ReviewRole of non-homologous end joining (NHEJ) in maintaining genomic integrity
Introduction
Of the various types of DNA damage that can occur within the mammalian cell, the DNA double strand break (DSB) is perhaps the most dangerous. DSBs are induced by ionizing radiation (X-rays or γ-rays) as well as by radiomimetic drugs used for chemotherapy. Moreover, the by products of cellular metabolism (reactive oxygen species or ROS) can also induce these breaks. In addition, programmed DBS are generated during regulated cellular processes such as V(D)J recombination. Estimates put the number of endogenous DSBs anywhere between 10 and 100 per nucleus per day. DNA double-strand breaks, as opposed to single-strand nicks or base modifications, can easily lead to gross chromosomal aberrations if not rejoined quickly. Even if repaired quickly, the repair process may be error-prone, and may eventually be detrimental to the organism. Mammalian cells, therefore, have mechanisms for quickly transmitting the damage signal to the cell cycle arrest or apoptotic machineries and mechanisms for DNA repair. Cell cycle arrest is necessary in order to give the cell enough time for repair and, in some case, it may be more prudent for the cell to undergo apoptosis when faced with excessive or unrepairable DNA damage and both these processes are seen as effective barriers to carcinogenesis. The other important barrier to genomic instability and carcinogenesis is DSB repair. Mammalian cells employ two mechanisms for DSB repair, non-homologous end joining (NHEJ) and homologous repair (HR). HR is operative only in the S/G2 phases of the cell cycle when a sister chromatid is available. NHEJ, which simply pieces together the broken DNA ends, can function in all phases of the cell cycle and is the predominant repair pathway in mammalian cells. In this review we will focus on the contributions of components of the NHEJ pathway in maintaining genomic stability and preventing cancer.
Section snippets
Genetic instability and cancer
It is now universally accepted that a strong link exists between genetic instability and the development of cancers and that cancer is a multi-step process during which mutations accumulate in genes controlling cell proliferation and apoptosis [1]. This genetic instability can manifest itself as small changes at the nucleotide level or can be cytogenetically observed as gross chromosomal alterations. Genetic changes associated with carcinogenesis can be broadly classified into four categories:
DNA double strand breaks and chromosomal aberrations
DSBs are typically induced by intrinsic sources such as the by products of cellular metabolism or by extrinsic sources such as X-rays or γ-rays. Both stimuli ultimately result in the generation of free oxygen radicals which can break the phosphodiester bonds in the DNA backbone; two such breaks on opposite strands of DNA, when present close enough to each other, result in a DSB [2]. Double strand breaks are also generated when a replication fork passes through a template with a nick. Finally,
DSBs provide selection pressure for the generation of cancer-promoting mutations
Genes that suppress carcinogenesis can be classified into gatekeepers and caretakers [8]. The gatekeepers prevent cancer by regulating cell proliferation and cell death while the caretakers essentially constitute DNA repair genes involved in maintaining the integrity of the genome. Carcinogenesis can be seen as a multi-step process wherein abrogation of both the gatekeeper and caretaker genes would lead to cancer. A direct link between DSBs, genomic instability, and cancer has been surmised by
Components of non-homologous end joining (NHEJ) pathway as caretakers of the mammalian genome
The two main mechanisms by which cells can repair DSBs are NHEJ and HR. During NHEJ, the two broken ends of DNA are simply pieced together, sometimes after limited processing of the DNA ends, resulting in quick, but error-prone, repair [13]. HR is a more accurate method of repair as here information is copied from an intact homologous DNA duplex; however, as HR requires the presence of an intact sister chromatid, this method of repair can only operate in the S/G2 phases of the cell cycle in
The DNA-dependent protein kinase (DNA-PK)
The DNA-dependent protein kinase (DNA-PK) is a key player in the NHEJ pathway of DSB repair and has additional functions in the mammalian cell including telomere maintenance and induction of apoptosis [15]. DNA-PK consists of an approximately 470-kDa catalytic subunit (DNA-PKcs) and a DNA-end binding component, Ku [16]. Ku is a heterodimer of two proteins of approximately 70 and 80 kDa termed Ku70 and Ku80, respectively [17]. Ku forms a ring-like structure and first binds to the DNA end and then
The DNA Ligase IV–XRCC4 complex
The coordinated assembly of Ku and DNA-PKcs on DNA ends is followed by recruitment of the DNA Ligase IV–XRCC4 complex that is responsible for the rejoining step [23]. This complex lies at the center of the NHEJ pathway and is present in all eukaryotes including yeast (which lacks DNA-PKcs but not Ku). Ligase IV exists in a tight complex with XRCC4; the latter stabilizes Ligase IV and stimulates its DNA ligation activity [24]. IR-induced phosphorylation of XRCC4 is dependent on DNA-PK in vivo
DNA end processing by Artemis prior to repair
In addition to NHEJ core components, several accessory factors have been implicated in NHEJ repair pathway in the end-processing step. End-processing is particularly important for the repair of IR-induced DSBs which may otherwise be unligatable with gaps and end-blocking groups, e.g. 3′ phosphate or phosphoglycolate (PG) and 5′-hydroxyl. Several enzymes have been implicated to function in this step of NHEJ. Of these enzymes, Artemis, a protein that is defective in patients with severe combined
The DNA-PK complex as a suppressor of genomic instability and cancer
DNA-PK has been proposed to function in maintaining genomic integrity by suppressing chromosomal rearrangements. Mouse embryonic fibroblasts (MEFs) deficient in Ku70 display gross genomic instability even in the absence of exogenous DNA damage and these include chromosomal fragmentation and non-reciprocal translocations involving several chromosomes [29]. Dermal fibroblasts from Ku80−/− mice have a high frequency of chromosome breaks while Ku80+/− mice have an intermediate frequency of
XRCC4–DNA Ligase IV and genomic stability
XRCC4 deficiency in mice leads to embryonic lethality associated with massive neuronal apoptosis [38]. The lethality of XRCC4 knockout mice can be rescued by p53 deficiency. However, though the XRCC4/p53 double knockout mice survive beyond birth, they eventually die from pro-Bcell lymphomas characterized by chromosomal translocations linking amplified c-myc oncogene and IgH locus sequences [39]. Like XRCC4 knockout mice, Ligase IV knockout mice also die during embryogenesis due to massive
Artemis as a protector of the genome
Artemis, the protein that provides the hairpin-opening activity in V(D)J recombination is associated with DNA-PKcs, is phosphorylated by DNA-PKcs, and is also involved in the processing of complex DNA ends prior to their rejoining. Artemis is mutated in a subset of human SCID (severe combined immunodeficiency) patients called RS-SCID [49] and in a second human SCID condition SCIDA [50]. MEFs from Artemis knockout mice exhibit significantly higher levels of spontaneous chromosomal aberrations
DSBs and genome stability in the context of chromatin
Our DNA is tightly wrapped around histones to form chromatin [56]. The importance of chromatin structure in DSB repair is just beginning to be understood. One of the earliest events that occur at the site of a break is the modification of Histone H2AX, a histone H2A variant that is rapidly phosphorylated in its unique C-terminal tail when DSBs are introduced into mammalian cells [57]. This phosphorylation is extensive (spanning about 100 kb) and occurs within seconds at the sites of DSBs
Influence of other pathways on NHEJ: Brca1 and precise NHEJ
One example of how other genome-maintenance mechanisms might impinge on NHEJ is the phosphorylation of Artemis (involved in repairing complex DSBs) by ATM (primarily involved in enforcing cell cycle checkpoints) [28]. Another recent example is that of the role of the tumor suppressor Brca1 (which primarily stimulates HR) in promoting precise NHEJ. Although NHEJ is the predominant pathway in mammalian cells and repairs DSBs with high efficiency, it is generally considered to be an error-prone
Conclusion
Both gatekeeper and caretaker genes are important for preventing cancer. It is clear now that all the components of the NHEJ pathway of DSB repair function as caretakers and are very important for maintaining genome integrity and suppressing carcinogenesis. It is also becoming clear that other gatekeeper and caretaker pathways may have an influence on NHEJ in preventing chromosomal aberrations. As the link between DSBs and carcinogenesis becomes stronger, it will become more important to
Acknowledgements
Our research is supported by grants from NIH (CA50519, CA86936, PO1-CA92584), Department of Defense BCRP (DAMD17-02-1-0439), NASA (NNA05CM04G, NNJ05HD36G) to DJC and a grant from NASA (NNA05CS97G) to SB.
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