CELLULAR MECHANISMS THAT MAINTAIN STABILITY
OF THE HUMAN GENOME

Dividing cells rely on three basic mechanisms to maintain genome stability. Two of these mechanisms, duplication of the genome by DNA replication and the segregation of each genome copy into dividing cells during mitosis, are mechanical in nature. These two bioengineering tasks require a large number of structural proteins that function together as complex machines. Superimposed on these two mechanical systems is a third component of quality control that monitors the integrity of the genome to ensure its accurate replication and distribution. Defects in any one of these three fundamental cellular processes lead to the accumulation of mutations in the genome that result in cell death or uncontrolled cell growth. An understanding of the biochemical basis of the three mechanisms that maintain genome stability is of utmost importance in developing new methods of cancer detection and treatment.

Our laboratory is focused on studying two of the three essential processes that involve:  1) a checkpoint system to monitor radiation-induced DNA damage, and 2) the biomechanical events that segregate chromosomes between two daughter cells during mitosis.

Our efforts to characterize how DNA damage activates a checkpoint control mechanism that results in cell cycle arrest or cell death is focused on the ataxia telangiectasia gene product. ATM (ataxia telangiectasia mutated) is a human autosomal recessive disease in which homozygous individuals die at an early age. ATM patients display symptoms of neuromuscular degeneration, impaired immune systems, high incidences of cancer, and extreme sensitivity to radiation and radiomimetic drugs. Cells cultured from ATM patients are unable to execute checkpoint induced cell cycle arrest when they accumulate damaged DNA. Ultimately, these cells either die or become cancerous.

Work by others has resulted in the identification and molecular cloning of the ATM gene. Analysis of the 9.3 kilobase (kb) ATM cDNA reveals that it encodes a protein of approximately 350 kilodaltons (kDa). The carboxyl terminal domain of ATM exhibits 50% identity with the kinase domain of phosphatidylinositol-3 kinase. Additional homology was found between the remaining carboxyl half of ATM and several proteins in yeast and Drosophila that are involved in sensing DNA damage. The combined data suggest that the ATM gene represents the human counterpart of an evolutionarily conserved gene that is critical for maintaining genome stability. The analysis of ATM function is crucial for understanding the biochemical basis of the disease, as well as the mechanism that induces cell cycle arrest in response to DNA damage.

Our primary goal is to characterize the biochemical properties of the ATM protein in normal cells and then compare these characteristics to cells that are defective for ATM. To achieve this, we have generated a panel of polyclonal anti-ATM antibodies. Affinity-purified ATM antibodies were used to detect the ATM protein in tissue culture cells by immunoblotting, as well as by immunoprecipitation of radiolabeled cell lysates. The antibodies recognized a high molecular weight protein of the appropriate size for the predicted 350 kDa ATM protein. Cell fractionation experiments indicate that ATM is a nuclear protein that is associated with chromatin and the nuclear matrix. Western blot analysis shows that ATM is constitutively expressed throughout the cell cycle. This finding is consistent with the notion that ATM acts at a very early step in the DNA damage response by detecting the presence of damaged DNA. Interestingly, ATM steady-state levels were unaltered after cells were exposed to ionizing radiation suggesting that an inherent activity of ATM, perhaps its kinase function, will be sensitive to the presence of DNA damage.

We have detected protein kinase activity in ATM immunoprecipitates by using the single-stranded DNA binding protein complex, replication protein A (RPA), as an in vitro substrate. Characterization of this activity showed that the p34 subunit of the RPA complex was phosphorylated only when double- or single-stranded DNA were present. The requirement of the ATM-associated kinase for double-stranded DNA may reflect its in vivo function. In the presence of double-stranded DNA breaks, ATM is recruited to the broken DNA and subsequently activates its protein kinase activity.

Currently, we are interested in identifying additional substrates for the ATM protein kinase. As DNA damage induces cell cycle arrest in G1, S and G2 phases of the cell cycle, our search for ATM substrates has focused on proteins that play essential roles in regulating cell cycle transition. Phosphorylation of the tumor suppressor, p53, or of cell cycle-dependent kinases by ATM might be a direct method by which DNA damage rapidly blocks cell cycle progression.

Our interest in understanding the mechanism of chromosome segregation has focused on a detailed molecular analysis of the protein components of the kinetochore. The kinetochore is situated at the centromere of chromosomes where it mediates the mechanical interactions with spindle microtubules. Our efforts led to the identification of centromere protein (CENP)-E, a kinetochore-associated motor protein. CENP-E belongs to the kinesin superfamily of microtubule-based motors, and was postulated to specify kinetochore microtubule interactions and to generate motive force for chromosome movement. We have previously demonstrated that CENP-E function at kinetochores is essential for proper chromosome alignment; CENP-E is the first molecular motor that was shown to be essential for chromosome alignment. We further demonstrated that the motor domain, which specifies movement along microtubules, was essential for chromosome alignment.

To understand the biochemical nature of the interactions between CENP-E and the kinetochore, we wish to identify the proteins that interact with CENP-E. As a first step towards this goal, we have identified a 430 amino acid domain that is responsible for targeting CENPE to kinetochores at mitosis. This kinetochore localization domain was identified by fusing various regions of CENPE to the green fluorescent protein (GFP) from the jellyfish Aequiroa victoria, which fluoresces upon excitation at the appropriate wavelength of light; this reporter protein normally does not bind to kinetochores. The intracellular distribution of various GFP:CENP-E fusions were then detected in transfected HeLa cells by fluorescence microscopy. By examining mitotic cells that express the GFP:CENP-E fusion proteins, we were able to identify the portion of CENP-E that specifies localization to kinetochores.

Using the kinetochore-targeting domain of CENP-E, we used the yeast two-hybrid system to identify a collection of proteins that potentially mediate interactions with CENP-E at the kinetochore. The proteins that bind to the kinetochore-targeting domain of CENP-E include CENP-E, CENP-F, and hBUBR1, a novel human kinase that exhibits strong similarities with the yeast BUB1 spindle checkpoint kinase. We confirmed the interaction between hBUBR1 and CENP-E in yeast by demonstrating complex formation in mitotic HeLa cells. We speculate that the hBUBR1/CENP-E complex may act as a mechanosensor, which links the mechanical activities at the kinetochore to the mitotic checkpoint pathway. This model implies that the biochemical activity of CENP-E is different between unaligned versus aligned kinetochores. A biochemical difference (motor activity, microtubule binding) may influence hBUBR1 in such a way that it either initiates the signal to block mitotic progression or extinguishes itself to relieve the mitotic delay imposed by the checkpoint.

Preliminary characterization of the hBUBR1 kinase showed that it is expressed throughout the cell cycle. In cells blocked in mitosis by microtubule destabilizing drugs, hBUBR1 is hyperphosphorylated. Interestingly, hBUBR1 kinase activity was only detectable in mitosis, indicating that mitosis-specific phosphorylations might be important for kinase activity in vitro. We are currently investigating the importance of the interaction between the CENP-E motor and hBUBR1 kinase at the kinetochore. As a checkpoint kinase, hBUBR1 kinase activity may be sensitive to the motor activity of CENP-E and act as a mechanosensor kinase that can detect whether chromosomes have achieved proper alignment at the spindle equator. This monitoring system is crucial for a cell to sense whether all of its chromosomes have formed a metaphase plate and can synchronously separate their chromatids to the dividing daughter cells at anaphase. The absence of such a checkpoint system would spell certain disaster for the cell, as it would produce progeny cells that were highly aneuploid.

We have previously demonstrated that CENP-E function at the kinetochore is essential for chromosomes to align properly at the spindle equator during mitosis. The kinetochore defects resulting from loss of CENP-E function caused cells to arrest in mitosis for extended periods of time. We postulated that the mitotic checkpoint identified the presence of unaligned chromosomes in these cells and, thus, invoked the mitotic arrest. As bipolar spindle formation was unaffected by the loss of CENP-E, it appeared that the checkpoint was sensitive to the defective kinetochore. To directly address this possibility, we examined whether hBUBR1 function was required for the mitotic arrest that was induced by the loss of CENP-E from kinetochores. To block endogenous CENP-E function, highly specific CENPE antibodies were microinjected into tissue culture cells and the fate of the injected cells was monitored. To test the importance of hBUBR1 activity in the mitotic checkpoint pathway, we performed two different experiments. We microinjected hBUBR1 antibodies into HeLa cells and then challenged the injected cells with a microtubule inhibitor; this challenge tested whether the cells were still capable of arresting in mitosis. We found that cells injected with hBUBR1 antibodies, in contrast to uninjected cells, failed to arrest in mitosis in the presence of microtubule inhibitors. These cells exited mitosis, re-replicated their DNA, and became highly polyploid. We next tested whether hBUBR1 function was required for cells to arrest in mitosis when CENP-E function was lost from kinetochores. HeLa cells were microinjected with CENP-E antibodies alone, or CENP-E and hBUBR1 antibodies. As previously shown, cells injected with CENP-E antibodies entered mitosis, but subsequently arrested with unaligned chromosomes. In contrast, when hBUBR1 function was also disrupted, cells with defective CENP-E function failed to arrest in mitosis. This finding demonstrates that one of the molecular signals at the kinetochore, which is monitored by hBUBR1, is CENP-E. This finding also supports our hypothesis that hBUBR1 is a component of the mitotic checkpoint and monitors the activity of CENP-E during chromosome alignment.

Genetic and biochemical studies in yeast, Drosophila, Xenopus and clam have shown that the transition from one cell cycle stage to the another is due to targeted proteolysis of proteins that act to block cell cycle transitions. In mitosis, the transition from metaphase to anaphase (chromosome alignment to chromosome separation) is mediated by the cyclosome/Anaphase Promoting Complex (APC), a multisubunit complex that ubiquinates substrates for ubiquitin-mediated proteolysis. As the cyclosome/APC has been shown to be a target of the mitotic checkpoint pathway, we wanted to determine whether the mitotic checkpoint kinase, hBUBR1, might induce mitotic delay by inhibiting the cyclosome/APC. We used a combination of immunoprecipitation assays and gel filtration chromatography to show that hBUBR1 associates with the cyclosome/APC. Significantly, this interaction was seen only when cells were blocked in mitosis; no interaction was detected in normal cycling cells. Current efforts are to examine the in vitro ubiquitin ligase activity of cyclosome/APC alone or in association with hBUBR1.

hBUBR1 is expressed throughout the cell cycle, yet we can only detect its kinase activity in mitotically blocked cells. In metaphase cells, where chromosomes have aligned, hBUBR1 kinase is only slightly elevated above background levels. If metaphase cells are treated with microtubule inhibitors to disrupt the spindle, the presence of unaligned chromosomes stimulates hBUBR1 kinase activity. As the kinase activity of hBUBR1 is likely to be important for establishing or maintaining the mitotic checkpoint pathway, understanding how hBUBR1 kinase is regulated is of significant interest to us. Efforts currently are underway to examine if phosphorylation of hBUBR1 during mitosis is essential for its kinase activity. In parallel, a series of known mitotic kinases are being tested for their ability to phosphorylate and activate hBUBR1. We envision that the mitotic checkpoint pathway may consist of a kinase cascade similar to conventional signal transduction pathways. The key difference for the mitotic checkpoint, however, is that the input signal or the signal transduction pathway is the mechanical force that is generated between kinetochores and microtubules during chromosome alignment.

The kinetochore complex is a structure detected only during mitosis. Although some of the components of this complex are visible as discrete foci in nuclei throughout the cell cycle, it is generally thought that a functional complex is assembled as cells prepare to enter mitosis. The mechanism that regulates the cell cycle-dependent assembly of kinetochores remains unexplored due to the lack of suitable reagents. Examination of the distribution of CENP-F at various times during the cell cycle suggests that this protein may be involved in the early steps of kinetochore assembly. CENP-F is not detectable in G1 cells, gradually accumulates in the nuclei of S phase cells and peak levels are reached during G2 where it is distributed uniformly throughout G2 nuclei; it is concentrated near centromere regions when cells near mitosis. When chromatin condensation is visible at prophase, CENP-F can be clearly localized to the centromere-kinetochore regions, where it persists until anaphase. The abrupt appearance of CENPF at centromeres during late stages of G2, coupled with the possibility that it may be a chromosome condensation protein, suggests that it may function in the early stages of centromere-kinetochore assembly. Characterization of CENP-F might reveal a segment of the assembly pathway that leads to a functional centromere-kinetochore complex.

Understanding the biochemical interactions between CENP-F and other proteins within the kinetochore first requires the identification of protein partners. Using the kinetochore targeting domain of CENP-F to screen an expression library by the yeast two-hybrid method, a large collection of candidate interacting proteins were identified. To verify that these proteins can mediate interactions with CENP-F, antibodies were generated against many of these interacting clones. These antibodies were then used to examine the subcellular distribution of the various interactors by immunofluorescence microscopy. This approach led to the identification of novel spindle and kinetochore proteins. One of these proteins, hBUB1, is a kinase that is related to hBUBR1. hBUB1 kinase is about 24% identical and 46% similar to hBUBR1 in sequence. To examine and compare these two related kinases, antibodies specific to each were generated. Immunofluorescence staining revealed that hBUB1 is a nuclear protein, while hBUBR1 is primarily cytoplasmic. Furthermore, hBUB1 assembled onto kinetochores during late G2 when hBUBR1 was still in the cytoplasm. By carefully comparing the kinetochore staining patterns of hBUB1, hBUBR1, CENP-E and CENP-F, we were able to show that these proteins assembled onto kinetochores in a sequential fashion. CENP-F assembles first, followed by hBUB1, hBUBR1 and CENP-E. The distinctive temporal patterns of kinetochore localization by these proteins suggest that kinetochore assembly follows a rigorously ordered pathway whereby the successful assembly of one component is required for subsequent assembly to occur. In addition, the sequential appearance of the hBUB1 and hBUBR1 kinases may also contribute to temporally regulating kinetochore assembly. Thus, these kinases might phosphorylate proteins at specified times so that other proteins can assemble. A detailed molecular and biochemical characterization of these proteins should reveal the molecular assembly pathway and also establish connections with mechanisms that temporally regulate their binding to the nascent kinetchore.

BASU, J., LOGARINHO, E., HERRMANN, S., BOUSBAA, H., LI, Z.X., CHAN, G.K.T., YEN, T.J, SUNKEL, C.E., GOLDBERG, M.L. Localization of the Drosophila checkpoint control protein Bub3 to the kinetochore requires Bub1 but not Zw10 or Rod. Chromosoma 107:376-385, 1998.

CHAN, D.W., GATELY, D.P., URBAN, S., GALLOWAY, A.M., LEES-MILLER, S.P., YEN, T., ALLALUNIS-TURNER, J. Lack of correlation between ATM protein expression and tumour cell radiosensitivity. Int. J. Radiat. Biol. 74:217-224, 1998.

CHAN, G.K.T., SCHAAR, B.T., YEN, T.J. Characterization of the kinetochore binding domain of CENP-E reveals interactions with the kinetochore proteins CENP-F and hBUBR1. J. Cell Biol. 143:49-63, 1998.

JABLONSKI, S.A., CHAN, G.K.T., COOKE, C.A., EARNSHAW, W.C., YEN, T.J. The hBUB1 and hBUBR1 kinases sequentially assemble onto kinetochores during prophase with hBUBR1 concentrating at the kinetochore places in mitosis. Chromosoma 107:386-396, 1998.

GATELY, D.P., HITTLE, J.C., CHAN, G.K.T., YEN, T. Characterization of ATM expression, localization and associated DNA-dependent protein kinase activity. Mol. Biol. Cell. 9:2361-2374, 1998.

^a   D.P. Gately: Present address--INEC, San Diego, CA

^b   B. Schaar: Department of Genetics, Stanford University, Stanford, CA 94305

Illustrations or unpublished data in these reports should not be used without permission of the author.

Fox Chase Cancer Center

Scientific Report 1998