A
The cell nucleus has a fundamental problem: a large amount of DNA has to be packed into an extremely small volume. Not only must the DNA be grossly compacted but it must remain readily accessible for the processes of transcription and replication. This is overcome by initially wrapping the DNA around histone cores to form nucleosomes, which are then arranged in higher order structures with the help of another histone, H1 (for review see ref. 1).Many years ago it was shown that the histones and DNA contain all the information required to assemble a correct nucleosome core by mixing the components (i.e. histones H2A, H2B, H3, and H4 with the DNA) at high ionic strengths and slowly removing the salt by Although the structure of the nucleosome particle, in terms of its histone content, appears to be well accepted (for review see ref. 5 ) , the fate of the nucleosomes during and after replication is the subject of much debate. When chromatin replicates, not only must the DNA be duplicated, but also the number of nucleosomes must be doubled to ensure that both daughter strands are capable of re-packing into a chromosome structure. The production of new histones is tightly coupled to the synthesis of DNA so that new nucleosomes can be made and assembled onto the DNA at the time of replication. Three models of nucleosome segregation can be envisaged (Fig. 1).The first (A), shows a completely random pattern, while (B) and (C) show two types of conservative arrangement, either between leading and lagging strand (B), or based on parental strand polarity (C) at the replication start site. Current opinion is that little mixing of the actual histones occurs between old and new nucleosomes‘, i.e. the histone octomers remain intact, but the distribution of the nucleosomes themselves is controversial (for example see refs 7-12). This controversy may in part be due to the variety of systems used, but nevertheless even basic points are not agreed. A further point of confusion is whether the nucleosomes completely dissociate from the DNA as a consequence of replication fork movement or whether they remain in contact throughout, and again evidence is available in the literature to support both views. The findin that nucleosomes will bind to single-stranded DNA‘l4 ! ) supports the non-dissociation argument. However, crosslinking experiments by Sogo et a1.(”) show that a nucleosome-free area exists directly behind the repli-
Fig. 1. Schematic representation of nucleosome segregation. A replication ‘bubble’ is depicted with old nucleosomes (open circles) on the unreplicated DNA (either end) and a mixture of old and new (filled circles) nucleosomes on the replicated DNA. A random mechanism is shown in A, and two forms of conservative segregation in B and C. B involves segregation according to the strand synthesis (leading or lagging) at the replication fork, C segregation depending o n the strand polarity at the replication start site. Reproduced from Dilworth and Dingwall (1988). Chromatin assembly in vitro and in vivo. BioEssays 9 , 44-49.
cation fork, leading them to propose that nucleosomes are transiently released from the DNA as the fork passes and these then reassociate with the newly replicated DNA. Both of these points have been addressed in a recent report from Bruce Alberts’ group published in Nature@). This work neatly circumvents many of the previously encountered problems because they base their studies on an in v i m replication system composed entirely of purified components. Previous work has shown that seven bacteriophage TCencoded proteins will catalyse coupled leading and lagging strand DNA synthesis on a double stranded, circular template which contains an M13 origin of replication. This can also be used to replicate a template with associated nucleosomes (made by the addition of purified histone octamers) by the inclusion of another T4-derived protein, the DNA-dependent ATPase, dda, although polymerase pausing occurs (see later). The mechanism of DNA replication in this system is via rolling circles (see Fig. 2), which has several distinct advantages. Firstly, the amount of replication that has occurred on each molecule can be determined by electron microscopy (E.M.), from the length of the tail produced. Secondly, the tail is formed entirely by lagging strand synthesis so, although a circular template is used, it is easy to determine how the nucleosomes are partitioning between the parental (circle) and daughter (tail) strands. The pattern of segregation of nucleosomes between these strands was addressed by treating with the DNA cross-linking agent psorolen after repli-
/----
3’
Fig. 2. Schematic representation of template replication by rolling circles.
cation, and studying the resultant spreads by E.M. Nucleosome positions were indicated by protection of the DNA from the cross-linker. Under the denaturing conditions used for E.M. preparation, the nucleosomeprotected DNA melts, and so appeared as singlestranded bubbles. Mapping the positions of these bubbles on a number of replicated molecules has led to certain conclusions. The first observation was that the molecules which had replicated most (i.e. had the longest tails) also had the fewest nucleosomes associated with the circle. From this they conclude that there is a more or less random distribution of nucleosomes to the two strands. Closer examination of the data also shows that the number of nucleosomes on each molecule does not fall as replication proceeds, thus indicating that the nucleosomes are inherited intact from the parent template (and also strengthens the nondissociation argument at the same time). By comparison with various theoretical partition coefficients they also calculate that nucleosomes segregate to the lagging strand at a ratio of 1in every 4 passes of the replication fork, i.e. there is a preference for nucleosomes to pass to the leading strand in this system. However, the choice is not completely determinate as a nucleosome can first partition to the leading strand (i.e. stay with the circle and therefore be subject to another passage of the fork) and then the same nucleosome can partition to the lagging strand (i.e. transfer to the tail) on the subsequent round of replication. The authors also note that at no time did they observe an accumuIation of nucleosomes near the fork, indicating that nucleosome sliding (caused by pushing at the fork) was not occurring. The question of whether or not nucleosomes are transiently displaced from the DNA was addressed in several ways. In theory, if all the nucleosomes are completely removed from the template by the passing of a fork, then nucleosome-caused-pausing of the polymerase, should only occur during the first round of replication, as subsequent rounds should be nucleosome free. However, pausing was found at later time points as well, thus indicating that the replicated DNA still had nucleosomes attached. To discount the possibility that nucleosomes were indeed displaced but then quickly rebound to the replicated DNA, replication was carried out in the presence of an excess of DNA that
lacked nucleosomes. This DNA could not be replicated and could only bind nucleosomes that were free in solution. By using 3H-tabeled histones and distinguishing the replicated DNA from non-replicated by digestion of the latter with Dpnl, they showed that the histones did not associate with the competitor DNA, thus indicating that at no time are the histone octamers released. Because the system is defined in its protein content, they were also able to show that the association of nucleosomes with the DNA is inherent to the nucleosome itself rather than due to ‘tethering’ proteins needed to ensure binding of the nucleosome to the DNA. However, as the distribution of nucleosomes is random in this system, they do not address the question of whether ‘positioned’ nucleosomes may require further protein involvement, The in vitro system described is not perfect - the authors point out that the number of nucleosomes is approx. five-fold lower than would be found in chromatin and that the system is independent of H1, thus giving a random arrangement of nucleosomes along the template. However, this paper gives good evidence to support both the non-detachment of the histone cores during fork movement and also a non-deterministic, albeit leading strand-biased, segregation of nucleosomes between the two resulting DNA helices. As with all fields that generate a large amount of conflicting data, the resulting picture could end up as a mosaic with each system having its own particular pattern of nucleosome segregation. Although this subject has in the past been dogged by artifacts due to E.M. sample preparation and the use of non-physiological ionic strengths, I would not be at all surprised if multiple mechanisms are indeed shown to exist. It is becoming increasingly apparent that chromatin structure plays a large part in the determination of gene expression by controlling the access of regulatory factors to the DNA. Therefore the transcriptional state of the DNA under study could be critical in the interpretation of the results obtained in the various systems with maybe several different mechanisms occurring within the same cell (for review see ref. 14). It is this that makes the purified system so attractive, as it enables one to study the inherent functions of nucleosomes. It is possible however, that in the more complex world of mature chromatin in viwo, other factors are involved in nucleosome segregation.
References 1 GASSER, S . M. AND LAEMMLI, U. K . (1987). A glimpse at chromosomal order. Trends Gene!. 3, 16-22. 2 AXEL,R., MELCHIOR, W.,STOLLNER-WEBB, B. AND FELSENFELD, G. (1974). Specific sites of interaction between histones and DNA in chromatin. Proc. Nad Acad. Sci. U S A 71, 4101-1105. 3 OUDET,P., GROSS-BELLARD, M. A N D CHAMBON, P. (1975). Electron microscope and biochemical evidence that chromatin structure is a repeating unit. Cell 4, 281-300. 4 CAMERINI-OTERO, R., SOLLNER-WEBB. 8. A N D FELSENFELO, G.(1976). The organization of histones and DNA in chromatin: Evidence for an arginine-rich histone kernel. Cell 8, 333-347.
5 VON HOLT,C. (1985). Histones in perspective. BioEssays 3 , 120-124. 6 BONNE-ANDREA, C., WONG,M. L. AND ALBERTS, B. (1990). In v i m replication through nucleosomes without histone displacement. Nature 343, 719-726. 7 RILEY, D . AND WEINTRAUBE, H . (1979). Conservative segregation of parental histones during replication in the presence of cycloheximide. Proc. Natl Acad. Sci. USA 76, 37.8-332. 8 SEIDMAN, M . , LEVENE, A. AND WEINTRAUBE, H. (1979). The asymmetric segregation of parental nucleosomes during chromosomal replication. Cell 18, 439-449. 9 JACKSON, V. AND CHALKEY, R. (1981). A reevaluation of new histone deposition on replicating chromatin. J . B i d . Chem. 265, 5095-5103. 10 LEFFAK. I. f 1984). Conservative segregation of nucleosome core histones. - Nature 307. 82-85. ’ 11 CUSICK, M. E., DEPAMPHILLIS, M. L. AND WASSARMAN, P. M. (1984).
Dispersive segregation of nucleosomes during replication of SV40 chromosomes. J . Mol. Biol. 178, 249-259. 12 SOGO,J. M., STAHL, H., KOLLER, T.AND KNIPPERS, R. (1986). Structure of replicating SV40 minichromosomes. The replication fork. core histone segregation and terminal structures. J . Molec. B i d . 189, 189-204. 13 PALTER, K., FOE,V . AND ALBERTS, B. (1979). Evidence for the formation of nucleosome-like histone conplexes on single stranded DNA. Cell 18, 451-467. 14 DILWORTH, S. M. AND DINGWALL, C. (1988). Chromatin assembly in virro and in vivo. BioEssays 9, 44-49.
M. P. Fairman is at the CRC Molecular Embryology Group, Department of Zoology, Downing St., Cambridge CB2 3EJ, UK.
The cell nucleus has a fundamental problem: a large amount of DNA has to be packed into an extremely small volume. Not only must the DNA be grossly compacted but it must remain readily accessible for the processes of transcription and replication. This is overcome by initially wrapping the DNA around histone cores to form nucleosomes, which are then arranged in higher order structures with the help of another histone, H1 (for review see ref. 1).Many years ago it was shown that the histones and DNA contain all the information required to assemble a correct nucleosome core by mixing the components (i.e. histones H2A, H2B, H3, and H4 with the DNA) at high ionic strengths and slowly removing the salt by Although the structure of the nucleosome particle, in terms of its histone content, appears to be well accepted (for review see ref. 5 ) , the fate of the nucleosomes during and after replication is the subject of much debate. When chromatin replicates, not only must the DNA be duplicated, but also the number of nucleosomes must be doubled to ensure that both daughter strands are capable of re-packing into a chromosome structure. The production of new histones is tightly coupled to the synthesis of DNA so that new nucleosomes can be made and assembled onto the DNA at the time of replication. Three models of nucleosome segregation can be envisaged (Fig. 1).The first (A), shows a completely random pattern, while (B) and (C) show two types of conservative arrangement, either between leading and lagging strand (B), or based on parental strand polarity (C) at the replication start site. Current opinion is that little mixing of the actual histones occurs between old and new nucleosomes‘, i.e. the histone octomers remain intact, but the distribution of the nucleosomes themselves is controversial (for example see refs 7-12). This controversy may in part be due to the variety of systems used, but nevertheless even basic points are not agreed. A further point of confusion is whether the nucleosomes completely dissociate from the DNA as a consequence of replication fork movement or whether they remain in contact throughout, and again evidence is available in the literature to support both views. The findin that nucleosomes will bind to single-stranded DNA‘l4 ! ) supports the non-dissociation argument. However, crosslinking experiments by Sogo et a1.(”) show that a nucleosome-free area exists directly behind the repli-
Fig. 1. Schematic representation of nucleosome segregation. A replication ‘bubble’ is depicted with old nucleosomes (open circles) on the unreplicated DNA (either end) and a mixture of old and new (filled circles) nucleosomes on the replicated DNA. A random mechanism is shown in A, and two forms of conservative segregation in B and C. B involves segregation according to the strand synthesis (leading or lagging) at the replication fork, C segregation depending o n the strand polarity at the replication start site. Reproduced from Dilworth and Dingwall (1988). Chromatin assembly in vitro and in vivo. BioEssays 9 , 44-49.
cation fork, leading them to propose that nucleosomes are transiently released from the DNA as the fork passes and these then reassociate with the newly replicated DNA. Both of these points have been addressed in a recent report from Bruce Alberts’ group published in Nature@). This work neatly circumvents many of the previously encountered problems because they base their studies on an in v i m replication system composed entirely of purified components. Previous work has shown that seven bacteriophage TCencoded proteins will catalyse coupled leading and lagging strand DNA synthesis on a double stranded, circular template which contains an M13 origin of replication. This can also be used to replicate a template with associated nucleosomes (made by the addition of purified histone octamers) by the inclusion of another T4-derived protein, the DNA-dependent ATPase, dda, although polymerase pausing occurs (see later). The mechanism of DNA replication in this system is via rolling circles (see Fig. 2), which has several distinct advantages. Firstly, the amount of replication that has occurred on each molecule can be determined by electron microscopy (E.M.), from the length of the tail produced. Secondly, the tail is formed entirely by lagging strand synthesis so, although a circular template is used, it is easy to determine how the nucleosomes are partitioning between the parental (circle) and daughter (tail) strands. The pattern of segregation of nucleosomes between these strands was addressed by treating with the DNA cross-linking agent psorolen after repli-
/----
3’
Fig. 2. Schematic representation of template replication by rolling circles.
cation, and studying the resultant spreads by E.M. Nucleosome positions were indicated by protection of the DNA from the cross-linker. Under the denaturing conditions used for E.M. preparation, the nucleosomeprotected DNA melts, and so appeared as singlestranded bubbles. Mapping the positions of these bubbles on a number of replicated molecules has led to certain conclusions. The first observation was that the molecules which had replicated most (i.e. had the longest tails) also had the fewest nucleosomes associated with the circle. From this they conclude that there is a more or less random distribution of nucleosomes to the two strands. Closer examination of the data also shows that the number of nucleosomes on each molecule does not fall as replication proceeds, thus indicating that the nucleosomes are inherited intact from the parent template (and also strengthens the nondissociation argument at the same time). By comparison with various theoretical partition coefficients they also calculate that nucleosomes segregate to the lagging strand at a ratio of 1in every 4 passes of the replication fork, i.e. there is a preference for nucleosomes to pass to the leading strand in this system. However, the choice is not completely determinate as a nucleosome can first partition to the leading strand (i.e. stay with the circle and therefore be subject to another passage of the fork) and then the same nucleosome can partition to the lagging strand (i.e. transfer to the tail) on the subsequent round of replication. The authors also note that at no time did they observe an accumuIation of nucleosomes near the fork, indicating that nucleosome sliding (caused by pushing at the fork) was not occurring. The question of whether or not nucleosomes are transiently displaced from the DNA was addressed in several ways. In theory, if all the nucleosomes are completely removed from the template by the passing of a fork, then nucleosome-caused-pausing of the polymerase, should only occur during the first round of replication, as subsequent rounds should be nucleosome free. However, pausing was found at later time points as well, thus indicating that the replicated DNA still had nucleosomes attached. To discount the possibility that nucleosomes were indeed displaced but then quickly rebound to the replicated DNA, replication was carried out in the presence of an excess of DNA that
lacked nucleosomes. This DNA could not be replicated and could only bind nucleosomes that were free in solution. By using 3H-tabeled histones and distinguishing the replicated DNA from non-replicated by digestion of the latter with Dpnl, they showed that the histones did not associate with the competitor DNA, thus indicating that at no time are the histone octamers released. Because the system is defined in its protein content, they were also able to show that the association of nucleosomes with the DNA is inherent to the nucleosome itself rather than due to ‘tethering’ proteins needed to ensure binding of the nucleosome to the DNA. However, as the distribution of nucleosomes is random in this system, they do not address the question of whether ‘positioned’ nucleosomes may require further protein involvement, The in vitro system described is not perfect - the authors point out that the number of nucleosomes is approx. five-fold lower than would be found in chromatin and that the system is independent of H1, thus giving a random arrangement of nucleosomes along the template. However, this paper gives good evidence to support both the non-detachment of the histone cores during fork movement and also a non-deterministic, albeit leading strand-biased, segregation of nucleosomes between the two resulting DNA helices. As with all fields that generate a large amount of conflicting data, the resulting picture could end up as a mosaic with each system having its own particular pattern of nucleosome segregation. Although this subject has in the past been dogged by artifacts due to E.M. sample preparation and the use of non-physiological ionic strengths, I would not be at all surprised if multiple mechanisms are indeed shown to exist. It is becoming increasingly apparent that chromatin structure plays a large part in the determination of gene expression by controlling the access of regulatory factors to the DNA. Therefore the transcriptional state of the DNA under study could be critical in the interpretation of the results obtained in the various systems with maybe several different mechanisms occurring within the same cell (for review see ref. 14). It is this that makes the purified system so attractive, as it enables one to study the inherent functions of nucleosomes. It is possible however, that in the more complex world of mature chromatin in viwo, other factors are involved in nucleosome segregation.
References 1 GASSER, S . M. AND LAEMMLI, U. K . (1987). A glimpse at chromosomal order. Trends Gene!. 3, 16-22. 2 AXEL,R., MELCHIOR, W.,STOLLNER-WEBB, B. AND FELSENFELD, G. (1974). Specific sites of interaction between histones and DNA in chromatin. Proc. Nad Acad. Sci. U S A 71, 4101-1105. 3 OUDET,P., GROSS-BELLARD, M. A N D CHAMBON, P. (1975). Electron microscope and biochemical evidence that chromatin structure is a repeating unit. Cell 4, 281-300. 4 CAMERINI-OTERO, R., SOLLNER-WEBB. 8. A N D FELSENFELO, G.(1976). The organization of histones and DNA in chromatin: Evidence for an arginine-rich histone kernel. Cell 8, 333-347.
5 VON HOLT,C. (1985). Histones in perspective. BioEssays 3 , 120-124. 6 BONNE-ANDREA, C., WONG,M. L. AND ALBERTS, B. (1990). In v i m replication through nucleosomes without histone displacement. Nature 343, 719-726. 7 RILEY, D . AND WEINTRAUBE, H . (1979). Conservative segregation of parental histones during replication in the presence of cycloheximide. Proc. Natl Acad. Sci. USA 76, 37.8-332. 8 SEIDMAN, M . , LEVENE, A. AND WEINTRAUBE, H. (1979). The asymmetric segregation of parental nucleosomes during chromosomal replication. Cell 18, 439-449. 9 JACKSON, V. AND CHALKEY, R. (1981). A reevaluation of new histone deposition on replicating chromatin. J . B i d . Chem. 265, 5095-5103. 10 LEFFAK. I. f 1984). Conservative segregation of nucleosome core histones. - Nature 307. 82-85. ’ 11 CUSICK, M. E., DEPAMPHILLIS, M. L. AND WASSARMAN, P. M. (1984).
Dispersive segregation of nucleosomes during replication of SV40 chromosomes. J . Mol. Biol. 178, 249-259. 12 SOGO,J. M., STAHL, H., KOLLER, T.AND KNIPPERS, R. (1986). Structure of replicating SV40 minichromosomes. The replication fork. core histone segregation and terminal structures. J . Molec. B i d . 189, 189-204. 13 PALTER, K., FOE,V . AND ALBERTS, B. (1979). Evidence for the formation of nucleosome-like histone conplexes on single stranded DNA. Cell 18, 451-467. 14 DILWORTH, S. M. AND DINGWALL, C. (1988). Chromatin assembly in virro and in vivo. BioEssays 9, 44-49.
M. P. Fairman is at the CRC Molecular Embryology Group, Department of Zoology, Downing St., Cambridge CB2 3EJ, UK.
