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Encircled: Large-Scale Purification of Replication Origins from Mammalian Chromosomes
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Molecular Cell 21, March 17, 2006 ª2006 Elsevier Inc.
DOI 10.1016/j.molcel.2006.03.002
Encircled: Large-Scale Purification of Replication Origins from Mammalian Chromosomes
A novel cloning strategy for sequences comprising mammalian replication origins, described by Mesner et al. (2006) in a recent issue of Molecular Cell, utilizes an origin trapping assay in which replication bubbles are selectively retained in agarose due to their circular nature.
In eukaryotic cells, DNA replication initiates at replication origins, specific sites in the genome that are recognized by the origin recognition complex (ORC; [Bell and Stillman, 1992]). The ORC functions as the initiator of replication by recruiting other factors, which facilitate the assembly of replication forks that move away from the origin in a bidirectional manner. In simple organisms like budding yeast, replication origins were first identified as autonomously replicating sequences (ARSs) because they conferred the ability to replicate to plasmid DNAs that otherwise could not replicate (Struhl et al., 1979). Further analysis of these ARSs revealed that they contained an ARS consensus sequence (ACS; [Palzkill et al., 1986]), which is part of the recognition site bound by ORC. Whereas ORC is evolutionarily conserved, the ACS is not, and thus, the nature of replication origins in metazoan genomes has remained elusive and highly controversial (Aladjem and Fanning, 2004). A limited number of eukaryotic origins has been identified by a two-dimensional (2D) gel replicon mapping technique that took advantage of the fact that bubbles exhibited a highly specific migration pattern that separated them effectively from other DNA structures when they were fractionated on agarose gels (Brewer and Fangman, 1987). While the 2D gel replicon mapping technique was instrumental in identifying replication origins in many multicellular organisms, including mammals, it was not amenable as a systematic screening tool for the large-scale identification of origin sequences. The same was true for the ARS assay, as virtually any DNA sequence that was several kilobases in size exhibited ARS activity when introduced into tissue culture cells (discussed in Mesner et al. [2006]). The field has struggled with this issue for almost two decades—until now. The team led by Joyce Hamlin at the University of Virginia has developed a strategy that appears to turn this long-standing quest into a more realistic endeavor. In a paper published in a recent issue of Molecular Cell, the authors describe a protocol to selectively trap replication bubbles in agarose due to their circular nature (Mesner et al., 2006). The separation is based on a technique described in 1973 as ‘‘the trapping of circular DNA in agarose gels’’ (Dean et al., 1973), applying a principle that was first described as ‘‘agar fixation’’ (Wada and Kishizaki, 1968): when DNA, containing
both linear fragments and circles, is solidified in an agar solution, then linear DNA can diffuse while circular DNA is trapped. Subsequent electrophoresis allows linear fragments to enter a gel, whereas circles remain topologically linked within the agarose mesh, thus unable to move. Mesner et al. convincingly demonstrate that the implementation of this trapping procedure yields highly purified preparations of replication bubbles. To generate a replication origin library, the authors utilized the Chinese hamster ovary cell line CHOC400, which harbors w1000 copies of a well-characterized origin located at the dihydrofolate reductase (DHFR) locus, which is activated early in S phase and served as a control in the study. DNA was harvested from cells that were synchronously released into early S phase at a time point at which the DHFR origin exhibits peak activation. Moreover, arresting cells at the onset of S phase also ensured that replication bubbles did not become too large in size, preventing them from being cleaved during the subsequent restriction digestion. After the partial exclusion of double-stranded, nonreplicating DNA, the preparation was mixed with liquid agarose and circle-containing fragments were retained by the trapping procedure outlined above. After isolation of the trapped DNA, the authors demonstrated that the material contained almost exclusively replication bubbles. The trapped fragments were finally cloned into a suitable vector to generate an origin library of w1000 clones after the removal of repetitive sequences. From this library, 15 individual clones were stochastically chosen, and all of them turned out to be active replication origins. Therefore, this new strategy allows for the efficient purification of origin sequences, greatly improving previous approaches (Todorovic et al., 2005). As the authors point out, with this high degree of enrichment, the cloned material might be suitable for microarray hybridization to conduct whole genome studies. Although it seems unlikely that such an approach will be developed for hamster cells in the near future, as the hamster genome has not been sequenced, there is no obvious reason to suspect that the strategy described by Mesner et al. (2006) should not be applicable to any other cellular organisms, including humans. It is also easy to envision variations on the protocol developed by Hamlin and coworkers to allow for the identification of both early- and latefiring origins rather than being limited to only those that fire early in S phase. For example, time course studies under conditions that allow for limited fork progression (by addition of hydroxyurea) and for the precocious activation of late-firing origins (by inhibition of S phase checkpoint kinases) might reveal the majority of all activated origins in a cell. This could significantly improve the resolution of current approaches that utilize the incorporation of bromodeoxyuridine into the DNA of replicating human cells (Jeon et al., 2005). In addition to presenting the details of their origin trapping assay, Mesner et al. (2006) also addressed another
Molecular Cell 736
important issue: the question of whether mammalian origins constitute large initiation zones composed of multiple initiation sites that are activated with low efficiency, similar to the DHFR origin. Indeed, all seven origins that were studied in more detail had striking similarities to the DHFR locus. However, it seems still premature to generalize these findings to all mammalian origins. After all, it remains unclear whether these examples are truly representative of all 1000 clones. Moreover, it cannot be excluded that certain features are specific to CHOC400 cells or to origins associated with the nuclear matrix (the DNA isolation procedure utilized in the study enriches for matrix associated sequences). It will be interesting to see whether other, more quantitative approaches will confirm the 2D gel replicon mapping results. One of the most exciting aspects about the development of the origin trapping assay is the premise of gaining access to a large number of origin sequences that can be analyzed with respect to their primary nucleotide composition, their chromosomal location, their ability to bind ORC in vivo, their proximity to promoters and known transcriptional regulators, and their degree of evolutionary conservation. The possibilities seem endless.
Anja-Katrin Bielinsky1 and Miruthubashini Raveendranathan1 1 Department of Biochemistry, Molecular Biology and Biophysics University of Minnesota Minneapolis, Minnesota 55455 Selected Reading Aladjem, M.I., and Fanning, E. (2004). EMBO Rep. 5, 686–691. Bell, S.P., and Stillman, B. (1992). Nature 357, 128–134. Brewer, B.J., and Fangman, W.L. (1987). Cell 51, 463–471. Dean, W.W., Dancis, B.M., and Thomas, C.A., Jr. (1973). Anal. Biochem. 56, 417–427. Jeon, Y., Bekiranov, S., Karnani, N., Kapranov, P., Ghosh, S., MacAlpine, D., Lee, C., Hwang, D.S., Gingeras, T.R., and Dutta, A. (2005). Proc. Natl. Acad. Sci. USA 102, 6419–6424. Mesner, L.D., Crawford, E.L., and Hamlin, J.L. (2006). Mol. Cell 21, 719–726. Palzkill, T.G., Oliver, S.G., and Newlon, C.S. (1986). Nucleic Acids Res. 14, 6247–6264. Struhl, K., Stinchcomb, D.T., Scherer, S., and Davis, R.W. (1979). Proc. Natl. Acad. Sci. USA 76, 1035–1039. Todorovic, V., Giadrossi, S., Pelizon, C., Mendoza-Maldonado, R., Masai, H., and Giacca, M. (2005). Mol. Cell 19, 567–575. Wada, A., and Kishizaki, A. (1968). Biochim. Biophys. Acta 166, 29–39.
Molecular Cell 21, March 17, 2006 ª2006 Elsevier Inc.
DOI 10.1016/j.molcel.2006.03.002
Encircled: Large-Scale Purification of Replication Origins from Mammalian Chromosomes
A novel cloning strategy for sequences comprising mammalian replication origins, described by Mesner et al. (2006) in a recent issue of Molecular Cell, utilizes an origin trapping assay in which replication bubbles are selectively retained in agarose due to their circular nature.
In eukaryotic cells, DNA replication initiates at replication origins, specific sites in the genome that are recognized by the origin recognition complex (ORC; [Bell and Stillman, 1992]). The ORC functions as the initiator of replication by recruiting other factors, which facilitate the assembly of replication forks that move away from the origin in a bidirectional manner. In simple organisms like budding yeast, replication origins were first identified as autonomously replicating sequences (ARSs) because they conferred the ability to replicate to plasmid DNAs that otherwise could not replicate (Struhl et al., 1979). Further analysis of these ARSs revealed that they contained an ARS consensus sequence (ACS; [Palzkill et al., 1986]), which is part of the recognition site bound by ORC. Whereas ORC is evolutionarily conserved, the ACS is not, and thus, the nature of replication origins in metazoan genomes has remained elusive and highly controversial (Aladjem and Fanning, 2004). A limited number of eukaryotic origins has been identified by a two-dimensional (2D) gel replicon mapping technique that took advantage of the fact that bubbles exhibited a highly specific migration pattern that separated them effectively from other DNA structures when they were fractionated on agarose gels (Brewer and Fangman, 1987). While the 2D gel replicon mapping technique was instrumental in identifying replication origins in many multicellular organisms, including mammals, it was not amenable as a systematic screening tool for the large-scale identification of origin sequences. The same was true for the ARS assay, as virtually any DNA sequence that was several kilobases in size exhibited ARS activity when introduced into tissue culture cells (discussed in Mesner et al. [2006]). The field has struggled with this issue for almost two decades—until now. The team led by Joyce Hamlin at the University of Virginia has developed a strategy that appears to turn this long-standing quest into a more realistic endeavor. In a paper published in a recent issue of Molecular Cell, the authors describe a protocol to selectively trap replication bubbles in agarose due to their circular nature (Mesner et al., 2006). The separation is based on a technique described in 1973 as ‘‘the trapping of circular DNA in agarose gels’’ (Dean et al., 1973), applying a principle that was first described as ‘‘agar fixation’’ (Wada and Kishizaki, 1968): when DNA, containing
both linear fragments and circles, is solidified in an agar solution, then linear DNA can diffuse while circular DNA is trapped. Subsequent electrophoresis allows linear fragments to enter a gel, whereas circles remain topologically linked within the agarose mesh, thus unable to move. Mesner et al. convincingly demonstrate that the implementation of this trapping procedure yields highly purified preparations of replication bubbles. To generate a replication origin library, the authors utilized the Chinese hamster ovary cell line CHOC400, which harbors w1000 copies of a well-characterized origin located at the dihydrofolate reductase (DHFR) locus, which is activated early in S phase and served as a control in the study. DNA was harvested from cells that were synchronously released into early S phase at a time point at which the DHFR origin exhibits peak activation. Moreover, arresting cells at the onset of S phase also ensured that replication bubbles did not become too large in size, preventing them from being cleaved during the subsequent restriction digestion. After the partial exclusion of double-stranded, nonreplicating DNA, the preparation was mixed with liquid agarose and circle-containing fragments were retained by the trapping procedure outlined above. After isolation of the trapped DNA, the authors demonstrated that the material contained almost exclusively replication bubbles. The trapped fragments were finally cloned into a suitable vector to generate an origin library of w1000 clones after the removal of repetitive sequences. From this library, 15 individual clones were stochastically chosen, and all of them turned out to be active replication origins. Therefore, this new strategy allows for the efficient purification of origin sequences, greatly improving previous approaches (Todorovic et al., 2005). As the authors point out, with this high degree of enrichment, the cloned material might be suitable for microarray hybridization to conduct whole genome studies. Although it seems unlikely that such an approach will be developed for hamster cells in the near future, as the hamster genome has not been sequenced, there is no obvious reason to suspect that the strategy described by Mesner et al. (2006) should not be applicable to any other cellular organisms, including humans. It is also easy to envision variations on the protocol developed by Hamlin and coworkers to allow for the identification of both early- and latefiring origins rather than being limited to only those that fire early in S phase. For example, time course studies under conditions that allow for limited fork progression (by addition of hydroxyurea) and for the precocious activation of late-firing origins (by inhibition of S phase checkpoint kinases) might reveal the majority of all activated origins in a cell. This could significantly improve the resolution of current approaches that utilize the incorporation of bromodeoxyuridine into the DNA of replicating human cells (Jeon et al., 2005). In addition to presenting the details of their origin trapping assay, Mesner et al. (2006) also addressed another
Molecular Cell 736
important issue: the question of whether mammalian origins constitute large initiation zones composed of multiple initiation sites that are activated with low efficiency, similar to the DHFR origin. Indeed, all seven origins that were studied in more detail had striking similarities to the DHFR locus. However, it seems still premature to generalize these findings to all mammalian origins. After all, it remains unclear whether these examples are truly representative of all 1000 clones. Moreover, it cannot be excluded that certain features are specific to CHOC400 cells or to origins associated with the nuclear matrix (the DNA isolation procedure utilized in the study enriches for matrix associated sequences). It will be interesting to see whether other, more quantitative approaches will confirm the 2D gel replicon mapping results. One of the most exciting aspects about the development of the origin trapping assay is the premise of gaining access to a large number of origin sequences that can be analyzed with respect to their primary nucleotide composition, their chromosomal location, their ability to bind ORC in vivo, their proximity to promoters and known transcriptional regulators, and their degree of evolutionary conservation. The possibilities seem endless.
Anja-Katrin Bielinsky1 and Miruthubashini Raveendranathan1 1 Department of Biochemistry, Molecular Biology and Biophysics University of Minnesota Minneapolis, Minnesota 55455 Selected Reading Aladjem, M.I., and Fanning, E. (2004). EMBO Rep. 5, 686–691. Bell, S.P., and Stillman, B. (1992). Nature 357, 128–134. Brewer, B.J., and Fangman, W.L. (1987). Cell 51, 463–471. Dean, W.W., Dancis, B.M., and Thomas, C.A., Jr. (1973). Anal. Biochem. 56, 417–427. Jeon, Y., Bekiranov, S., Karnani, N., Kapranov, P., Ghosh, S., MacAlpine, D., Lee, C., Hwang, D.S., Gingeras, T.R., and Dutta, A. (2005). Proc. Natl. Acad. Sci. USA 102, 6419–6424. Mesner, L.D., Crawford, E.L., and Hamlin, J.L. (2006). Mol. Cell 21, 719–726. Palzkill, T.G., Oliver, S.G., and Newlon, C.S. (1986). Nucleic Acids Res. 14, 6247–6264. Struhl, K., Stinchcomb, D.T., Scherer, S., and Davis, R.W. (1979). Proc. Natl. Acad. Sci. USA 76, 1035–1039. Todorovic, V., Giadrossi, S., Pelizon, C., Mendoza-Maldonado, R., Masai, H., and Giacca, M. (2005). Mol. Cell 19, 567–575. Wada, A., and Kishizaki, A. (1968). Biochim. Biophys. Acta 166, 29–39.