Penelope A. Jeggo

DNA repair is limiting for haematopoietic stem cells during ageing

Accumulation of DNA damage leading to adult stem cell exhaustion has been proposed to be a principal mechanism of ageing. Here we address this question by taking advantage of the highly specific role of DNA ligase IV in the repair of DNA double-strand breaks by non-homologous end-joining, and by the discovery of a unique mouse strain with a hypomorphic Lig4Y288C mutation. The Lig4Y288C mouse, identified by means of a mutagenesis screening programme, is a mouse model for human LIG4 syndrome, showing immunodeficiency and growth retardation. Diminished DNA double-strand break repair in the Lig4Y288C strain causes a progressive loss of haematopoietic stem cells and bone marrow cellularity during ageing, and severely impairs stem cell function in tissue culture and transplantation. The sensitivity of haematopoietic stem cells to non-homologous end-joining deficiency is therefore a key determinant of their ability to maintain themselves against physiological stress over time and to withstand culture and transplantation.

The life and death of DNA-PK

Double-strand breaks (DSBs) arise endogenously during normal cellular processes and exogenously by genotoxic agents such as ionizing radiation (IR). DSBs are one of the most severe types of DNA damage, which if left unrepaired are lethal to the cell. Several different DNA repair pathways combat DSBs, with nonhomologous end-joining (NHEJ) being one of the most important in mammalian cells. Competent NHEJ catalyses repair of DSBs by joining together and ligating two free DNA ends of little homology (microhomology) or DNA ends of no homology. The core components of mammalian NHEJ are the catalytic subunit of DNA protein kinase (DNA-PKcs), Ku subunits Ku70 and Ku80, Artemis, XRCC4 and DNA ligase IV. DNA-PK is a nuclear serine/threonine protein kinase that comprises a catalytic subunit (DNA-PKcs), with the Ku subunits acting as the regulatory element. It has been proposed that DNA-PK is a molecular sensor for DNA damage that enhances the signal via phosphorylation of many downstream targets. The crucial role of DNA-PK in the repair of DSBs is highlighted by the hypersensitivity of DNA-PK−/− mice to IR and the high levels of unrepaired DSBs after genotoxic insult. Recently, DNA-PK has emerged as a suitable genetic target for molecular therapeutics such as siRNA, antisense and novel inhibitory small molecules. This review encompasses the recent literature regarding the role of DNA-PK in the protection of genomic stability and focuses on how this knowledge has aided the development of specific DNA-PK inhibitors, via both small molecule and directed molecular targeting techniques. This review promotes the inhibition of DNA-PK as a valid approach to enhance the tumor-cell-killing effects of treatments such as IR.

DNA breakage and repair.

For many years it has been evident that mammalian cells differ dramatically from yeast and rejoin the majority of their DNA DSBs by a nonhomologous mechanism, recently termed NHEJ. In the last few years a number of genes and proteins have been identified that operate in the pathway providing insights into the mechanism. These proteins include the three components of DNA-PK, DNA ligase IV, and XRCC4. In yeast Sir2, -3, and -4 proteins are also involved in the process and therefore are likely to play a role in higher organisms. Studies with yeast suggest that NHEJ is an error-free mechanism. Although the process is far from understood, it is likely that the DNA-PK complex or Ku alone acts in a complex with the Sir proteins possibly protecting the ends and preventing random rejoining. Further work is required to establish the details of this mechanism and to determine whether this represents an accurate rejoining process for a complex break induced by ionizing radiation. It will be intriguing to discover how the cell achieves efficient and accurate rejoining without the use of homology. Interactions between the components of DNA-PK and other proteins playing a central role in damage response mechanisms are beginning to emerge. Interestingly, there is evidence that DNA repair and damage response mechanisms overlap in lower organisms. The overlapping defects of the yeast Ku mutants, tell mutants, and AT cell lines in telomere maintenance further suggest overlapping functions or interacting mechanisms. A challenge for the future will be to establish how these different damage response mechanisms overlap and interact.

53BP1-dependent robust localized KAP-1 phosphorylation is essential for heterochromatic DNA double-strand break repair

DNA double-strand breaks (DSBs) trigger ATM (ataxia telangiectasia mutated) signalling and elicit genomic rearrangements and chromosomal fragmentation if misrepaired or unrepaired. Although most DSB repair is ATM-independent, ∼15% of ionizing radiation (IR)-induced breaks persist in the absence of ATM-signalling. 53BP1 (p53-binding protein 1) facilitates ATM-dependent DSB repair but is largely dispensable for ATM activation or checkpoint arrest. ATM promotes DSB repair within heterochromatin by phosphorylating KAP-1 (KRAB-associated protein 1, also known as TIF1β, TRIM28 or KRIP-1; ref. 2). Here, we show that the ATM signalling mediator proteins MDC1, RNF8, RNF168 and 53BP1 are also required for heterochromatic DSB repair. Although KAP-1 phosphorylation is critical for 53BP1-mediated repair, overall phosphorylated KAP-1 (pKAP-1) levels are only modestly affected by 53BP1 loss. pKAP-1 is transiently pan-nuclear but also forms foci overlapping with γH2AX in heterochromatin. Cells that do not form 53BP1 foci, including human RIDDLE (radiosensitivity, immunodeficiency, dysmorphic features and learning difficulties) syndrome cells, fail to form pKAP-1 foci. 53BP1 amplifies Mre11–NBS1 accumulation at late-repairing DSBs, concentrating active ATM and leading to robust, localized pKAP-1. We propose that ionizing-radiation induced foci (IRIF) spatially concentrate ATM activity to promote localized alterations in regions of chromatin otherwise inhibitory to repair.

KAP-1 phosphorylation regulates CHD3 nucleosome remodeling during the DNA double-strand break response

KAP-1 poses a substantial barrier to DNA double-strand break (DSB) repair within heterochromatin that is alleviated by ATM-dependent KAP-1 phosphorylation (pKAP-1). Here we address the mechanistic consequences of pKAP-1 that promote heterochromatic DSB repair and chromatin relaxation. KAP-1 function involves autoSUMOylation and recruitment of nucleosome deacetylation, methylation and remodeling activities. Although heterochromatin acetylation or methylation changes were not detected, radiation-induced pKAP-1 dispersed the nucleosome remodeler CHD3 from DSBs and triggered concomitant chromatin relaxation; pKAP-1 loss reversed these effects. Depletion or inactivation of CHD3, or ablation of its interaction with KAP-1SUMO1, bypassed pKAP-1s role in repair. Though KAP-1 SUMOylation was unaffected after irradiation, CHD3 dissociated from KAP-1SUMO1 in a pKAP-1–dependent manner. We demonstrate that KAP-1Ser824 phosphorylation generates a motif that directly perturbs interactions between CHD3′s SUMO-interacting motif and SUMO1, dispersing CHD3 from heterochromatin DSBs and enabling repair.

The Repair and Signaling Responses to DNA Double-Strand Breaks

A DNA double-strand break (DSB) has long been recognized as a severe cellular lesion, potentially representing an initiating event for carcinogenesis or cell death. The evolution of DSB repair pathways as well as additional processes, such as cell cycle checkpoint arrest, to minimize the cellular impact of DSB formation was, therefore, not surprising. However, the depth and complexity of the DNA damage responses being revealed by current studies were unexpected. Perhaps the most surprising finding to emerge is the dramatic changes to chromatin architecture that arise in the DSB vicinity. In this review, we overview the cellular response to DSBs focusing on DNA repair pathways and the interface between them. We consider additional events which impact upon these DSB repair pathways, including regulated arrest of cell cycle progression and chromatin architecture alterations. Finally, we discuss the impact of defects in these processes to human disease.

The C Terminus of Ku80 Activates the DNA-Dependent Protein Kinase Catalytic Subunit

ABSTRACT Ku is a heterodimeric protein with double-stranded DNA end-binding activity that operates in the process of nonhomologous end joining. Ku is thought to target the DNA-dependent protein kinase (DNA-PK) complex to the DNA and, when DNA bound, can interact and activate the DNA-PK catalytic subunit (DNA-PKcs). We have carried out a 3′ deletion analysis of Ku80, the larger subunit of Ku, and shown that the C-terminal 178 amino acid residues are dispensable for DNA end-binding activity but are required for efficient interaction of Ku with DNA-PKcs. Cells expressing Ku80 proteins that lack the terminal 178 residues have low DNA-PK activity, are radiation sensitive, and can recombine the signal junctions but not the coding junctions during V(D)J recombination. These cells have therefore acquired the phenotype of mouse SCID cells despite expressing DNA-PKcs protein, suggesting that an interaction between DNA-PKcs and Ku, involving the C-terminal region of Ku80, is required for DNA double-strand break rejoining and coding but not signal joint formation. To gain further insight into important domains in Ku80, we report a point mutational change in Ku80 in the defective xrs-2 cell line. This residue is conserved among species and lies outside of the previously reported Ku70-Ku80 interaction domain. The mutational change nonetheless abrogates the Ku70-Ku80 interaction and DNA end-binding activity.

Phosphoproteomic analysis reveals that PP4 dephosphorylates KAP-1 impacting the DNA damage response

Protein phosphatase PP4C has been implicated in the DNA damage response (DDR), but its substrates in DDR remain largely unknown. We devised a novel proteomic strategy for systematic identification of proteins dephosphorylated by PP4C and identified KRAB‐domain‐associated protein 1 (KAP‐1) as a substrate. Ionizing radiation leads to phosphorylation of KAP‐1 at S824 (via ATM) and at S473 (via CHK2). A PP4C/R3β complex interacts with KAP‐1 and silencing this complex leads to persistence of phospho‐S824 and phospho‐S473. We identify a new role for KAP‐1 in DDR by showing that phosphorylation of S473 impacts the G2/M checkpoint. Depletion of PP4R3β or expression of the phosphomimetic KAP‐1 S473 mutant (S473D) leads to a prolonged G2/M checkpoint. Phosphorylation of S824 is necessary for repair of heterochromatic DNA lesions and similar to cells expressing phosphomimetic KAP‐1 S824 mutant (S824D), or PP4R3β‐silenced cells, display prolonged relaxation of chromatin with release of chromatin remodelling protein CHD3. Our results define a new role for PP4‐mediated dephosphorylation in the DDR, including the regulation of a previously undescribed function of KAP‐1 in checkpoint response.

Impaired lymphocyte development and antibody class switching and increased malignancy in a murine model of DNA ligase IV syndrome

Hypomorphic mutations in DNA ligase IV (LIG4) cause a human syndrome of immunodeficiency, radiosensitivity, and growth retardation due to defective DNA repair by the nonhomologous end-joining (NHEJ) pathway. Lig4-null mice are embryonic lethal, and better mouse models are needed to study human LigIV syndrome. We recently identified a viable mouse strain with a Y288C hypomorphic mutation in the Lig4 gene. Lig4Y288C mice exhibit a greater than 10-fold reduction of LigIV activity in vivo and recapitulate the immunodeficiency and growth retardation seen in human patients. Here, we have demonstrated that the Lig4Y288C mutation leads to multiple defects in lymphocyte development and function, including impaired V(D)J recombination, peripheral lymphocyte survival and proliferation, and B cell class switch recombination. We also highlight a high incidence of thymic tumors in the Lig4Y288C mice, suggesting that wild-type LigIV protects against malignant transformation. These findings provide explanations for the complex lymphoid phenotype of human LigIV syndrome.

ATM Localization and Heterochromatin Repair Depend on Direct Interaction of the 53BP1-BRCT2 Domain with γH2AX

Summary 53BP1 plays multiple roles in mammalian DNA damage repair, mediating pathway choice and facilitating DNA double-strand break repair in heterochromatin. Although it possesses a C-terminal BRCT2 domain, commonly involved in phospho-peptide binding in other proteins, initial recruitment of 53BP1 to sites of DNA damage depends on interaction with histone post-translational modifications—H4K20me2 and H2AK13/K15ub—downstream of the early γH2AX phosphorylation mark of DNA damage. We now show that, contrary to current models, the 53BP1-BRCT2 domain binds γH2AX directly, providing a third post-translational mark regulating 53BP1 function. We find that the interaction of 53BP1 with γH2AX is required for sustaining the 53BP1-dependent focal concentration of activated ATM that facilitates repair of DNA double-strand breaks in heterochromatin in G1.