ATR-mediated DNA Damage Response Research Articles

Lycopene inhibits Helicobacter pylori-induced ATM/ATR-dependent DNA damage response in gastric epithelial AGS cells

Oxidative stress linked to DNA damage is involved in the pathogenesis of Helicobacter pylori-associated gastric diseases. The DNA damage response (DDR) coordinates cell-cycle transitions, DNA repair, and apoptosis through the activation of ataxia–telangiectasia-mutated (ATM) and ATM and Rad3-related (ATR) and their target proteins. However, neither H. pylori-induced DDR nor the effects of antioxidants on the DNA damage have been established. This study aimed to investigate the detailed process of H. pylori-induced DNA damage and to examine whether lycopene, a natural antioxidant, inhibits DNA damage and cellular response of gastric epithelial AGS cells infected with H. pylori. AGS cells were cultured with H. pylori in Korean isolates and treated with or without lycopene. Cell viability, DNA damage indices, levels of 8-OH-dG, and reactive oxygen species (ROS) as well as cell-cycle distributions were determined. The activation of ATM, ATR, Chk1, and Chk2; histone H2AX focus formation; activation and induction of p53; and levels of Bax and Bcl-2 and poly(ADP-ribose) polymerase-1 (PARP-1) were assessed. The results showed that H. pylori induced apoptosis in AGS cells with increased Bax and decreased Bcl-2 expression as well as PARP-1 cleavage. Culture with H. pylori led to increases in intracellular ROS, 8-OH-dG, double-strand DNA breaks (DSBs), and DNA fragmentation. H. pylori induced activation of the ATM/Chk2 and ATR/Chk1 pathways, phosphorylation of H2AX and p53, and a delay in the progression of the cells entering the S phase. Lycopene inhibited H. pylori-induced increases in ROS, apoptosis, alterations in cell-cycle distribution, DSBs, and ATM- and ATR-mediated DDR in AGS cells. In conclusion, lycopene may be beneficial for treatment of H. pylori-induced gastric diseases associated with oxidative DNA damage.

New players in end-protection: LIM-domain proteins associate with the shelterin complex.

One of the vital activities performed by telomeres is the protection of chromosome ends. This allows cells to distinguish the tip of linear chromosomes from sites of DNA damage, and to prevent end-to-end chromosome fusions. The protective function of telomeres is lost when telomeres become critically short; a condition that is frequently found in aging cells [1]. In mammals end-protection is achieved by the localization of a six-protein complex, termed shelterin [2], to the terminal telomeric DNA. Of the six members that constitute this complex three bind directly to DNA: TRF1, TRF2 and POT1 while the others; TIN2, TPP1 and RAP1 are recruited to chromosome ends through protein-protein interactions [3]. Depletion of individual components of this complex is sufficient to render telomeres dysfunctional resulting in the activation of a DNA damage response [4-8]. Interestingly, components of this complex show a high degree of specialization being able to suppress different branches of the DNA damage response machinery. TRF2 is primarily involved in suppressing ATM kinase activity at chromosome ends [9], while TRF1 protects telomeres during replication suppressing the activation of a ATR-mediated DNA damage response [8]. POT1 on the other hand suppresses ATR activation throughout the cell cycle [9]. The major question, which for the most part remains to be addressed, is how can these proteins suppress the DNA damage response? A key step towards decoding this puzzle is the identification of additional proteins involved in end-protection. It seems likely that the shelterin complex does not act alone but needs to recruit accessory proteins in order to protect chromosome ends. Indeed, one such accessory protein was identified in the nuclease Apollo that is recruited to telomeres by TRF2 [10] and plays a critical role in end protection [11, 12]. In this issue of Aging Sheppard and Loayza report on a new family of protein that associate with the shelterin complex and might play a critical role in end-protection [13]. TRIP6, a member of the Zyxin family of proteins was identified as a POT1 interacting protein in a yeast two-hybrid screen. TRIP6 binds to several members of the shelterin complex including TRF2 and TIN2. Intriguingly, TRIP6 is not the only member of this family of proteins to interact with the shelterin complex since Sheppard and Loayza also report that an other member of the Zyxin family, called LPP, interacts with the same shelterin components. Using knockdown experiments Sheppard and Loayza report that inhibition of either TRIP6 or LPP result in loss of end-protection with the accumulation of DNA damage proteins at telomeres. This body of work suggests that members of the Zyxin family of proteins associate transiently with telomeres and play a role in end-protection. Additional work is required to establish what is the precise role of these proteins at chromosome ends. However, given the role of this family of proteins in other cellular processes, it is likely these proteins at telomeres serve as recruitment platforms for enzymatic activities. This would parallel the role of the Zyxin family member Ajuba in transcriptional repression. Ajuba is able to recruit to RAREs arginine methylase activity that in turns is essential to achieve transcriptional repression. In a similar manner TRIP6 and LPP might recruit enzymatic activities required to suppress the DNA damage response at chromosome ends. As such, Zyxin family members would bring at chromosome ends key tools required to disguise the tips of chromosomes end and to hide them from the DNA damage machinery. Future work will define what are the relative contribu-tion and the mechanism of action of this new family of proteins involved in end-protection.

HIV-1 Vpr Triggers Natural Killer Cell–Mediated Lysis of Infected Cells through Activation of the ATR-Mediated DNA Damage Response

Natural killer (NK) cells are stimulated by ligands on virus-infected cells. We have recently demonstrated that NK cells respond to human immunodeficiency virus type-1 (HIV-1)-infected autologous T-cells, in part, through the recognition of ligands for the NK cell activating receptor NKG2D on the surface of the infected cells. Uninfected primary CD4pos T-cell blasts express little, if any, NKG2D ligands. In the present study we determined the mechanism through which ligands for NKG2D are induced on HIV-1-infected cells. Our studies reveal that expression of vpr is necessary and sufficient to elicit the expression of NKG2D ligands in the context of HIV-1 infection. Vpr specifically induces surface expression of the unique-long 16 binding proteins (ULBP)-1 and ULBP-2, but not ULBP-3, MHC class I-related chain molecules (MIC)-A or MIC-B. In these studies we also demonstrated that Vpr increases the level of ULBP-1 and ULBP-2 mRNA in primary CD4pos T-cell blasts. The presence of ULBP-1 and ULBP-2 on HIV-1 infected cells is dependent on the ability of Vpr to associate with a protein complex know as Cullin 4a (Cul4a)/damaged DNA binding protein 1 (DDB1) and Cul4a-associated factor-1(DCAF-1) E3 ubiquitin ligase (Cul4aDCAF-1). ULBP-1 and -2 expression by Vpr is also dependent on activation of the DNA damage sensor, ataxia telangiectasia and rad-3-related kinase (ATR). When T-cell blasts are infected with a vpr-deficient HIV-1, NK cells are impaired in killing the infected cells. Thus, HIV-1 Vpr actively triggers the expression of the ligands to the NK cell activation receptor.

Single- and double-stranded DNA: building a trigger of ATR-mediated DNA damage response: Figure 1.

The DNA damage signaling pathways mediated by the ataxia-telangiectasia mutated (ATM) and the ATM and Rad3-related (ATR) kinases play crucial roles in the maintenance of genomic integrity and may function as an anti-cancer barrier during early tumorigenesis. Although the ATM and ATR pathways share some of their downstream functions, the DNA damage that evoke these two pathways are distinct. While ATM plays a primary role in the response to double-stranded DNA breaks (DSBs), ATR controls the response to a much broader spectrum of DNA damage, including many that interfere with DNA replication. And, unlike ATM, ATR is crucial for maintaining genomic integrity during S phase of the cell cycle, and is indispensable for cell survival. Clearly, revealing the DNA structure that elicits the ATR pathway would be a critical step toward understanding the essential function of ATR and the genomic instability that it counters. The versatility of the ATR pathway in DNA damage response suggests that this pathway is likely able to sense a common signal generated by different types of DNA damage and genomic instability. Two simple structures commonly generated at sites of DNA repair and stressed DNA replication forks are single-stranded DNA coated with replication protein A (RPA-ssDNA) and junctions of singleand doublestranded DNA. Both of these structures have been implicated in the activation of ATR checkpoint by a number of studies using different model organisms. In this issue of Genes & Development, Cimprich and colleagues (MacDougall et al. 2007) report that circular singlestranded DNA (ssDNA) annealed with primers specifically triggers the ATR-mediated checkpoint responses in Xenopus egg extracts, revealing the first defined DNA structure sufficient to activate the ATR checkpoint pathway.

Mass Spectrometric Identification of Lysines Involved in the Interaction of Human Replication Protein A with Single-Stranded DNA

Human replication protein A (hRPA), a heterotrimeric single-stranded DNA (ssDNA) binding protein, is required for many cellular pathways including DNA damage repair, recombination, and replication as well as the ATR-mediated DNA damage response. While extensive effort has been devoted to understanding the structural relationships between RPA and ssDNA, information is currently limited to the RPA domains, the trimerization core, and a partial cocrystal structure. In this work, we employed a mass spectrometric protein footprinting method of single amino acid resolution to investigate the interactions of the entire heterotrimeric hRPA with ssDNA. In particular, we monitored surface accessibility of RPA lysines with NHS-biotin modification in the contexts of the free protein and the nucleoprotein complex. Our results not only indicated excellent agreement with the available crystal structure data for RPA70 DBD-AB-ssDNA complex but also revealed new protein contacts in the nucleoprotein complex. In addition to two residues, K263 and K343 of p70, previously identified by cocrystallography as direct DNA contacts, we observed protection of five additional lysines (K183, K259, K489, K577, and K588 of p70) upon ssDNA binding to RPA. Three residues, K489, K577, and K588, are located in ssDNA binding domain C and are likely to establish the direct contacts with cognate DNA. In contrast, no ssDNA-contacting lysines were identified in DBD-D. In addition, two lysines, K183 and K259, are positioned outside the putative ssDNA binding cleft. We propose that the protection of these lysines could result from the RPA interdomain structural reorganization induced by ssDNA binding.

Activation of a DNA damage checkpoint response in a TAF1-defective cell line.

Although the link between transcription and DNA repair is well established, defects in the core transcriptional complex itself have not been shown to elicit a DNA damage response. Here we show that a cell line with a temperature-sensitive defect in TBP-associated factor 1 (TAF1), a component of the TFIID general transcription complex, exhibits hallmarks of an ATR-mediated DNA damage response. Upon inactivation of TAF1, ATR rapidly localized to subnuclear foci and contributed to the phosphorylation of several downstream targets, including p53 and Chk1, resulting in cell cycle arrest. The increase in p53 expression and the G(1) phase arrest could be blocked by caffeine, an inhibitor of ATR. In addition, dominant negative forms of ATR but not ATM were able to override the arrest in G(1). These results suggest that a defect in TAF1 can elicit a DNA damage response.

Activation of the ATR-mediated DNA Damage Response by the HIV-1 Viral Protein R

DNA damage is a universal inducer of cell cycle arrest at the G2 phase. Infection by the human immunodeficiency virus type 1 (HIV-1) also blocks cellular proliferation at the G2 phase. The HIV-1 accessory gene vpr encodes a conserved 96-amino acid protein (Vpr) that is necessary and sufficient for the HIV-1-induced block of cellular proliferation. In the present study, we examined a recently identified DNA damage-signaling protein, the ATM- and Rad3-related protein, ATR, for its potential role in the induction of G2 arrest by Vpr. We show that inhibition of ATR by pharmacological inhibitors, by expression of the dominant-negative form of ATR, or by RNA interference inhibits Vpr-induced cell cycle arrest. As with DNA damage, activation of ATR by Vpr results in phosphorylation of Chk1. This study provides conclusive evidence of activation of the ATR-initiated DNA damage-signaling pathway by a viral gene product. These observations are important toward understanding how HIV infection promotes cell cycle disruption, cell death, and ultimately, CD4+ lymphocyte depletion.