From the University of Konstanz, Division of Biology,
Universitätsstr.10, 78457 Konstanz, Federal Republic of Germany,
and the § Laboratory of Molecular Carcinogenesis, NCI,
National Institutes of Health, Bethesda, Maryland 20892
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ABSTRACT |
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The effect of chromosomal protein HMG-17 on the replication of a chromatin template was studied with minichromosomes containing the SV40 origin of replication. The minichromosomes were assembled from M13 DNA in Xenopus egg extracts in either the absence or presence of HMG-17. Structural data show that HMG-17 was efficiently incorporated into the chromatin and induced an extended chromatin structure. Using an in vitro SV40 replication system, we find that minichromosomes containing HMG-17 replicate with higher efficiency than minichromosomes deficient of HMG-17. The replicational potential of chromatin was enhanced only when HMG-17 was incorporated into the template during, but not after, chromatin assembly. HMG-17 stimulated replication only from a chromatin template, but not from protein-free DNA. Thus, HMG-17 protein enhances the rate of replication of a chromatin template by unfolding the higher order chromatin structure and increasing the accessibility of target sequences to components of the replication machinery.
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INTRODUCTION |
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The eukaryotic genome is packaged by histone and non-histone proteins into a highly condensed complex termed chromatin (reviewed in Refs. 1 and 2). The packaging of eukaryotic DNA in nucleosomes presents an obstacle to the processes of transcription (reviewed in Refs. 3-8) and replication (reviewed in Ref. 9). As a consequence, efficient transcription and replication require mechanisms that relieve the repressive activity of the chromatin structure. Recently, several ATP-dependent nucleosome remodelling factors, which seem to be required for the initiation of transcription from nucleosomally organized promotor sequences have been identified (10-15). Furthermore, it is well documented that post-translational acetylation of histone amino termini cores is associated with an increased rate of transcriptional (reviewed in Refs. 16-19) and replicational (20) activity. One of the features commonly associated with transcriptionally active chromatin is an increase in the content of non-histone chromosomal proteins (1, 21, 22).
The high mobility group (HMG)1 chromosomal proteins are among the most abundant and ubiquitous non-histone proteins found in nuclei of all higher eukaryotes (reviewed in Refs. 23 and 24). The HMG-14/-17 subgroup is the only known class of nuclear proteins which bind specifically to the nucleosomal core particle, each nucleosome contains two binding sites for either chromosomal protein HMG-14 or HMG-17 (25). In the cell nucleus, the amount of these proteins is smaller than the number of nucleosomes. Consequently, HMG-14 and HMG-17 are associated with only a subset of the nucleosomes.
A variety of experiments have shown that HMG-14/-17 are preferentially associated with chromatin subunits containing transcribed genes. The role of chromosomal proteins HMG-14 and HMG-17 in the generation of transcriptionally active chromatin was studied in Xenopus egg extracts with a chromatin template carrying the 5 S rRNA gene (26, 27). The experiments showed that HMG-14/-17 enhance the transcriptional potential of RNA polymerase III genes by increasing the turnover of transcriptionally active templates. The ability of HMG proteins to stimulate transcription from chromatin templates was further demonstrated with SV40 minichromosomes, isolated from CV-1 cells overexpressing HMG-14. These experiments revealed that elevated levels of HMG-14 stimulate the rate of transcriptional elongation by RNA polymerase II but not the level of transcriptional initiation (28). In addition, Paranjape et al. (29) demonstrated that HMG-17, in conjunction with the sequence-specific activator GAL4-VP16, stimulates the initiation of transcription.
Although the mechanism whereby HMG-14/-17 stimulate transcription from chromatin templates is not fully understood, recent experiments suggest that the proteins unfold the higher order chromatin structure (27, 30). Thus, HMG-containing minichromosomes sediment slower in sucrose gradients and are digested faster by various nucleases than minichromosomes lacking HMG-14/-17 (27). In addition, neutron scattering experiments on the binding of HMG-14/-17 to salt-washed chromatin suggest that the proteins decrease the mass per unit length of the chromatin fiber (30). Thus, changes in higher order structure seem to be the main mechanism whereby HMG-14/-17 enhance the transcriptional potential of chromatin. A reduction in compactness may facilitate access of various components of the transcriptional machinery to their target.
Changes in chromatin structure also influence the processes of chromatin replication (9). A stimulation of replication has thus been shown to be accompanied by a relaxation of the underlying chromatin structure (31, 32). So far, little is known about the influence of HMG proteins on the replication efficiency of chromatin templates. To this end, we have investigated the replication of minichromosomes assembled in the absence or presence of HMG-17 proteins. We found that minichromosomes assembled in the presence of HMG-17 have a more extended chromatin structure than minichromosomes devoid of HMG-17 and that these minichromosomes replicate with significantly higher efficiency compared with minichromosomes deficient of HMG proteins.
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EXPERIMENTAL PROCEDURES |
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Xenopus Egg Extracts-- Xenopus laevis unfertilized egg extract was prepared as described previously (33, 34).
Chromatin Assembly-- The HindIII/SphI fragment from pSVori (35) was cloned into double-stranded M13 DNA. Single-stranded M13 DNA, containing the SV40 origin, was assembled into chromatin during complementary DNA strand synthesis for 2 h at 22 °C under previously described conditions (36, 37) in the absence of exogenously added ATP and MgCl2. Recombinant human HMG-17 proteins (38) were added at the beginning of the chromatin assembly reaction in a molar ratio of HMG:nucleosome core of 70:1 or to reconstituted chromatin in a ratio of 1:1, 2:1, 5:1, and 10:1. Assembled chromatin was purified on 10-30% sucrose gradients (20 mM Hepes-KOH, pH 7.5, 1 mM EDTA, pH 8, 0.25% Triton X-100) containing either 70 or 400 mM KCl and centrifuged in an SW 40 rotor for 2.5 h at 40,000 rpm at 4 °C (26). Chromatin-containing fractions were identified by agarose gel electrophoresis, pooled, and concentrated by centrifugation over a 30% sucrose cushion (SW 55 rotor, 16 h, 31,000 rpm, 4 °C). Chromatin was resuspended in hypotonic buffer (20 mM Hepes-KOH, pH 7.8, 5 mM KCl, 0.5 mM MgCl2, 0.1 mM dithiothreitol).
Sedimentation Behavior of Assembled Chromatin--
1 µg of
single-stranded M13ori DNA was assembled into chromatin in the presence
of 5 µCi [-32P]dATP for 2 h at 22 °C in the
absence or presence of HMG protein and purified on sucrose gradients.
Chromatin-containing fractions were identified by trichloroacetic
acid precipitation of the individual fractions and agarose gel
electrophoresis, followed by autoradiography.
Supercoiling Assay-- An aliquot of the assembly reaction was removed after the indicated time points and stopped with stop solution (20 mM EDTA, pH 8, 0.25% SDS). Samples were deproteinized by proteinase K treatment and phenol extraction, loaded on a 0.8% agarose gel, and visualized by autoradiography. For topoisomer analysis, samples were separated on 0.8% agarose gels containing 20 µM chloroquine.
In Vitro Replication of Assembled Chromatin--
SV40 T-Ag was
prepared from insect cells (Sf9) infected with a recombinant
baculovirus (39) by immunoaffinity chromatography (40). Cytosolic HeLa
S100 extracts were prepared exactly as described (41). In standard
experiments, 300 ng of chromatin, assembled in the absence or presence
of HMG proteins, were incubated with 1 µg of T-Ag and 250 µg of
cytosolic extract proteins in a 50-µl reaction for 2 h at
37 °C exactly as described (42). For determination of the
incorporated nucleotides, of the replication assay was
precipitated with 10% trichloroacetic acid. For restriction analysis,
the replication products were purified as described (42) and digested
for 1 h at 37 °C with 5 units each of the restriction enzymes
ClaI, AvaII, and MscI in the
recommended buffer (Biolabs). The restriction fragments were analyzed
on 1.5% agarose gels in 1× TBE (43) and visualized by
autoradiography.
Electron Microscopy-- Purified minichromosomes were diluted into triethanolamine buffer (10 mM, pH 7.5) and fixed with glutaraldehyde (final concentration of 0.1%). Samples were processed by using the benzyldimethylalkylammonium chloride spreading technique of Vollenweider et al. (44) as described in detail before (45).
Western Analysis-- 300 ng of the sucrose gradient purified minichromosomes preparations were separated on a 15% SDS-PAGE (46) and blotted to a Teflon membrane. HMG-17 antibodies (47) were diluted 1:1000 in Tris-buffered saline with Tween buffer. All the other steps were exactly done as described in the protocol for ECL Western blotting from Amersham Pharmacia Biotech.
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RESULTS |
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Characterization of Chromosomal Templates Containing HMG-17-- To investigate the influence of HMG-17 on DNA replication in the context of a chromatin template, single-stranded circular M13 DNA containing the SV40 origin sequences was used as a template for complementary strand synthesis in high speed supernatants of Xenopus egg extracts. Complementary DNA strands are synthesized on added single-stranded DNA concomitantly with nucleosome assembly (48). Assembled minichromosomes were then used as template in the SV40 in vitro replication system. Complementary DNA strand synthesis was done for 2 h at 22 °C in Xenopus egg extracts in the absence or presence of HMG-17. Purified HMG-17 was added at the beginning of the reaction at a level comparable with that of the histones in the Xenopus extract. As has been shown before, HMG 17 is not present in Xenopus egg extracts (26). Prior to carrying out the replicational analysis in the SV40 in vitro replication system, it was essential to determine whether HMG-17 was incorporated efficiently and properly into the chromatin. We therefore purified assembled chromatin on sucrose gradients containing 70 mM KCl and analyzed it first by immunoblotting with HMG-17 specific antibodies (Fig. 1A). By comparison with the intensities of HMG marker proteins, the amount of HMG incorporated into the assembled chromatin corresponded to the physiological ratio of 2 HMG molecules per nucleosome. The data is in agreement with mobility shift assays which had demonstrated that in this assembly system the HMG proteins are associated with nucleosomes (26). Likewise, polyacrylamide gel analysis of the proteins present in the purified chromatin confirmed our previous results, indicating that the assembled chromatin contains a full complement of histones and HMG (data not shown). Thus, HMG-17 was properly assembled into chromatin, and the presence of HMG-17 did not affect the content of histones in the assembled template.
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HMG-17 Enhances the Rate of Replication of
Minichromosomes--
Equal amounts of minichromosomes that were
assembled in either the absence or presence of HMG-17 and purified over
sucrose gradients containing 70 or 400 mM KCl were used as
templates in the SV40 in vitro replication system. In these
experiments, the minichromosomes were incubated for 2 h in the
presence of the SV40 T-Ag and [-32P]dATP to label the
newly synthesized DNA. Replication products were purified and analyzed
by agarose gel electrophoresis and autoradiography (Fig.
3, A and C). The
incorporation of radioactive nucleotides was determined by
trichloroacetic acid precipitation (Fig. 3, B and
D). When we purified minichromosomes on gradients containing
70 mM salt, we found that the replication efficiency of
minichromosomes containing HMG-17 was ~3-fold higher than that of
minichromosomes lacking HMG-17. The increase in replication efficiency
is evident from the higher amounts of both replicative intermediates
(Fig. 3A, RI) and of completely replicated
molecules (Fig. 3A, between form II and
form I). Additionally, in the presence of HMG-17, more
topoisomers are visible in the replicated DNA. This is due to a limited
chromatin assembly in this system, which is not sufficient to package
higher amounts of replicated DNA completely into chromatin (42, 49),
and therefore distinct topoisomers appear between forms I and II DNA.
Control experiments were performed in the absence of the SV40 T-Ag, and
in this case, no incorporation of nucleotides was measured, which
demonstrates that the observed incorporation is not due to repair
synthesis (data not shown). When we used minichromosomes, which were
purified on sucrose gradients containing 400 mM salt as
template for in vitro replication, differences in
replication efficiency between minichromosomes assembled in the absence
or presence of HMG-17 disappeared (Fig. 3, C and
D). This demonstrates that the observed differences in
replication efficiency (Fig. 3, A and B) are due to the presence of HMG-17 on chromatin.
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DISCUSSION |
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Our major finding is that chromosomal protein HMG-17 enhances the rate of replication of a chromatin template, but not that of a DNA template. We present evidence that the timing of deposition of the HMG into the chromatin template is an important step in the generation of a chromatin template with an increased rate of replication. Addition of HMG-17 to chromatin does not increase the rate of replication. Based on the data presented here, and previously published results, we suggest that HMG-17 protein functions as an architectural element and unfolds the chromatin template. Assembly of the protein into nucleosomes reduces the overall compactness and therefore the repressive activity of the chromatin fiber. As demonstrated here, the unfolded chromatin fiber is more efficiently replicated and, also as was demonstrated elsewhere, is a better substrate for transcription by both RNA polymerase II and polymerase III.
Chromatin was generated from a single-stranded circular DNA template by a coupled DNA replication-chromatin assembly process that was carried out, with Xenopus egg extracts, in either the absence or presence of HMG-17 protein. Analysis of the replication kinetics of these minichromosomes in the SV40 in vitro replication system showed that incorporation of nucleotides into the chromatin region containing the origin of replication is higher in minichromosomes containing HMG-17 as compared with minichromosomes devoid of this protein. What is the mechanism whereby the presence of HMG-17 stimulates the initiation of replication only in chromatin and not on DNA? Since in this system a nucleosome at the origin inhibits the initiation of replication (42, 50), it is conceivable that the presence of HMG allows initiation from a nucleosomal origin. We feel that this is unlikely because we have demonstrated before that neither the acetylation of the core histones (20) nor the complete removal of the amino-terminal domains of the histones (31) release the block imposed by nucleosomes on the SV40 origin. Thus, it seems unlikely that association of HMG-17 with the nucleosome is sufficient for opening of a nucleosomal origin to the replication machinery. We favor a second possibility, namely that the HMG-17 protein unfolds the higher order chromatin structure of the minichromosomes and thereby induces a more extended chromatin structure that is more accessible to the replication machinery. This results in a higher rate of initiation events of minichromosomes with a nucleosome-free origin.
More importantly, the HMG-17-dependent stimulation of the rate of elongation of replication of the chromatin template is also in full agreement with the suggestion that the effect of HMG-17 is mediated through unfolding of the chromatin template. Recent experiments have shown that a more extended chromatin structure facilitates elongation of replication. Thus, minichromosomes where the amino-terminal histone domains were completely removed by trypsin treatment (31) have a more extended chromatin structure and replicate with higher efficiency than control chromatin. Furthermore, minichromosomes with hyperacetylated core histones (20) showed a higher rate of replication fork movement than control minichromosomes. Recent experiments have shown that the chromatin structure can regulate the accessibility for topoisomerase I and topoisomerase II to the chromatin and thus the replication efficiency of the template (51). One possibility for the increased replication efficiency of HMG-containing chromatin could be the facilitated access of topoisomerases to this chromatin.
What might be the in vivo function of HMG-17 during DNA replication? HMG proteins are preferentially associated with chromatin subunits containing transcribed genes (52, 53). Transcriptionally active chromatin is preferentially replicated early in S-phase (54). Conceivably, early replicating DNA could contain a higher amount of HMG proteins than late replicating DNA. In addition, transcriptional active chromatin is enriched in hyperacetylated histones which has been shown to facilitate the access of transcription factors to their target sequences (55, 56) and to cause an increase in transcriptional initiation and elongation on chromatin templates (57). Furthermore SV40 minichromosomes containing hyperacetylated histones replicate with higher efficiency than control minichromosomes in vitro (20). Thus, the interesting possibility arises that the association of chromatin with HMG proteins together with core histone acetylation may contribute to the regulation of early S-phase replication by facilitating the passage of the replication machinery.
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ACKNOWLEDGEMENTS |
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We thank Rolf Knippers and Lothar Halmer for helpful discussions and reviewing of the manuscript. We are especially grateful to Kai Treuner for help and advice with electron microscopy.
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FOOTNOTES |
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* This work was supported by Grant Gr 1201/2-1 from the Deutsche Forschungsgemeinschaft (to C. G.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
¶ To whom correspondence should be addressed. Tel.: 49 7531 882125; Fax: 49 7531 884036; E-mail: cg2@chclu.chemie.uni- konstanz.de.
1 The abbreviation used is: HMG, high mobility group.
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REFERENCES |
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