation (IR) is its clustered character that leads to the formation of complex lesions challenging the cellular repair mechanisms. The most widely investigated such complex lesion is the DNA double strand break (DSB). DSBs undermine chromatin stability and challenge the repair machinery because an intact template strand is lacking to assist restoration of integrity and sequence in the DNA molecule. Therefore, cells have evolved a sophisticated machinery to detect DSBs and coordinate a response on the basis of inputs from various sources. A central function of cellular responses to DSBs is the coordination of DSB repair. Two conceptually different mechanisms can in principle remove DSBs from the genome of cells of higher eukaryotes. Homologous recombination repair (HRR) uses as template a homologous DNA molecule and is therefore error-free; it functions preferentially in the S and G2 phases. Non-homologous end joining (NHEJ), on the other hand, simply restores DNA integrity by joining the two ends, is error prone as sequence is only fortuitously preserved and active throughout the cell cycle. The basis of DSB repair pathway choice remains unknown, but cells of higher eukaryotes appear programmed to utilize preferentially NHEJ. Recent work suggests that when the canonical DNA-PK dependent pathway of NHEJ (D-NHEJ), becomes compromised an alternative NHEJ pathway and not HRR substitutes in a quasi-backup function (B-NHEJ). Here, we outline aspects of DSB induction by IR and review the mechanisms of their processing in cells of higher eukaryotes. We place particular emphasis on backup pathways of NHEJ and summarize their increasing significance in various cellular processes, as well as their potential contribution to carcinogenesis.

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 OHradical dot 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).

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