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Response to Letter from Renata Maas
DNA Repair 9 (2010) 1024–1025
Contents lists available at ScienceDirect
DNA Repair journal homepage: www.elsevier.com/locate/dnarepair
Letter to the Editor
Response to Letter from Renata Maas Dear Editor, DNA replication has been thought to be semi-conservative for many years, a certainty if there ever was one. Now, in a restriction digest meant to confirm that the Escherichia coli host of a plasmid lacks dcm methyltransferase, Maas notes that only one half of the plasmid is fully digested by a methylation-sensitive restriction enzyme. These same dcm-methylation motifs are methylated de novo as part of the activation-of-replication reaction by dammethyltransferase, and de novo motifs remain unmethylated in progeny strands until the next round of replication. Because the original strands can be distinguished from newly synthesized strands by their methylation, these data suggest DNA is replicated conservatively. Moreover, Maas points out that the conformation of activated de novo methylated DNA assumes a conformation that equilibrates in cesium gradients as a distinct band above the non-activated B-DNA band. Such issues may have confounded the Meselson-Stahl experiments, which originally demonstrated semi-conservative DNA replication using N15 labeling and cesiumdensity shift experiments to distinguish between original and newly replicated strands. These are important observations, which are of interest to all biologists. Further studies should to be done to reinvestigate DNA replication. In particular, it will be necessary to extend these studies in other bacteria and eukaryotes to see if the replication of DNA is conservative per se, or if this is a bizarre feature of E. coli. It would be of interest to apply different approaches to strengthen these results, for instance, the use of tritiated thymidine or electron microscopy might also provide confirmatory evidence. The conservation of DNA replication is of particular interest in stem cell biology, chiefly because the inheritance of dissimilar DNA strands could influence cell fate and function. The nonrandom segregation of chromatids was first proposed following studies that used pulses of thymidine analogs to label the DNA of dividing cells. A wide range of eukaryotes were shown to retain label in constitutively dividing cells [1–3], and eventually an explanation of these results, the Immortal Strand Hypothesis, was conceived by Cairns [4]. As DNA replication was agreed to be semi-conservative, the retention of DNA label was attributed to only half of each chromosome. Cairns proposed that stem cells retained one copy of each original strand, from each chromosome, during asymmetric divisions. Label would thus be shed in two divisions if stem cells were dividing only asymmetrically, or retained in asymmetrically dividing stem cells if they selected labeled chromosomes as “Immortal” strands during a preceding symmetric division. Cairns hypothesized that the retention of older DNA templates occurred in order to lower the mutation load of stem cells which divide asymmetrically throughout the lifetime of the animal. More recent work has shown the asymmetric assortment of specific DNA strands in S. poombe 1568-7864/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.dnarep.2010.07.016
[5] and embryonic stem cells [6] during periods of differentiation. These cases do not involve the retention of all original DNA strands, rather a subset of the strands present, and are thought to occur in order to specify a different cell fate to the daughter cells following division. While the mechanisms of these findings remain elusive, new results in intestinal stem cells [7,8] continue to raise interest in these controversial issues. Is fully conservative DNA replication consistent with these findings? Following a thymidine analog pulse, a positive signal in a cell nucleus would be a result of a cell containing some fully labeled chromosomes and some completely unlabeled chromosomes. If a non-stem cell always segregated chromatids randomly, both its uptake of label and its subsequent diminishment would occur similarly as in semi-conservative replication. If a stem cell segregated some chromatids non-randomly, during consecutive divisions, the positive signal in its nucleus would deviate from the diminishment of label predicted – because all chromatids would be segregated randomly except the one(s) selected for retention. However, if a stem cell retained all of its original DNA templates (a stronger version of the Immortal Strand Hypothesis) then such a cell would remain label-negative, even in the presence of analog, unless it was caught at some point during S-Phase. Thus far such a case has not been observed – seemingly all dividing cells are labeled during these pulse periods, hence supporting a model where stem cell retain only a subset of their chromosomes rather than one copy of each original strand. This would seem to cast some doubt on the Immortal Strand Hypothesis, unless stem cells retaining all of its original templates are rare and have been overlooked. The live imaging of stem cells in a thymidine analog would be an excellent way to answer this question. The conservative replication of DNA has important implications for understanding stem cell division asymmetry. If the original strands are epigenetically marked, conservative replication would result in one cell inheriting epigenetic signatures while its sister might not inherit such marks at all. If these notions hold true, they may provide important insights into the maintenance of the stem cell state and the concomitant differentiation of stem cell progeny. Maas’ provocative hypothesis warrants a reexamination of DNA replication to see if our most fundamental ideas are in need of revision. References [1] K.G. Lark, R.A. Consigli, H.C. Minocha, Segregation of sister chromatids in mammalian cells, Science 154 (1966) 1202–1205. [2] R.F. Rosenberger, M. Kessel, Nonrandom sister chromatid segregation and nuclear migration in hyphae of Aspergillus nidulans, J. Bacteriol. 96 (1968) 1208–1213. [3] S.P. Tomasovic, M.C. Mix, Cell renewal in the gill of the freshwater mussel Margaritifera margaritifera: an autoradiographic study using high specific activity tritiated thymidine, J. Cell Sci. 14 (1974) 561–569. [4] J. Cairns, Mutation selection and the natural history of cancer, Nature 255 (1975) 197–200.
Letter to the Editor / DNA Repair 9 (2010) 1024–1025 [5] J.Z. Dalgaard, A.J. Klar, Does S. pombe exploit the intrinsic asymmetry of DNA synthesis to imprint daughter cells for mating-type switching? Trends Genet. 17 (2001) 153–157. [6] A. Armakolas, A.J. Klar, Cell type regulates selective segregation of mouse chromosome 7 DNA strands in mitosis, Science 311 (2006) 1146–1149. [7] E. Falconer, E.A. Chavez, A. Henderson, S.S. Poon, S. McKinney, L. Brown, D.G. Huntsman, P.M. Lansdorp, Identification of sister chromatids by DNA template strand sequences, Nature 463 (2010) 93–97. [8] A.J. Quyn, P.L. Appleton, F.A. Carey, R.J. Steele, N. Barker, H. Clevers, R.A. Ridgway, O.J. Sansom, I.S. Nathke, Spindle orientation bias in gut epithelial stem cell compartments is lost in precancerous tissue, Cell Stem Cell 6 (2010) 175–181.
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Phillip Karpowicz Department of Genetics, Harvard Medical School, 77 Avenue Louis Pasteur, NRB 339, Boston, MA 02115, USA E-mail address: [email protected]
Contents lists available at ScienceDirect
DNA Repair journal homepage: www.elsevier.com/locate/dnarepair
Letter to the Editor
Response to Letter from Renata Maas Dear Editor, DNA replication has been thought to be semi-conservative for many years, a certainty if there ever was one. Now, in a restriction digest meant to confirm that the Escherichia coli host of a plasmid lacks dcm methyltransferase, Maas notes that only one half of the plasmid is fully digested by a methylation-sensitive restriction enzyme. These same dcm-methylation motifs are methylated de novo as part of the activation-of-replication reaction by dammethyltransferase, and de novo motifs remain unmethylated in progeny strands until the next round of replication. Because the original strands can be distinguished from newly synthesized strands by their methylation, these data suggest DNA is replicated conservatively. Moreover, Maas points out that the conformation of activated de novo methylated DNA assumes a conformation that equilibrates in cesium gradients as a distinct band above the non-activated B-DNA band. Such issues may have confounded the Meselson-Stahl experiments, which originally demonstrated semi-conservative DNA replication using N15 labeling and cesiumdensity shift experiments to distinguish between original and newly replicated strands. These are important observations, which are of interest to all biologists. Further studies should to be done to reinvestigate DNA replication. In particular, it will be necessary to extend these studies in other bacteria and eukaryotes to see if the replication of DNA is conservative per se, or if this is a bizarre feature of E. coli. It would be of interest to apply different approaches to strengthen these results, for instance, the use of tritiated thymidine or electron microscopy might also provide confirmatory evidence. The conservation of DNA replication is of particular interest in stem cell biology, chiefly because the inheritance of dissimilar DNA strands could influence cell fate and function. The nonrandom segregation of chromatids was first proposed following studies that used pulses of thymidine analogs to label the DNA of dividing cells. A wide range of eukaryotes were shown to retain label in constitutively dividing cells [1–3], and eventually an explanation of these results, the Immortal Strand Hypothesis, was conceived by Cairns [4]. As DNA replication was agreed to be semi-conservative, the retention of DNA label was attributed to only half of each chromosome. Cairns proposed that stem cells retained one copy of each original strand, from each chromosome, during asymmetric divisions. Label would thus be shed in two divisions if stem cells were dividing only asymmetrically, or retained in asymmetrically dividing stem cells if they selected labeled chromosomes as “Immortal” strands during a preceding symmetric division. Cairns hypothesized that the retention of older DNA templates occurred in order to lower the mutation load of stem cells which divide asymmetrically throughout the lifetime of the animal. More recent work has shown the asymmetric assortment of specific DNA strands in S. poombe 1568-7864/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.dnarep.2010.07.016
[5] and embryonic stem cells [6] during periods of differentiation. These cases do not involve the retention of all original DNA strands, rather a subset of the strands present, and are thought to occur in order to specify a different cell fate to the daughter cells following division. While the mechanisms of these findings remain elusive, new results in intestinal stem cells [7,8] continue to raise interest in these controversial issues. Is fully conservative DNA replication consistent with these findings? Following a thymidine analog pulse, a positive signal in a cell nucleus would be a result of a cell containing some fully labeled chromosomes and some completely unlabeled chromosomes. If a non-stem cell always segregated chromatids randomly, both its uptake of label and its subsequent diminishment would occur similarly as in semi-conservative replication. If a stem cell segregated some chromatids non-randomly, during consecutive divisions, the positive signal in its nucleus would deviate from the diminishment of label predicted – because all chromatids would be segregated randomly except the one(s) selected for retention. However, if a stem cell retained all of its original DNA templates (a stronger version of the Immortal Strand Hypothesis) then such a cell would remain label-negative, even in the presence of analog, unless it was caught at some point during S-Phase. Thus far such a case has not been observed – seemingly all dividing cells are labeled during these pulse periods, hence supporting a model where stem cell retain only a subset of their chromosomes rather than one copy of each original strand. This would seem to cast some doubt on the Immortal Strand Hypothesis, unless stem cells retaining all of its original templates are rare and have been overlooked. The live imaging of stem cells in a thymidine analog would be an excellent way to answer this question. The conservative replication of DNA has important implications for understanding stem cell division asymmetry. If the original strands are epigenetically marked, conservative replication would result in one cell inheriting epigenetic signatures while its sister might not inherit such marks at all. If these notions hold true, they may provide important insights into the maintenance of the stem cell state and the concomitant differentiation of stem cell progeny. Maas’ provocative hypothesis warrants a reexamination of DNA replication to see if our most fundamental ideas are in need of revision. References [1] K.G. Lark, R.A. Consigli, H.C. Minocha, Segregation of sister chromatids in mammalian cells, Science 154 (1966) 1202–1205. [2] R.F. Rosenberger, M. Kessel, Nonrandom sister chromatid segregation and nuclear migration in hyphae of Aspergillus nidulans, J. Bacteriol. 96 (1968) 1208–1213. [3] S.P. Tomasovic, M.C. Mix, Cell renewal in the gill of the freshwater mussel Margaritifera margaritifera: an autoradiographic study using high specific activity tritiated thymidine, J. Cell Sci. 14 (1974) 561–569. [4] J. Cairns, Mutation selection and the natural history of cancer, Nature 255 (1975) 197–200.
Letter to the Editor / DNA Repair 9 (2010) 1024–1025 [5] J.Z. Dalgaard, A.J. Klar, Does S. pombe exploit the intrinsic asymmetry of DNA synthesis to imprint daughter cells for mating-type switching? Trends Genet. 17 (2001) 153–157. [6] A. Armakolas, A.J. Klar, Cell type regulates selective segregation of mouse chromosome 7 DNA strands in mitosis, Science 311 (2006) 1146–1149. [7] E. Falconer, E.A. Chavez, A. Henderson, S.S. Poon, S. McKinney, L. Brown, D.G. Huntsman, P.M. Lansdorp, Identification of sister chromatids by DNA template strand sequences, Nature 463 (2010) 93–97. [8] A.J. Quyn, P.L. Appleton, F.A. Carey, R.J. Steele, N. Barker, H. Clevers, R.A. Ridgway, O.J. Sansom, I.S. Nathke, Spindle orientation bias in gut epithelial stem cell compartments is lost in precancerous tissue, Cell Stem Cell 6 (2010) 175–181.
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Phillip Karpowicz Department of Genetics, Harvard Medical School, 77 Avenue Louis Pasteur, NRB 339, Boston, MA 02115, USA E-mail address: [email protected]