The Quest for Protectors of Genomic Stability

Courtesy of Linda B. Schultz CHECK POINT: The 53Bp1-dependent checkpoint pathway. DNA double-strand breaks are caused by irradiation and other geotoxic events. The ATM [ataxia-telangiectasia mutated] molecule phosphorylates H2AX at or near the break, an event required for 53Bp1 phosphorylation and localization into nuclear foci. Nearly 15 years ago, Saccharomyces cerevisiae researchers at the University of Washington discovered the function of Rad9, the first DNA damage-checkpoint protein.1 The finding's im-portance was not lost on cancer researchers: The Rad9 protein protects cells from genomic instability, a hallmark of cancer cells, by delaying progression into the cell cycle until DNA is repaired. Since then, numerous mammalian homologs of these checkpoint proteins have been identified, including ataxia telangiectasia mutated (ATM), which responds swiftly to a critical type of DNA damage--the double-strand break (DSB)--by phosphorylating key proteins in numerous signaling pathways.2 But a big question, until now, had remained unanswered: What regulates the protectors of genomic stability? Recent work demonstrates that 53Bp1 is one such regulator. It transduces the DNA damage signal to p53, which is a checkpoint protein mutated in many cancers. This protein also is required for checkpoint responses, and regulates partial ATM substrate phosphorylation.3-5 Moreover, murine studies suggest a role in DNA repair and tumor suppressor functions for this protein.6,7 Researchers have their work cut out for them. "We are quickly learning that checkpoint signaling and genome repair are highly intertwined. 53Bp1, as a critical regulator of ATM-dependent checkpoint signaling, will undoubtedly crosstalk to the DNA repair machinery," says Robert Abraham, at the Burnham Institute in La Jolla, Calif. While 53Bp1 is swimming in a milieu of other signaling and repair proteins, all data point in one direction: 53Bp1 plays a direct role in maintaining genomic stability, thereby potentially preventing cancer. DNA damage, which occurs through oxidative stress from normal metabolic processes, threatens cellular homeostasis and life. Cellular responses include initiating DNA repair and activating numerous checkpoint-signaling pathways. The focus of basic science cancer researchers lies in teasing apart these networks. Stan Fields, while at State University of New York at Stony Brook, first identified 53Bp1 nine years ago in a screen for p53-interacting proteins.8 Originally characterized as an activator of p53-dependent gene transcription, researchers found that 53Bp1 contained tandem C-terminal BRCT (BRCA1 C-terminal) motifs that were highly homologous to those in Rad9. "It's an interesting protein because for years it was considered an illegitimate child of the yeast two-hybrid system," says Phil Carpenter, University of Texas Health Science Center in Houston, "And then all of a sudden people started to look at it because of its tandem BRCT repeats." Some believe it is the mammalian Rad9 homolog. The first evidence that 53Bp1 was a damage-response participant came when researchers found that it relocalized into discrete nuclear foci upon exposure of cells to ionizing radiation (IR).9 These foci colocalized with known DNA damage-response proteins, such as the phosphorylated form of H2AX (g-H2AX), Mre11/NBS, BRCA1, and Rad51 at sites of DNA double-strand breaks. Functional data, however, were elusive until four groups created 53Bp1-deficient cellular models.3,4,6,7 The collective findings of these reports demonstrate the role of 53Bp1 in the DNA damage-response pathway. THEORIES AND EVIDENCE Where 53Bp1 functions in the DNA damage pathway is another key question. One hypothesis is that it could function as a scaffolding protein by recruiting substrates to ATM at DNA double-strand break sites. This idea is credible because it is similar to Rad9's role in budding yeast. Here, the yeast ATM equivalent, Mec1, phosphorylates Rad9 following DNA damage. Rad9 then binds to and recruits Rad53, a Mec1 substrate and human Chk2 kinase homolog. It is a likely scenario if 53Bp1 is the Rad9 homolog. Another theory: Because it's needed for at least a subset of ATM-dependent phosphorylation events, 53Bp1 might function upstream of ATM in response to DNA DSBs. Supporting evidence: the protein's foci form in AT cells, in which ATM is mutated and, therefore, nonfunctional. Others believe just the opposite, that 53Bp1 functions downstream of the kinase ATM, because ATM is required for the protein's phosphorylation. The relationship may not be that clear. Junjie Chen, at the Mayo Clinic in Rochester, Minn., explains the controversy: "It's a component of the pathway and there are functional interactions between [all] these proteins, but I wouldn't go beyond that. [People] want to see a one-to-one pathway, but in reality, it doesn't exist." Most agree that even if 53Bp1 is a Rad9 homolog, that does not preclude it from having a role upstream of the kinase; it may function in multiple places. Adds Thanos Halazonetis of the Wistar Institute in Philadelphia: "53Bp1 could be needed for ATM activation and as a scaffold protein ... these models don't preclude each other." Data supporting the downstream hypothesis suggest that 53Bp1 regulates p53 function because its induction is substantially decreased in 53Bp1-inhibited cells. Moreover, the phosphorylation of Chk2, a checkpoint protein that regulates p53, is decreased in the same cells. "These data suggest that 53Bp1 is a central transducer of the DNA damage signal to p53," wrote Stephen Elledge in Science.3 "It is involved in DNA damage signaling and ... it controls p53. Not only is it a responder to [DNA] damage, but it transduces the information to control other proteins too." Says Elledge in an interview: "Those are the most important issues. Knowing it has a role in signaling is important. What people really need [now] is biochemistry, the hard stuff." While its functional location remains unsure, data are clear that 53Bp1 is a DNA damage-checkpoint protein. "Because of its homology with Rad9, and other criteria, we always thought it would be a checkpoint protein. We were gratified that our initial hypothesis turned out to be true," says Halazonetis. The Elledge team also showed the protein's role in the G2-M checkpoint and in intra-S phase checkpoint. Since publication of the original papers, other unpublished results have confirmed this finding. Jiri Bartek, Institute of Cancer Biology, Copenhagen, says, "For sure we think it's involved in both the S-phase and the G2-phase checkpoints." Experiments in B-cells from 53Bp1 -/- mice had similar defects. "We found that 53Bp1 knockout cells exhibited a defect in the G2-M checkpoint. It's a mild effect in response to low-dose irradiation, which is more physiological anyway," says Andre Nussenzweig, National Cancer Institute, Bethesda, Md. Courtesy of Oscar Fernandez-Capetillo and Andre Nussenzweig GETTING DOSED: 53Bp1 foci colocalized with g-H2AX. Mouse embryo fibroblasts irradiated with a dose of 1 Gray and immuno-stained for 53Bp1 (red) and g-H2AX (green). The surrounding white frame outlines the location of the cellular nucleus (Merge). A POTENTIAL TUMOR SUPPRESSOR In contrast to the checkpoint defects reported for 53Bp1-deficient cell lines and B cells from 53Bp1-deficient mice, mouse embryo fibroblasts from the same mice do not exhibit checkpoint defects.5-7 The explanation, says Chen, is that there may be "tissue-specific functions of 53Bp1, since the experiments were performed with different cell types." Alternatively, many redundant pathways could exist. 53Bp1-deficient mice are growth retarded, immune deficient, radiation sensitive and cancer prone, suggesting that 53Bp1 may be involved in DNA repair.6,7 Carpenter describes his team's findings: "We were excited because the mice were sensitive to IR, which, to me, clearly suggests it has something to do with the repair process." 53Bp1-deficient mice have various immune defects, including decreases in thymus cellularity, thymocytes, and peripheral blood CD4+ T lymphocytes. These data suggest that this checkpoint protein participates in T-cell development, a process that requires DNA repair proteins. The immune deficiencies, he says, "are telling us something, and that something is that 53Bp1 functions in the repair that takes place in those cell types." Cells from these animals exhibit chromatid breaks and gaps, tetraploidy, and anueploidy, signifying that mutant cells have an intrinsic genomic stability defect that could contribute to tumorigenesis. Some mice had massive lymphomas. "To us, the most significant finding is that 53Bp1 is a potential tumor suppressor, since 53Bp1-deficient mice are extremely radiation sensitive and develop spontaneous tumors," says Chen. While 53Bp1 could be involved in DNA repair, another explanation exists. Checkpoint defects can mimic repair flaws by allowing DNA replication and/or mitosis to proceed with a damaged genome. Alternatively, 53Bp1 could be multifunctional. One thing is certain, says Abraham, "The networking of checkpoint signaling and repair processes ... suggests that whether 53Bp1 is directly involved in DNA repair or not, the genome will be destabilized by loss of 53Bp1," a significant event in the road to cancer. Linda B. Schultz (lbschultz@sciwriting.com) is a freelance science writer in Suwanee, Ga.; she obtained her PhD in Thanos Halazonetis' laboratory. References 1. T. Weinert, L. Hartwell, "The RAD9 gene controls the cell cycle response to DNA damage in Saccharomyces cerevisiae," Science, 241:317-22,1988. 2. Y. Shiloh, "ATM (ataxia telangiectasia mutated): expanding roles in the DNA damage response and cellular homeostasis," Biochem Soc Trans, 29:661-6, 2001. 3. B. Wang et al., "53Bp1, a mediator of the DNA damage checkpoint," Science, 298:1435-8, 2002. 4. R.A. DiTullio et al., "53Bp1 functions in an ATM-dependent checkpoint pathway that is constitutively activated in human cancer," Nat Cell Biol, 4:998-1002, 2002. 5. O. Fernandez-Capetillo et al., "DNA damage-induced G2-M checkpoint activation by histone H2AX and 53Bp1," Nat Cell Biol, 4:993-7, 2002. 6. I.M. Ward et al., "53Bp1 is required for DNA damage responses and tumor suppression in mice," Mol Cell Biol, 7:2556-63, 2003. 7. J.C. Morales et al., "Role for the BRCT protein 53BP1 in maintaining genomic stability," J Biol Chem, 278:14971-7,2003. 8. K. Iwabuchi et al., "Two cellular proteins that bind to wild-type but not mutant p53," Proc Natl Acad Sci, 91:6098-102,1994. 9. L.B. Schultz et al., "p53 binding protein 1 (53BP1) is an early participant in the cellular response to DNA double-strand breaks," J Cell Biol, 151:1381-90, 2000. function sendData() { document.frm.pathName.value = location.pathname; result = false if (document.frm.score[0].checked) result = true; if (document.frm.score[1].checked) result = true; if (document.frm.score[2].checked) result = true; if (document.frm.score[3].checked) result = true; if (document.frm.score[4].checked) result = true; if (!result) alert("Please select") return result; } .myradio{background-color : #94bee5} Please indicate on a 1 - 5 scale how strongly you would recommend this article to your colleagues? 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The Quest for Protectors of Genomic Stability

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