Mini-reviewAssembly and function of DNA double-strand break repair foci in mammalian cells
Introduction
DNA damage arises continuously as the result of intracellular metabolism and upon the exposure of cells to a multitude of genotoxic agents [1], [2]. If left unrepaired, such insults can be life-threatening for cells and organisms as they alter the content and organization of the genetic material. To overcome this challenge to genomic stability, cells have evolved a global signaling network known as the DNA damage response (DDR) that senses different types of genotoxic stress to mount a coordinated and multi-faceted response, which includes modulation of cell cycle transitions and transcriptional processes, and stimulation of DNA repair [3], [4], [5]. Accordingly, the DNA damage response functions as a major cellular defence mechanism against the accumulation of genetic changes associated with diseases such as cancer and neurodegenerative disorders [4], [6]. Besides being activated by DNA damaging agents, the DDR constitutes a key surveillance mechanism that monitors the quality of DNA replication. At the molecular level, the DDR is organized into an elaborate network of interacting pathways, the constituents of which can be grouped into three major classes of proteins that act in concert to translate the signal of damaged DNA into the appropriate downstream response. These comprise (1) sensors, proteins that recognize abnormally structured DNA and initiate the signaling response, and (2) transducers, factors that relay and amplify the damage signal to (3) effector proteins in numerous downstream pathways [3], [7].
DNA can be damaged in many ways, ranging from relatively innocuous single base or nucleotide modifications and single-strand breaks to highly cytotoxic lesions such as interstrand crosslinks and double-strand breaks (DSBs) [2], [8]. The former types of lesions occur spontaneously in vast numbers during the cell cycle, and normally these are swiftly repaired without eliciting full-blown activation of the DDR. Rather, this phenomenon appears to be restricted to conditions of massive replication problems or the presence of DSBs, a particularly destructive type of DNA lesion [2]. DSBs arise from a number of endogenous and exogenous sources, such as ionizing radiation, oxidative stress, and replication of damaged DNA. In addition, intentional DSBs are formed during genetically programmed processes such as meiotic recombination and V(D)J recombination in developing lymphocytes [2]. Persistent or inappropriately repaired DSBs can lead to mutations or more gross chromosomal aberrations such as deletions and chromosome loss or translocations. DSB repair occurs via two principal mechanisms: non-homologous end-joining (NHEJ) [9] and homologous recombination (HR) [10]. Most non-replication-associated DSBs are repaired by NHEJ, the predominant mode of DSB repair in G0/G1 cells, in which the broken DNA ends are simply pieced together in an efficient but error-prone fashion. In contrast, HR repairs DSBs in an error-free fashion, but because this requires an intact sister chromatid as a template, this mode of DSB repair only takes place in S/G2 phase cells.
While most of the DDR components are present at all times in the cell, activation of the DDR is accompanied by a dramatic increase in the availability of these factors. In bacteria such as Escherichia coli, this is ensured by the SOS network, a transcriptional program activated by DNA damage that mediates the rapid production of various DNA repair factors [11]. In eukaryotes, the total amount of DNA repair factors is not regulated by DNA damage on a global scale; rather, the local concentration and availability of these proteins is increased. The purpose, however, is the same: to markedly boost the ability of the DDR to faithfully reestablish genomic integrity. The local up-concentration of DDR proteins into so-called IRIF (Ionizing Radiation-Induced Foci) is a highly regulated yet dynamic process, where numerous proteins are recruited to sites of DSBs by a range of intricate mechanisms [12], [13], [14] (Fig. 1A). In yeast, multiple DSBs can be mobilized into the same repair focus (referred to as a DNA repair factory). In mammalian cells, on the other hand, DSBs are generally immobile and hence IRIF largely reflects protein accumulation at single DSBs [13]. The molecular mechanisms underpinning the structure and generation of IRIF have been the subject of intense investigation over the last decade, and while it is clear that the formation of these structures is of great importance for a successful DDR, our understanding of their biological function is still surprisingly limited. In this review, we discuss recent advances in our understanding of the mechanisms that govern IRIF formation, and the physiological importance of these structures.
Section snippets
DNA damage induces compartmentalization of the nucleus
The complex protein aggregates at sites of DNA damage that we refer to as IRIF contain all the common hallmarks of nuclear domains and bodies, such as PML and Cajal bodies, Polycomb regions, and replication factories [15], [16]. Thus, IRIF can be viewed as an affinity platform for a substantial number of proteins, allowing for the local concentration of these factors. The involved proteins generally do not bind stably and constantly to the affinity platform; rather, they are highly dynamic and
γ-H2AX: the most proximal marker of IRIF formation
The assembly of proteins at the DSB-flanking chromatin occurs in a highly ordered, strictly hierarchical, and rapid fashion (Fig. 1, Fig. 2). Key to the formation of IRIF is the DNA damage-induced phosphorylation of H2AX, a histone H2A variant that comprises 10–15% of total cellular H2A in higher eukaryotes, on S139 (to form γ-H2AX) [23], [25]. While several of the PI3K-like kinases, including ATM, ATR, and DNA-PK, seem capable of performing this function [23], [26], ATM has emerged as the
Sequential H2A poly-ubiquitylation by RNF8 and RNF168
The discovery that histone ubiquitylation plays a key role in promoting the retention of DDR factors at sites of DNA damage was spurred by the identification of RNF8 as a novel and critically important DDR protein. Since then, an overwhelming amount of new discoveries have highlighted the regulatory complexity and biological importance of this seemingly simple ubiquitylation reaction. Following the identification of RNF8 as a central regulator of the DSB response, two independent genome-wide
SUMOylation-mediated regulation of IRIF formation
Recent evidence has implicated post-translational modification of DDR proteins by the ubiquitin-like modifier protein SUMO as a signaling mechanism which, analogous to and in parallel with protein ubiquitylation, plays an important role in the execution of the chromatin response that governs IRIF formation. Thus, the SUMO E3 ligases PIAS1 and PIAS4 were shown to be required for recruitment of BRCA1 and 53BP1 to IRIF, respectively, and both SUMO1 and SUMO2/3 accumulate in IRIF [92], [93].
What are the biological functions of IRIF?
Despite our growing insight into the constituents of IRIF and the molecular mechanisms that govern their formation, our understanding of the physiological relevance of these structures still remains limited. The accumulation of DDR factors in DSB-containing chromatin regions may help to shelter the broken DNA ends from decay, and prevent illegitimate repair processes, such as those that lead to chromosomal translocations [95], [96]. Another simplified explanation is that higher local
IRIF and human disease
Highlighting its central importance for the cellular response to DSBs, several links between IRIF formation and the biology of human disease have been found. As an example, the activity of the DDR and viral proteins that inhibit the response is known to be an evolutionary battlefield in virus–host interactions [107]. For example, several strains of adenoviruses can inactivate the MRN complex either through its degradation or sequestration into inactive inclusion bodies [108], [109]. Such
Conflict of interest
The authors declare no conflict of interest.
Acknowledgements
We thank Dr. Jiri Lukas (Danish Cancer Society, Copenhagen, Denmark) for comments on the manuscript. Work in the laboratory of the authors is funded by the Novo Nordisk Foundation, Danish Medical Research Council, the Danish Cancer Society, and the Lundbeck Foundation.
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