Vol. 43, No. 3
MICROBIOLOGICAL REVIEWS, Sept. 1979, p. 297-319
0146-0749/79/03-0297/23$02.00/0
Chromatin Replication Revealed by Studies of Animal Cells and Papovaviruses (Simian Virus 40 and Polyoma Virus) CHANTAL CRlMISIt Unite des Virus oncogenes, Dipartement de Biologie monculaire, Institut Pasteur, 75015 Paris, France
INTRODUCTION ................... .......................................... 297 RELATIONSHIP BETWEEN PROTEIN AND DEOXYRIBONUCLEIC ACID (DNA) OF CHROMATIN DURING REPLICATION ...... ............... 299 Synchronization Between DNA and Histone Syntheses ...................... 299 Histone Turnover ................... 299 Ratio of Protein to DNA During Replication .... ... 299 Cellular chromatin ....................................................... 299 Viral chromatin ..............................2.....................9.. .29 STRUCTURE OF REPLICATING CHROMATIN AT THE LEVEL OF THE REPLICATING FORK ..................................... ........ 300
Microseopic Approach ..............................
300 Biochemical Approach: Use of Micrococcal Nuclease and Pancreatic Deoxyribonuclease I ........... ........... ... ..... 301 Cellular and viral chromatin synthesized in vivo .... ......... 301 Conclusions .......................................... 303 Digestion of chromatin synthesized in isolated nuclei ..................... 304 HISTONE DISTRIBlTION DURING REPLICATION .......................... 305 Presentation of Models ..................................................... 305 Totally conservative, or cis, distribution ................................ 305 Truns distribution ......................... 305 Semiconservative di8tribution ... .................................. . ... 305 Random segregation .................. ......... ... ........ 305 Assembly of Histones Inside the Octamer ...... ... .......... 305 Segregation of Histones with Respect to DNA ....... 306 L lization of newly synthesized histones ............................... 306 (i) Studies on celular chromatin ........................................ 306 (ii) Studies on viral chromatin .......................................... 307 (ifl) Conclusions and comments ................... ........... 307 Distribution of histones according to DNA strand ...... 307 (i) Results ....... ...................... ...... ...... .... ..... 308 (ii) Conclusion ................................ 309 Segregation of histones with respect to the two daughter strands of DNA 309 (i) Effect of protein synthesis inhibitors on DNA replication ... 309 (ii) Studies on cellular chromatin: nuclease sensitivity .............. 309 (iii) Studies on cellular chromatin: electron microscopic studies ...... 309 (iv) Viral chromatin studies ..................... .......... ... .... 310 (v) Comments and conclusions ......................................... 312 ORIGIN OF REPLICATION AND POSITIONING OF NUCLEOSOMES .... .. 313 CONCLUSIONS ....................................................... 314 LITERATURE CITED ............................................ 316 INTRODUCTION In the eucaryotic cell, deoxyribonucleic acid (DNA) is never found naked, but it is complexed with proteins, histone and nonhistone, and small ribonucleic acid molecules, the total ensemble constituting chromatin. The primary structure of chromatin consists of a flexible chain punctuated with repetitive structural units (nucleosomes) of about 10 nm in diameter and containing, on average, 200 base pairs of DNA cont
Presnt address: Fred Hutchinson Cancer Research Cen-
ter, Seattle,
WA 98104.
densed around a protein core of eight histone molecules (two of each of the four principal types) (17, 39). This chain is condensed in a superstructure in which histone Hi plays an essential role (19, 105). This structure is not static, but is, on the contrary, very dynamic, changing so as to accommodate replicative and transcriptional processes. Although it is not yet possible to describe the replicative process in its entirety, we do understand certain fundamentals of the mechanism: histone synthesis is intimately coupled to that of DNA, and the synthesis of nonhistone proteins takes place for the 297
298
CREMISI
most part throughout the whole cell cycle (14, 15, 89). It has been demonstrated that DNA replication is bidirectional, starting from a given origin, and proceeds in a semiconservative mode. The 3'-*5' strand is preferentially replicated in a discontinuous fashion, whereas the 5'-*3' strand is preferentially replicated in a continuous fashion. It must, however, be stated that it has not yet been demonstrated that there is any specificity for initiation of Okazaki fragments, and the average size of such fragments in eucaryotic cells is 150 to 200 base pairs (14, 34, 61, 82; Fig. 1). In this review I attempt to compile the data conceming the coupling of all of these synthetic processes. Because studies concerning nonhistone proteins are far outnumbered by those con-
MICROBIOL. REV.
cerning histones, I shall limit myself to a study of the distribution of histones during chromatin replication and to the structure of the replicating chromatin. Although all cells of an organism possess the same genome, they nevertheless express different phenotypes, that is, different spectra of proteins. When one considers mechanisms by which a given phenotypic state may be transmitted to daughter cells during cell division and how this state can be stably maintained for many generations, one is led to consider the possibility that the structure of the chromatin may carry the information for these events. Actually, it is likely that a part of the information programing the control of the genetic activity is mediated by chromosomal proteins at the moment of chromosome division (1, 91). In fact, although there are only a few histone species and although they show considerable sequence conservation in BIDIRECTIONAL widely differing species, they are nevertheless among the most highly post-translationally modified proteins known (15). It is therefore not unreasonable, despite the lack of direct evidence, to think that these proteins could intervene at some point in the regulation of genetic expression, perhaps even by relaying signals to the nonhistone proteins. This has led us to consider the existence of a strict mechanism for the transmission of phenotypic information from the mother cells to the daughter cells during replication. Clearly, a complete understanding of this mechanism requires an understanding of the structural organization of chromatin, and it is in this framework that the studies presented below have been oriented. Very often references will be made to studies with the papovaviruses polyoma virus and simian virus 40 (SV40). These viruses multiply in the nuclei of certain eucaryotic cells in close relationship with cellular chromatin (80). The viral DNA, like the cellular DNA, is associated with cellular histones in a nucleosome structure comparable to that of the cellular chromatin (3, 11, 25). Infection by these viruses stimulates simultaneously cellular DNA synthesis, histone synthesis, and those functions TERMINUS FIG. 1. Physical map ofpolyoma virus DNA show- required for DNA replication (45, 104). Whating strand polarities for DNA synthesis. The HpaII ever the stage of infection, the synthesis of hisfragments are ordered on polyoma DNA. The strand tones is always coupled to that of DNA, be it polarities are derived from the polarity determined cellular or viral (48). With the exception of T for the transcription of late mRNA. By convention, antigen, a specific viral protein involved in the the strand from which late mRNA is transcribed is initiation of viral DNA replication (45, 87), the designated L. In DNA replication, the growing forks virus utilizes the cellular machinery for its mulmove bidirectionally away from the unique origin of tiplication. Its replication follows the same replication. The continuous arrowed lines indicate mechanism as that for cellular DNA; it is bidithe progeny strands which, in theory, can grow continuously; the broken arrowed lines indicate the rectional, starting at a given origin, and proceeds strand which has to grow discontinuously. (Repro- by a semiconservative mechanism (14,34,38,61, duced from reference 34 with pernission from the 82). There is a quantitative difference in size beM.I.T. Press, Cambridge, Mass.)
VOL. 43, 1979
tween viral genomes and replication units of the cell. The former are of the order of 3 kilobases, whereas the latter can be as large as 300 kilobases (30). However, no qualitative functional difference has been found between the two genome types. Therefore, the papovaviruses are often considered a model system for the study of chromatin and DNA replication (14, 82).
CHROMATIN REPLICATION
299
Ratio of Protein to DNA During Replication Cellular chromatin. Several laboratories have measured the density of chromatin undergoing replication (16, 47, 80). The newly synthesized DNA made during a 30-s interval is associated with a deoxyribonucleoprotein complex which has a buoyant density in cesium chloride after treatment with formaldehyde which is approximately 1% lower than that of RELATIONSHIP BETWEEN PROTEIN the nucleoprotein complex containing mature AND DEOXYRIBONUCLEIC ACID DNA (16). The replicative DNA is therefore (DNA) OF CHROMATIN DURING associated with additional proteins required for REPLICATION replication. A similar conclusion has been drawn on the basis of studies on chromatin extracted Synchronization Between DNA and sea urchin embryo and centrifuged to equifrom Histone Syntheses librium in metrizamide, thereby obviating the It is now very well established that there is need to fix the material before centrifugation very tight coupling between the replication of (47). DNA and the synthesis of histones (15, 65, 68). Viral chromatin. The replicating viral chroSuch coupling is manifested in both directions: matins of polyoma virus and SV40 are easily inhibition of DNA synthesis affects the synthesis separated by sucrose gradient sedimentation of histones, and, similarly, inhibition of the syn- from the mature chromatins (11, 24, 50, 80). It is thesis of histones results in a diminution of cel- therefore possible to compare the density of the lular DNA synthesis. Histone messenger ribo- mature chromatin with that of every stage of nucleic acids appear to be synthesized through- the replicating chromatin, from molecules early out the cell cycle (54), but it is only during S in replication to those late in replication but phase that they are processed and transported whose daughter molecules have not yet sepavery rapidly into the cytoplasm, that is, within rated. All of the studies agree and demonstrate several minutes after their synthesis, a relatively that whatever the type of equilibrium centrifushort time in comparison with other messenger gation used, be it metfizamide (11, 50) or cesium ribonucleic acids (71). chloride (24, 80), and whatever the stage of During S phase, at least 20,000 molecules of replication (80), the ratio of protein to DNA of histones are synthesized per min per cell, and these viral chromatin molecules is conserved they are transported very rapidly (within 10 s) throughout DNA replication and is of the order to the nucleus (95), which therefore finds itself of 1:1 (11). Analyses of proteins of the mature constantly fed with newly made histones during and replicative complexes, on polyacrylamide S phase. There appears to be no significant gels containing sodium dodecyl sulfate and urea, extranuclear pool of free histones (estimated at reveal a large similarity between their protein 0.2% of the total [60]). Such intimate coupling contents (9). The histones are notably associated between DNA replication and histone synthesis with the two types of DNA, the replicating form has suggested to certain authors (95) that his- (circular) and form I (supercoiled). Not only tones could play a role in the process of elonga- does the replicating DNA not lose its parental tion during replication. histones, but it remains constantly associated with a new lot of histones, depending upon its Histone Turnover degree of replication. This fact is in accord with The turnover of histones and of nonhistone earlier work which demonstrated that the newly proteins in the course of the cell cycle has been synthesized DNA is immediately protected from studied in HeLa celLs (27, 73). It has been dem- attack by deoxyribonuclease (DNase) 1 (96). The onstrated unambiguously that there is no signif- discrepancy between the studies on cellular icant irreversible dissociation of histones from chromatin and those on viral chromatin conthe chromatin during several division cycles in cerning the ratio of protein to DNA (there being vitro. These data are in accord with the results a slight increase in the first case and no change of a previous study made in vivo in which no in the second) probably arises from the differturnover of histone was observed (5). Having ences in labeling times used for the two cases. demonstrated the absence of turnover, it is The viral DNA was marked for 5 to 10 min, therefore necessary to know if, during the repli- which is too long to detect such a change in cation, the histones dissociate temporarily from density of newly synthesized chromatin. This the DNA and if the ratio of protein to DNA in change is very transient, and it was detected the chromatin is changed during this stage. with a half-life of 2 min in HeLa cells (16).
300
CRRMISI
STRUCTURE OF REPLICATING CHROMATIN AT THE LEVEL OF THE REPLICATION FORK In the above paragraphs, it has been shown that the histones remain associated with DNA during replication, but this result has no implications for nucleosomal structure. This structure may be modified or completely lost during replication. This problem will be dealt with this section. Microscopic Approach Several groups have analyzed the structure of replicating chromatin with the electron microscope, in particular, the viral chromatin of SV40 (9, 81) and the embryonic chromatin of Drosophila melanogaster (53). Globular particles resembling nucleosomes are visible on both arms
MICROBIOL. REV.
of the replicating fork (Fig. 2). Their size and their periodicity are similar to those of the nonreplicating chromatin. With the chromatin of SV40, it is possible to see that the contraction of the replicating DNA is essentially the same as that for the DNA which has not yet been replicated (9). In addition, it appears from polyacrylamide gel analysis of the protein content of replicating minichromosomes of SV40 that the four histones H3, H4, H2a, and H2b are present. Immunomicroscopic examination of the chromatin of the embryo of Drosophila, using an antibody to histones H3 and H2b, has demonstrated the presence of these two histones on the two daughter chromatids (53). If one refers to the observations provided by electron microscopy, all of these data suggest that the beadlike particles present on the two arms of the repli-
FIG. 2. High-magnification photographs of D. melanogaster replicon configurations. (Note the beadlike appearance of newly replicated and prereplicative chromatid regions.) The micrographs were taken from samples prepared from cellular blastoderm embryos exhibiting between 2 and 6 tum of inward membrane synthesis. The arrow in (b) depicts the smaller fragment (PMB-9) of HindIIT-digested Xlr-101 plasmid DNA. The PMB-9 fragments contain -5.6 kilobases, as determined by polyacrylamide gel electrophoresis, and measure slightly more than 2.0 pm, thus equaling the expected B-form length. Bars = 1.0 pm. (Reproduced from reference 53 with permission from the M.I.T. Press.)
VOL. 43, 1979
CHROMATIN REPLICATION
301
sized material, examined after brief labeling (90 s), was twice as sensitive to nuclease as was control chromatin. The size distribution of the newly synthesized DNA fragments released by micrococcal nuclease was comparable to that of the DNA fragments obtained from mature chromatin. The authors concluded that in the course of DNA replication the histones are always distributed to the daughter chromosomes in their oligomeric form, in such a fashion that the newly synthesized DNA carries only one-half of the protein content of the mature chromatin. Hence, the period of 10 to 20 min required for maturation of the resistance to nucleases is interpreted as the time necessary to complete one replicon Biochemical Approach: Use of Micrococcal by the addition of chromosomal proteins (74). Nuclease and Pancreatic These conclusions are all based on the hypothDeoxyribonuclease I esis of a decrease in protein content in replicatCellular and viral chromatin synthesized ing chromatin. However, two points should be noted. (i) An in vivo. Nucleases, such as micrococcal nuclease and pancreatic DNase I, have been used exten- increase in the sensitivity of the replicating chrosively in the last few years for the study of the matin to nucleases is not sufficient to permit the structure of chromatin (17, 39). The digestion of conclusion that there is a decline in protein chromatin by micrococcal nuclease, which at- content. (ii) Also, if one supposes that the protacks internucleosomal DNA, reveals a repeti- tein content is reduced by one-half, 50% of the tive pattern of approximately 200 base pairs of DNA should become naked, and in this case one DNA which appears to be relatively unaltered should then obtain biphasic curves in the course during transcription of the chromatin (2, 22, 99). of digestion with the nucleases. In such a situaDigestion by DNase I, which attacks the inter- tion, the ratio of protein to DNA in the replicatnucleosomal DNA as well as the nucleosomal ing chromatin would be very greatly reduced, DNA, reveals a characteristic repetitive pattern that is to say, on gradients of cesium chloride, of approximately 10 base pairs which, in contrast replicating chromatin should have a density to the preceding digestion, appears to be selec- higher than that of the control. However, this has never been observed. The ratio of protein to tive for actively transcribed genes (99). These nucleases have also been used to study DNA was found to be conserved throughout newly synthesized chromatin. Differential sen- replication (11, 24, 80) or to be slightly higher sitivity of mature and replicating chromatin to (16, 47, 79). These approaches have been further extended pancreatic DNase I was first examined in nuclei of chick erythroblasts (96), where it seemed that by other studies (75) on Chinese hamster ovary the newly synthesized chromatin was more re- cells (33), chick erythrocytes (31), the minichrosistant to DNase I. It was suggested that part of mosome of SV40 (E. Fanning, K. H. Klempthis resistance was due to elements required for nauer, B. Otto, and I. Baumgartner, Tumor Vireplication, notably, the histones. However, this rus Meeting, Cold Spring Harbor, N.Y., 1978; T. result has not been confirmed by other authors H. Herman, M. L. De Pamphilis, and P. M. Wassermann, Tumor Virus Meeting, Cold using the same experimental approach. A similar approach has been applied to HeLa Spring Harbor, N.Y., 1978), and sea urchin emcells (74), using DNase I and the micrococcal bryos (46). Pulse-labeled chromatin is much nuclease. The cells were labeled with tritiated more sensitive to micrococcal nuclease than is thymidine for periods of 30 s to 32 min, and the the mature chromatin (Fig. 3; Table 1), and we nuclease sensitivity of the labeled material was know that this effect becomes more significant examined. The shorter the labeling time (30 s to as the incubation period for labeling is 1 min), the more sensitive was the labeled ma- shortened. The initial rate of digestion with miterial to attack by DNase I. However, the in- crococcal nuclease is several times higher for creased sensitivity was of brief duration; after 10 nascent DNA than for mature DNA (Fig. 3). to 20 min, the newly synthesized chromatin was This difference in rate of digestion decreases and no more sensitive than was bulk chromatin. The disappears when the incubation periods are results are qualitatively the same as those ob- longer (Fig. 3). These facts are in accord with tained with micrococcal nuclease; newly synthe- those already cited (74, 75, 79). These studies cating fork are nucleosomes indistinguishable from the nucleosomes present on the nonreplicating chromatin. In addition, these results suggest that the histones remain associated with the DNA throughout replication; if they do undergo dissociation, they must reassociate rapidly (see Histone Distribution During Replication). It must be noted that conclusions drawn from these studies are not unequivocal, in the sense that one cannot exclude a slight modification of nucleosome structure during replication. This has been shown by the work of numerous laboratories on the study of the sensitivity of replicating chromatin to different nucleases.
CREMISI
302
MICROBIOL. REV.
,A)f 3H - ---- --
60
An examination of the sizes of DNA synthesized during a pulse of 30 s in Chinese hamster ovary cells reveals that 50% of the DNA sediments at 4S (a size similar to that of the eucaryotic Okazaki fragments [33]). These results suggest that the newly replicated DNA is rapidly assembled into the repetitive subunits of chromatin even before the Okazaki fragments mature into intermediates of larger molecular weight (33). In addition, it has been verified that the nascent DNA fragments, both monomeric and multiy .. which meric, _ ...... are resistant to micrococcal nuclease do not have single-stranded regions. To demonstrate this, DNA derived from digestion with micrococcal nucleases was denatured by heat and fractionated on polyacrylamide-urea gels or on regular polyacrylamide gels after having been treated in both cases with nuclease Si, specific for single-strand DNA (46).
.
MICROBIOLOGICAL REVIEWS, Sept. 1979, p. 297-319
0146-0749/79/03-0297/23$02.00/0
Chromatin Replication Revealed by Studies of Animal Cells and Papovaviruses (Simian Virus 40 and Polyoma Virus) CHANTAL CRlMISIt Unite des Virus oncogenes, Dipartement de Biologie monculaire, Institut Pasteur, 75015 Paris, France
INTRODUCTION ................... .......................................... 297 RELATIONSHIP BETWEEN PROTEIN AND DEOXYRIBONUCLEIC ACID (DNA) OF CHROMATIN DURING REPLICATION ...... ............... 299 Synchronization Between DNA and Histone Syntheses ...................... 299 Histone Turnover ................... 299 Ratio of Protein to DNA During Replication .... ... 299 Cellular chromatin ....................................................... 299 Viral chromatin ..............................2.....................9.. .29 STRUCTURE OF REPLICATING CHROMATIN AT THE LEVEL OF THE REPLICATING FORK ..................................... ........ 300
Microseopic Approach ..............................
300 Biochemical Approach: Use of Micrococcal Nuclease and Pancreatic Deoxyribonuclease I ........... ........... ... ..... 301 Cellular and viral chromatin synthesized in vivo .... ......... 301 Conclusions .......................................... 303 Digestion of chromatin synthesized in isolated nuclei ..................... 304 HISTONE DISTRIBlTION DURING REPLICATION .......................... 305 Presentation of Models ..................................................... 305 Totally conservative, or cis, distribution ................................ 305 Truns distribution ......................... 305 Semiconservative di8tribution ... .................................. . ... 305 Random segregation .................. ......... ... ........ 305 Assembly of Histones Inside the Octamer ...... ... .......... 305 Segregation of Histones with Respect to DNA ....... 306 L lization of newly synthesized histones ............................... 306 (i) Studies on celular chromatin ........................................ 306 (ii) Studies on viral chromatin .......................................... 307 (ifl) Conclusions and comments ................... ........... 307 Distribution of histones according to DNA strand ...... 307 (i) Results ....... ...................... ...... ...... .... ..... 308 (ii) Conclusion ................................ 309 Segregation of histones with respect to the two daughter strands of DNA 309 (i) Effect of protein synthesis inhibitors on DNA replication ... 309 (ii) Studies on cellular chromatin: nuclease sensitivity .............. 309 (iii) Studies on cellular chromatin: electron microscopic studies ...... 309 (iv) Viral chromatin studies ..................... .......... ... .... 310 (v) Comments and conclusions ......................................... 312 ORIGIN OF REPLICATION AND POSITIONING OF NUCLEOSOMES .... .. 313 CONCLUSIONS ....................................................... 314 LITERATURE CITED ............................................ 316 INTRODUCTION In the eucaryotic cell, deoxyribonucleic acid (DNA) is never found naked, but it is complexed with proteins, histone and nonhistone, and small ribonucleic acid molecules, the total ensemble constituting chromatin. The primary structure of chromatin consists of a flexible chain punctuated with repetitive structural units (nucleosomes) of about 10 nm in diameter and containing, on average, 200 base pairs of DNA cont
Presnt address: Fred Hutchinson Cancer Research Cen-
ter, Seattle,
WA 98104.
densed around a protein core of eight histone molecules (two of each of the four principal types) (17, 39). This chain is condensed in a superstructure in which histone Hi plays an essential role (19, 105). This structure is not static, but is, on the contrary, very dynamic, changing so as to accommodate replicative and transcriptional processes. Although it is not yet possible to describe the replicative process in its entirety, we do understand certain fundamentals of the mechanism: histone synthesis is intimately coupled to that of DNA, and the synthesis of nonhistone proteins takes place for the 297
298
CREMISI
most part throughout the whole cell cycle (14, 15, 89). It has been demonstrated that DNA replication is bidirectional, starting from a given origin, and proceeds in a semiconservative mode. The 3'-*5' strand is preferentially replicated in a discontinuous fashion, whereas the 5'-*3' strand is preferentially replicated in a continuous fashion. It must, however, be stated that it has not yet been demonstrated that there is any specificity for initiation of Okazaki fragments, and the average size of such fragments in eucaryotic cells is 150 to 200 base pairs (14, 34, 61, 82; Fig. 1). In this review I attempt to compile the data conceming the coupling of all of these synthetic processes. Because studies concerning nonhistone proteins are far outnumbered by those con-
MICROBIOL. REV.
cerning histones, I shall limit myself to a study of the distribution of histones during chromatin replication and to the structure of the replicating chromatin. Although all cells of an organism possess the same genome, they nevertheless express different phenotypes, that is, different spectra of proteins. When one considers mechanisms by which a given phenotypic state may be transmitted to daughter cells during cell division and how this state can be stably maintained for many generations, one is led to consider the possibility that the structure of the chromatin may carry the information for these events. Actually, it is likely that a part of the information programing the control of the genetic activity is mediated by chromosomal proteins at the moment of chromosome division (1, 91). In fact, although there are only a few histone species and although they show considerable sequence conservation in BIDIRECTIONAL widely differing species, they are nevertheless among the most highly post-translationally modified proteins known (15). It is therefore not unreasonable, despite the lack of direct evidence, to think that these proteins could intervene at some point in the regulation of genetic expression, perhaps even by relaying signals to the nonhistone proteins. This has led us to consider the existence of a strict mechanism for the transmission of phenotypic information from the mother cells to the daughter cells during replication. Clearly, a complete understanding of this mechanism requires an understanding of the structural organization of chromatin, and it is in this framework that the studies presented below have been oriented. Very often references will be made to studies with the papovaviruses polyoma virus and simian virus 40 (SV40). These viruses multiply in the nuclei of certain eucaryotic cells in close relationship with cellular chromatin (80). The viral DNA, like the cellular DNA, is associated with cellular histones in a nucleosome structure comparable to that of the cellular chromatin (3, 11, 25). Infection by these viruses stimulates simultaneously cellular DNA synthesis, histone synthesis, and those functions TERMINUS FIG. 1. Physical map ofpolyoma virus DNA show- required for DNA replication (45, 104). Whating strand polarities for DNA synthesis. The HpaII ever the stage of infection, the synthesis of hisfragments are ordered on polyoma DNA. The strand tones is always coupled to that of DNA, be it polarities are derived from the polarity determined cellular or viral (48). With the exception of T for the transcription of late mRNA. By convention, antigen, a specific viral protein involved in the the strand from which late mRNA is transcribed is initiation of viral DNA replication (45, 87), the designated L. In DNA replication, the growing forks virus utilizes the cellular machinery for its mulmove bidirectionally away from the unique origin of tiplication. Its replication follows the same replication. The continuous arrowed lines indicate mechanism as that for cellular DNA; it is bidithe progeny strands which, in theory, can grow continuously; the broken arrowed lines indicate the rectional, starting at a given origin, and proceeds strand which has to grow discontinuously. (Repro- by a semiconservative mechanism (14,34,38,61, duced from reference 34 with pernission from the 82). There is a quantitative difference in size beM.I.T. Press, Cambridge, Mass.)
VOL. 43, 1979
tween viral genomes and replication units of the cell. The former are of the order of 3 kilobases, whereas the latter can be as large as 300 kilobases (30). However, no qualitative functional difference has been found between the two genome types. Therefore, the papovaviruses are often considered a model system for the study of chromatin and DNA replication (14, 82).
CHROMATIN REPLICATION
299
Ratio of Protein to DNA During Replication Cellular chromatin. Several laboratories have measured the density of chromatin undergoing replication (16, 47, 80). The newly synthesized DNA made during a 30-s interval is associated with a deoxyribonucleoprotein complex which has a buoyant density in cesium chloride after treatment with formaldehyde which is approximately 1% lower than that of RELATIONSHIP BETWEEN PROTEIN the nucleoprotein complex containing mature AND DEOXYRIBONUCLEIC ACID DNA (16). The replicative DNA is therefore (DNA) OF CHROMATIN DURING associated with additional proteins required for REPLICATION replication. A similar conclusion has been drawn on the basis of studies on chromatin extracted Synchronization Between DNA and sea urchin embryo and centrifuged to equifrom Histone Syntheses librium in metrizamide, thereby obviating the It is now very well established that there is need to fix the material before centrifugation very tight coupling between the replication of (47). DNA and the synthesis of histones (15, 65, 68). Viral chromatin. The replicating viral chroSuch coupling is manifested in both directions: matins of polyoma virus and SV40 are easily inhibition of DNA synthesis affects the synthesis separated by sucrose gradient sedimentation of histones, and, similarly, inhibition of the syn- from the mature chromatins (11, 24, 50, 80). It is thesis of histones results in a diminution of cel- therefore possible to compare the density of the lular DNA synthesis. Histone messenger ribo- mature chromatin with that of every stage of nucleic acids appear to be synthesized through- the replicating chromatin, from molecules early out the cell cycle (54), but it is only during S in replication to those late in replication but phase that they are processed and transported whose daughter molecules have not yet sepavery rapidly into the cytoplasm, that is, within rated. All of the studies agree and demonstrate several minutes after their synthesis, a relatively that whatever the type of equilibrium centrifushort time in comparison with other messenger gation used, be it metfizamide (11, 50) or cesium ribonucleic acids (71). chloride (24, 80), and whatever the stage of During S phase, at least 20,000 molecules of replication (80), the ratio of protein to DNA of histones are synthesized per min per cell, and these viral chromatin molecules is conserved they are transported very rapidly (within 10 s) throughout DNA replication and is of the order to the nucleus (95), which therefore finds itself of 1:1 (11). Analyses of proteins of the mature constantly fed with newly made histones during and replicative complexes, on polyacrylamide S phase. There appears to be no significant gels containing sodium dodecyl sulfate and urea, extranuclear pool of free histones (estimated at reveal a large similarity between their protein 0.2% of the total [60]). Such intimate coupling contents (9). The histones are notably associated between DNA replication and histone synthesis with the two types of DNA, the replicating form has suggested to certain authors (95) that his- (circular) and form I (supercoiled). Not only tones could play a role in the process of elonga- does the replicating DNA not lose its parental tion during replication. histones, but it remains constantly associated with a new lot of histones, depending upon its Histone Turnover degree of replication. This fact is in accord with The turnover of histones and of nonhistone earlier work which demonstrated that the newly proteins in the course of the cell cycle has been synthesized DNA is immediately protected from studied in HeLa celLs (27, 73). It has been dem- attack by deoxyribonuclease (DNase) 1 (96). The onstrated unambiguously that there is no signif- discrepancy between the studies on cellular icant irreversible dissociation of histones from chromatin and those on viral chromatin conthe chromatin during several division cycles in cerning the ratio of protein to DNA (there being vitro. These data are in accord with the results a slight increase in the first case and no change of a previous study made in vivo in which no in the second) probably arises from the differturnover of histone was observed (5). Having ences in labeling times used for the two cases. demonstrated the absence of turnover, it is The viral DNA was marked for 5 to 10 min, therefore necessary to know if, during the repli- which is too long to detect such a change in cation, the histones dissociate temporarily from density of newly synthesized chromatin. This the DNA and if the ratio of protein to DNA in change is very transient, and it was detected the chromatin is changed during this stage. with a half-life of 2 min in HeLa cells (16).
300
CRRMISI
STRUCTURE OF REPLICATING CHROMATIN AT THE LEVEL OF THE REPLICATION FORK In the above paragraphs, it has been shown that the histones remain associated with DNA during replication, but this result has no implications for nucleosomal structure. This structure may be modified or completely lost during replication. This problem will be dealt with this section. Microscopic Approach Several groups have analyzed the structure of replicating chromatin with the electron microscope, in particular, the viral chromatin of SV40 (9, 81) and the embryonic chromatin of Drosophila melanogaster (53). Globular particles resembling nucleosomes are visible on both arms
MICROBIOL. REV.
of the replicating fork (Fig. 2). Their size and their periodicity are similar to those of the nonreplicating chromatin. With the chromatin of SV40, it is possible to see that the contraction of the replicating DNA is essentially the same as that for the DNA which has not yet been replicated (9). In addition, it appears from polyacrylamide gel analysis of the protein content of replicating minichromosomes of SV40 that the four histones H3, H4, H2a, and H2b are present. Immunomicroscopic examination of the chromatin of the embryo of Drosophila, using an antibody to histones H3 and H2b, has demonstrated the presence of these two histones on the two daughter chromatids (53). If one refers to the observations provided by electron microscopy, all of these data suggest that the beadlike particles present on the two arms of the repli-
FIG. 2. High-magnification photographs of D. melanogaster replicon configurations. (Note the beadlike appearance of newly replicated and prereplicative chromatid regions.) The micrographs were taken from samples prepared from cellular blastoderm embryos exhibiting between 2 and 6 tum of inward membrane synthesis. The arrow in (b) depicts the smaller fragment (PMB-9) of HindIIT-digested Xlr-101 plasmid DNA. The PMB-9 fragments contain -5.6 kilobases, as determined by polyacrylamide gel electrophoresis, and measure slightly more than 2.0 pm, thus equaling the expected B-form length. Bars = 1.0 pm. (Reproduced from reference 53 with permission from the M.I.T. Press.)
VOL. 43, 1979
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sized material, examined after brief labeling (90 s), was twice as sensitive to nuclease as was control chromatin. The size distribution of the newly synthesized DNA fragments released by micrococcal nuclease was comparable to that of the DNA fragments obtained from mature chromatin. The authors concluded that in the course of DNA replication the histones are always distributed to the daughter chromosomes in their oligomeric form, in such a fashion that the newly synthesized DNA carries only one-half of the protein content of the mature chromatin. Hence, the period of 10 to 20 min required for maturation of the resistance to nucleases is interpreted as the time necessary to complete one replicon Biochemical Approach: Use of Micrococcal by the addition of chromosomal proteins (74). Nuclease and Pancreatic These conclusions are all based on the hypothDeoxyribonuclease I esis of a decrease in protein content in replicatCellular and viral chromatin synthesized ing chromatin. However, two points should be noted. (i) An in vivo. Nucleases, such as micrococcal nuclease and pancreatic DNase I, have been used exten- increase in the sensitivity of the replicating chrosively in the last few years for the study of the matin to nucleases is not sufficient to permit the structure of chromatin (17, 39). The digestion of conclusion that there is a decline in protein chromatin by micrococcal nuclease, which at- content. (ii) Also, if one supposes that the protacks internucleosomal DNA, reveals a repeti- tein content is reduced by one-half, 50% of the tive pattern of approximately 200 base pairs of DNA should become naked, and in this case one DNA which appears to be relatively unaltered should then obtain biphasic curves in the course during transcription of the chromatin (2, 22, 99). of digestion with the nucleases. In such a situaDigestion by DNase I, which attacks the inter- tion, the ratio of protein to DNA in the replicatnucleosomal DNA as well as the nucleosomal ing chromatin would be very greatly reduced, DNA, reveals a characteristic repetitive pattern that is to say, on gradients of cesium chloride, of approximately 10 base pairs which, in contrast replicating chromatin should have a density to the preceding digestion, appears to be selec- higher than that of the control. However, this has never been observed. The ratio of protein to tive for actively transcribed genes (99). These nucleases have also been used to study DNA was found to be conserved throughout newly synthesized chromatin. Differential sen- replication (11, 24, 80) or to be slightly higher sitivity of mature and replicating chromatin to (16, 47, 79). These approaches have been further extended pancreatic DNase I was first examined in nuclei of chick erythroblasts (96), where it seemed that by other studies (75) on Chinese hamster ovary the newly synthesized chromatin was more re- cells (33), chick erythrocytes (31), the minichrosistant to DNase I. It was suggested that part of mosome of SV40 (E. Fanning, K. H. Klempthis resistance was due to elements required for nauer, B. Otto, and I. Baumgartner, Tumor Vireplication, notably, the histones. However, this rus Meeting, Cold Spring Harbor, N.Y., 1978; T. result has not been confirmed by other authors H. Herman, M. L. De Pamphilis, and P. M. Wassermann, Tumor Virus Meeting, Cold using the same experimental approach. A similar approach has been applied to HeLa Spring Harbor, N.Y., 1978), and sea urchin emcells (74), using DNase I and the micrococcal bryos (46). Pulse-labeled chromatin is much nuclease. The cells were labeled with tritiated more sensitive to micrococcal nuclease than is thymidine for periods of 30 s to 32 min, and the the mature chromatin (Fig. 3; Table 1), and we nuclease sensitivity of the labeled material was know that this effect becomes more significant examined. The shorter the labeling time (30 s to as the incubation period for labeling is 1 min), the more sensitive was the labeled ma- shortened. The initial rate of digestion with miterial to attack by DNase I. However, the in- crococcal nuclease is several times higher for creased sensitivity was of brief duration; after 10 nascent DNA than for mature DNA (Fig. 3). to 20 min, the newly synthesized chromatin was This difference in rate of digestion decreases and no more sensitive than was bulk chromatin. The disappears when the incubation periods are results are qualitatively the same as those ob- longer (Fig. 3). These facts are in accord with tained with micrococcal nuclease; newly synthe- those already cited (74, 75, 79). These studies cating fork are nucleosomes indistinguishable from the nucleosomes present on the nonreplicating chromatin. In addition, these results suggest that the histones remain associated with the DNA throughout replication; if they do undergo dissociation, they must reassociate rapidly (see Histone Distribution During Replication). It must be noted that conclusions drawn from these studies are not unequivocal, in the sense that one cannot exclude a slight modification of nucleosome structure during replication. This has been shown by the work of numerous laboratories on the study of the sensitivity of replicating chromatin to different nucleases.
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An examination of the sizes of DNA synthesized during a pulse of 30 s in Chinese hamster ovary cells reveals that 50% of the DNA sediments at 4S (a size similar to that of the eucaryotic Okazaki fragments [33]). These results suggest that the newly replicated DNA is rapidly assembled into the repetitive subunits of chromatin even before the Okazaki fragments mature into intermediates of larger molecular weight (33). In addition, it has been verified that the nascent DNA fragments, both monomeric and multiy .. which meric, _ ...... are resistant to micrococcal nuclease do not have single-stranded regions. To demonstrate this, DNA derived from digestion with micrococcal nucleases was denatured by heat and fractionated on polyacrylamide-urea gels or on regular polyacrylamide gels after having been treated in both cases with nuclease Si, specific for single-strand DNA (46).
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