Overview
Mammalian cells possess sophisticated and intricate mechanisms to detect and repair DNA double-strand breaks, serious genetic lesions that can cause a cell to die or become cancerous. Our laboratory is exploring the mechanisms that maintain genome stability and how they relate to human pathology. We are investigating the two predominant double-strand break repair pathways: nonhomologous end joining, which repairs two broken ends without respect to sequence integrity, and homologous recombination, which uses a template to catalyze high fidelity repair. Another goal is to clarify the mechanisms of oncogenic chromosomal translocations, where the exchange of genetic material between chromosomes leads to tumorigenesis. We are examining how nuclear architecture, genome structure and DNA repair mechanisms all influence this process as well as working with a mouse model of human leukemia/lymphoma to better understand the molecular pathobiology of the human disease. We are also investigating the connection between DNA damage and natural variations in aging, using the mouse as a model system to explore how genome stability control changes with age.
Research details
Genome stability and cancer
Nonhomologous end joining
Nonhomologous end joining (NHEJ) is a critical DNA repair pathway, with proposed tumor suppression functions in many tissues. Mutations in the NHEJ factor ART/DCLRE1C cause radiation sensitive severe combined immunodeficiency (RS-SCID) in humans and may increase susceptibility to lymphoma in some settings. We have found that lack of Art/Dclre1c provokes loss of heterozygosity (LOH) for Trp53, which encodes the critical tumor suppressor p53, leading to the formation of lymphomas. We also discovered that the lack of Art/Dclre1c can accelerate tumor formation in several other tissues, showing for the first time the predicted tumor suppression function of NHEJ outside the immune system. Together these findings have led us to a model for NHEJ-mediated tumor suppression, where the interplay between NHEJ and Trp53 LOH influences the sequence and timing of multi-hit oncogenesis, in several different cell types. Our ongoing work is focused on testing this model and determining the mechanisms of NHEJ-mediated tumor suppression in both lymphoid and non-lymphoid cells.
Homologous recombination
It has long been recognized that genomic instability represents one of the hallmarks of a wide variety of tumor types, including lymphoid tumors. However, the cellular functions that normally prevent such instability, and the precise role of instability in driving tumor formation, remain poorly understood. Several lines of evidence have implicated NHEJ as a key suppressor of oncogenic genome instability, but the contribution of homologous recombination (HR) has been less well characterized. In this context, recurrent translocations or deletions affecting Chromosome 7q32-36 are often noted in myeloid and lymphoblastic leukemias, especially those with complex karyotypes. The X-ray cross-complementing 2 (Xrcc2) gene is an intriguing candidate at 7q36, as a member of the Rad51-family of HR factors required for chromosomal stability, ionizing radiation resistance, and embryonic viability in mice. Defects in Rad51 complex-mediated DNA repair are implicated in several human cancers, but the molecular basis is unknown. We are taking several related approaches to elucidate the function of Xrcc2 in preventing genome instability, preserving normal lymphocytes, and preventing lymphoma/leukemia. We are developing a conditional allele of Xrcc2 that will be used to test the effects of inactivation in specific cell types or at specific developmental stages. To complement these studies, we have also developed siRNA reagents to permit more facile manipulation of Xrcc2 status, both in vitro and in vivo. Finally, we are using mice harboring a gene-targeted null mutation to probe the role of Xrcc2 in lymphoid development and neoplasia.
Nuclear architecture
In addition to the genetics, we are also interested in the molecular and biophysical mechanisms behind oncogenic genome instability. Following chromosomal breakage, 3-D proximity of translocation donor and target sites must occur, at least transiently, for inappropriate chromosomal recombination to occur. In this context, it has become increasingly clear that interphase chromosomes are organized into discrete chromosome territories (CTs) and that CTs may occupy characteristic positions within the nucleus. While chromosome territory proximity has been postulated as a key determinant of translocation susceptibility, this hypothesis has remained controversial. We discovered that, in primary lymphocytes, CT positioning is non-random and favors groupings of heterologous, rather than homologous, CTs. We went on to demonstrate that the composition of heterologous CT groupings directly relates to the susceptibility to translocation between specific chromosomes. This was an important finding because it has confirmed a long-standing hypothesis that the characteristic proximity of chromosomes in interphase nuclei is sufficient to determine their inherent likelihood of translocation. We are now combining high-resolution microscopy and innovative new image analysis approaches with our mouse models to investigate the influence of nuclear architecture on oncogenic genome instability.
The aging-cancer connection
One theory of aging posits that accumulation of DNA damage or chromosomal abnormalities with age leads to decline or derangement of normal cellular processes. In support of this hypothesis, mice deficient for DNA damage response or repair often show phenotypes resembling accelerated aging, especially tumor development. However, natural aging and age-specific pathologies, such as cancer, are complex and variable phenomena that may be considered quantitative traits.
As an outgrowth of the lab's interest in genome instability, we are investigating the connection between DNA damage and natural variations in aging. The mouse represents an excellent model system to explore how genome stability control changes with age. In this regard, we have a unique opportunity to exploit a comprehensive collection of aging inbred mouse strains available at TJL. This strain collection, developed and maintained with funding from The Ellison Foundation and the NIH Nathan Shock Center of Excellence in the Basic Biology of Aging, provides us with nearly 30 strains of well-characterized, genetically defined young, middle age, and old mice. This resource is providing us with a novel opportunity to investigate the natural variability in DNA damage responsiveness and the extent to which it influences natural aging or known pathologies of age representing a set of unique fixed complements of allelic variation, just as each human has a unique assortment of polymorphisms, that sum to determine complex phenotypes. For our part in this large collaborative undertaking, we are probing for strain specific differences in DNA damage susceptibility, DNA repair, and chromosome instability with age. We have already uncovered some intriguing strain-related differences, and are continuing to examine this aging panel in detail. We will then exploit these inbred strains to identify the relevant genes which may control genome stability, aging, and cancer.
Lab staff
Assistant Professor: Kevin Mills, Ph.D.
Postdoctoral Fellows: Sarah Maas, Ph.D., Muneer Hasham, Ph.D.
Research Assistants II: Sarah Wright, B.A.
Research Assistant I: Travis Alley, B.S.
Research Administrative Assistant: Maxine Friend
Publication listings
Caddle LB, Hasham MG, Schott W, Shirley BJ, Mills KD. (2008) Homologous recombination is necessary for normal lymphocyte development. Mol Cell Biol (In Press).
Caddle LB, Grant JL, Szatkiewicz J, van Hase J, Shirley BJ, Bewersdorf J, Cremer C, Arneodo A, Khalil A, Mills KD. 2007. Chromosome neighborhood composition determines translocation outcomes after exposure to high-dose radiation in primary cells. Chromosome Res. 15:1061-1073.
Hess ST, Gould TJ, Gudheti MV, Maas SA, Mills KD, Zimmerberg J. 2007. Dynamic clustered distribution of hemagglutinin resolved at 40 nm in living cell membranes discriminates between raft theories. Proc Natl Acad Sci USA. 104:17370-17375.
Khalil A, Grant JL, Caddle LB, Atzema E, Mills KD, Arneodo A. 2007. Chromosome territories have a highly nonspherical morphology and nonrandom positioning. Chromosome Res. 15:899-916.
Woo Y, Wright SM, Maas SS, Alley TL, Caddle LB, Kamdar S, Affourtit J, Foreman O, Akeson EC, Shaffer D, Bronson RT, Morse 3rd, Roopenian D, Mills KD. 2007. The Nonhomologous end joining factor Artemis suppresses multi-tissue tumor formation and prevents loss of hetrozygosity. Oncogene 26:6010-6020.
Yan CT, Kaushal D, Murphy M. Zhang Y, Datta A, Chen C, Monroe B, Mostoslavsky G, Coakley K, Gao Y, Mills KD, Fazeli AP, Tepsuporn S, Hall G, Mulligan R, Rox E, Bronson R, De Girolami U, Lee C, Alt FW. 2006. XRCC4 suppresses medulloblastomas with recurrent translocations in p53-deficient mice. Proc Natl Acad Sci USA. 103:7378-7383.
Morales JC, Franco S, Murphy MM, Bassing CH, Mills KD, Adams MM, Walsh NC, Manis JP, Rassidakis GZ, Alt FW, Carpenter PB. 2006. 53BP1 and p53 synergize to suppress genomic instability and lymphomagenesis. Proc Natl Acad Sci USA. 103:3310-3315.
Mostoslavsky R, Chua KF, Lombard DB, Pang WW, Fischer MR, Gellon L, Liu P, Mostoslavsky G, Franco S, Murphy MM, Mills KD, Patel P, Hsu JT, Hong AL, Ford E, Cheng HL, Kennedy C, Nunez N, Bronson R, Frendewey D, Auerbach W, Valenzuela D, Karow M, Hottiger MO, Hursting S, Barrett JC, Guarente L, Mulligan R, Demple B, Yancopoulos GD, Alt FW. 2006. Genomic instability and aging-like phenotype in the absence os mammalian SIRT6. Cell 124:315-329.
Couedel C, Mills KD, Marco B, Shen L, Olshen A, Johnson RD, Nussenzweig A, Essers J, Kanaar R, Li GC, Alt FW, Jasin M. 2004. Collaboration of homologous recombination and nonhomologous end-joining factors for the survival and integrity of mice and cells. Genes Dev 18:1293-1304.
Mills KD, Ferguson DO, Essers J, Eckersdorff M, Kanaar R, Alt FW. 2004. Rad54 and DNA Ligase IV cooperate to maintain mammalian chromatid stability. Genes Dev 18:1283-1292
