Chapter 2 DNA-PK: The Means to Justify the Ends?
Introduction
DNA is the blueprint for all living organisms; accordingly, all organisms have evolved numerous mechanisms to ensure maintenance of an exact copy of their genomes for propagation. In somatic cells of multicellular organisms, genomic maintenance is critical to the organism to ensure cellular viability and prevent oncogenesis. Given its importance to life, it is somewhat surprising that evolution has allowed DNA to be so labile, being quite sensitive to various forms of damage including oxidation, hydrolysis, and methylation. Efficient DNA repair systems have evolved to repair base damage (base excision repair, BER), nucleotide damage (nucleotide excision repair, NER), single strand breaks (single strand break repair, SSBR), and double strand breaks (double strand break repair, DSBR). Double strand DNA breaks (DSBs) are perhaps the most lethal form of DNA damage. Two major DNA repair pathways [nonhomologous end joining (NHEJ) and homologous recombination (HR)] repair DSBs in all eukaryotes. NHEJ (the primary pathway in higher eukaryotes) is active throughout the cell cycle (Critchlow and Jackson, 1998, Lieber, 1999, Pastink et al., 2001) whereas HR is generally limited to S and G2 when a sister chromatid is available as a repair template (Rothkamm et al., 2003). Emerging data provide strong evidence that an additional repair pathway(s) may also contribute to resolution of double strand breaks, especially in the absence of NHEJ, although the composition of this pathway as well as its role are not well understood (Audebert et al., 2004, Corneo et al., 2007, Udayakumar et al., 2003, Yan et al., 2006).
Although NHEJ has long been labeled “error prone,” it should be emphasized that NHEJ functions efficiently to preserve DNA ends. Furthermore, direct ligation of two DNA ends is a preferable repair choice (over “error free” HR) in G0/G1 when HR using the homologous chromosome or nonallelic homologous sequences could result in deletions, duplications, or loss of heterozygosity. Since most of the genome (in higher eukaryotes) is noncoding, error prone rejoining of DSBs by NHEJ generally has minimal deleterious consequences. Rejoining of DSBs in coding regions obviously has the potential to introduce functionally important coding changes, a mechanism exploited by the vertebrate adaptive immune system when it commandeered the NHEJ system (evolutionarily) to resolve breaks during immune receptor assembly. In fact, this exploitation of the error prone nature of NHEJ by the immune system to generate literally billions of unique receptors is (arguably) one of the most clever of all biologic mechanisms. Many excellent recent reviews have provided comprehensive comparisons of the factors and functions of these two pathways, as well as NHEJ's role in VDJ recombination, and will not be exhaustively reviewed here (Jung et al., 2006, Lees-Miller et al., 1990, Lieber, 2008, Weterings and Chen, 2008).
Briefly, NHEJ involves the direct ligation of DNA ends without a requirement for homology. Seven molecules have been shown to be required for NHEJ: Ku70, Ku86, DNA-PKcs, XRCC4, DNA ligase IV, Artemis, and XLF. XRCC4 is a 37-kDa protein that interacts with, and catalytically stimulates, the activity of DNA ligase IV (Grawunder et al., 1997, Li et al., 1995, Modesti et al., 1999). The XRCC4/DNA ligase IV complex carries out the final end-joining step in NHEJ. Artemis is the only nuclease known to function in NHEJ. The function of a newly discovered factor, XLF (Ahnesorg et al., 2006, Buck et al., 2006)) that interacts with the XRCC4/ligase IV complex is only now being discerned but in its absence, NHEJ is severely impaired. Additionally, X family polymerases λ and μ (Nick McElhinny and Ramsden, 2003, Ma et al., 2004, Mahajan et al., 2002) as well as polynucleotide kinase provide activities not entirely essential for NHEJ (Chappell et al., 2002).
Three of the essential factors comprise the DNA-dependent protein kinase (DNA-PK) complex, a serine/threonine protein kinase that must be physically associated with DNA to be active (Lees-Miller et al., 1990, Meek et al., 2004). Although DNA-PK has been implicated in a variety of other processes—from activation of innate immunity (Chu et al., 2000) to regulation of gene expression (Mo and Dynan, 2002)—its primary role in cellular metabolism is to initiate NHEJ. As discussed above, the primary role of NHEJ is to resolve DNA double strand breaks. Emerging data from our laboratories and others implicate DNA-PK as a central regulator of DNA end access. The focus of this review will be how DNA-PK mechanistically regulates DNA end access (primarily via autophosphorylation) to promote end joining with minimal loss of sequence information. Additionally, it is becoming apparent that DNA-PK may affect other repair pathways, potentially by limiting access of DNA ends to other repair factors. This may have particularly important sequelae in species that express very high levels of DNA-PK and may thus partially explain why DNA-PK may play varying roles in different species.
During publication, a hypomorphic DNA-PKcs mutation that affects Artemis activation (but not kinase assembly or enzymatic activity) has been described in a human radiosensitive SCID patient (D. van Gent, personal communication).
Section snippets
Composition
DNA-PK is composed of the DNA end binding heterodimer, Ku, and the large catalytic subunit, DNA-PKcs. Ku, initially discovered as an autoantigen, consists of two subunits of 70 and 86 kDa (Mimori et al., 1986). Its DNA end binding activity prompted early speculation that Ku might function in DNA repair (Mimori et al., 1986) although this was not formally proven until studies emerged implicating Ku as the defective factor in cells hypersensitive to DNA damaging agents (Liang et al., 1996).
DNA Binding and Kinase Activation
Ku binds DNA ends in a sequence-independent manner. Ku completely encircles bound DNA; its binding site encompasses approximately two turns of the helix, but only the central 3–4 base pairs are completely surrounded by Ku. When Ku is bound (in the absence of DNA-PKcs), the extreme DNA terminus is bound in an accessible channel (Walker et al., 2001). Ku has strong avidity for DNA with a variety of end structures [including blunt, over-hanged, hair-pinned, and damaged]. Ku can also recognize
Structural Studies of DNA-PK
As noted above, the Ku heterodimer exists as a ring structure that completely encircles the DNA, binding the extreme terminus in an accessible channel (Spagnolo et al., 2006, Walker et al., 2001). Association of DNA-PKcs with Ku bound DNA results in translocation of Ku to a more interior location on the DNA (Yoo and Dynan, 1999). Studies from Llorca and colleagues have provided a low-resolution electron microscopy (EM) structure of a DNA-PK synapse (Spagnolo et al., 2006); previous work from
Targets of DNA-PK's Enzymatic Activity
DNA-PK's enzymatic activity is clearly requisite to its role in NHEJ (Kienker et al., 2000, Kurimasa et al., 1999), and significant efforts have been made to define functionally relevant targets of DNA-PK that could explain why. DNA-PK (and the related PIKK family members, ATR and ATM) preferentially targets serines and threonines followed by a glutamine (S-T/Q sites) though other target sites (S-T/hydrophobic residues) have also been reported (Lees-Miller and Meek, 2003). There is a very long
DNA-PK's Autophosphorylation is Functionally Complex
Early work in the Lees-Miller laboratory demonstrated that autophosphorylation of DNA-PK results in kinase inactivation and dissociation of the kinase's catalytic subunit (DNA-PKcs) from DNA end bound Ku (Chan et al., 1999, Douglas et al., 2007). Kinase dissociation is certainly the most well-studied and well-accepted consequence of DNA-PK's autophosphorylation. However, more recent work has shown that autophosphorylation of DNA-PKcs occurs on many sites (probably more than 30 of the 4,129
Autophosphorylation Within Two Clusters Reciprocally Regulates DNA End Access
The ABCDE and PQR clusters contain six and five conserved sites, respectively. These sites have been studied by generating phospho-ablating (alanine) or phospho-mimicking (aspartic acid) mutants at each site. Published data regarding the function of different DNA-PKcs phosphorylation sites is summarized in Table 2.1. Ablation of only 1 or 2 sites in either cluster has little or no functional effect. However, complete ablation of ABCDE phosphorylation (by alanine substitution of all six sites)
Further DNA-PK Autophosphorylation is Required During NHEJ
An additional relevant in vivo autophosphorylation site resides within the activation loop (or “T” loop) of the kinase (threonine 3950, termed T) (Douglas et al., 2007). Mimicking phosphorylation at the T site inactivates the kinase, but does not reduce affinity of DNA-PKcs for DNA bound Ku. A DNA-PKcs mutant with the ABCDE, PQR, and T sites substituted to alanine (13 altogether) still substantially autophosphorylates in vitro (50% of wild-type levels) and still undergoes
Model of DNA-PK Activation
A current model of DNA-PK's activation is as follows (Fig. 2.2). Ku initially binds the two ends of a double strand break, each of which then recruits a DNA-PKcs molecule. In vitro, if DNA-PKcs is not recruited to an end, it is possible for multiple Ku heterodimers to be loaded onto the end (Calsou et al., 1999). It is not known whether this occurs in living cells. Structural studies revealed the presence of distinct channels within the palm domain of DNA-PKcs that likely accommodate DNA. These
End Processing to Promote End Conservation
Recent studies reveal several interesting aspects of NHEJ's DNA end processing activities that promote DNA sequence conservation. Artemis is the only nuclease unequivocally involved in NHEJ; it likely contributes to only a subset of DNA breaks including endonucleolytic opening of hair-pinned coding ends during VDJ recombination (Goodarzi et al., 2006, Ma et al., 2005a, Ma et al., 2002, Ma et al., 2005b, Niewolik et al., 2006, Riballo et al., 2004, Yannone et al., 2008). Physical association of
Does DNA-PK Regulate Dsbr Repair Pathway Choice?
How a cell chooses whether to repair a DNA double strand break by NHEJ or by HR is a fundamental and largely unanswered question. There is abundant evidence for competition between HR and NHEJ. This includes reports showing: (1) competition of the two pathways for repair of the same lesions (Frank-Vaillant and Marcand, 2002, Fukushima et al., 2001, Kim et al., 2005, Takata et al., 1998), (2) sensitivity to MMC-induced damage [that is dependent on HR for repair] in paired cell strains based on
Why is DNA-PK So Abundant?
A long-standing question in this field is why primate cells express such high levels of DNA-PK. While DNA-PK is fairly abundant in all mammalian cells, primate cells express ∼50 times more DNA-PK activity than rodent cells (Finnie et al., 1995). The other NHEJ factors are not so highly expressed in human cells. The high levels of DNA-PK in human cells are somewhat paradoxical in that this does not impart any increased ability to repair DNA damage (Allen et al., 2003). If DNA-PK does not afford
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