Stimulation of Replication Efficiency of a Chromatin Template by Chromosomal Protein HMG-17*

Birgit VestnerDagger , Michael Bustin§, and Claudia GrussDagger

From the Dagger  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

    ABSTRACT
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Abstract
Introduction
Procedures
Results
Discussion
References

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.

    INTRODUCTION
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Abstract
Introduction
Procedures
Results
Discussion
References

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.

    EXPERIMENTAL PROCEDURES
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Abstract
Introduction
Procedures
Results
Discussion
References

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 [alpha -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, <FR><NU>1</NU><DE>10</DE></FR> 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.

    RESULTS
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Abstract
Introduction
Procedures
Results
Discussion
References

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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Fig. 1.   Characterization of reconstituted minichromosomes. A, Western analysis. Minichromosomes, reconstituted in the absence (-) or presence (+) of HMG-17 were purified on sucrose gradients containing either 70 or 400 mM KCl. 300 ng of chromatin were separated on a 15% PAGE and analyzed by immunoblotting with HMG-17-specific antibodies. Increasing amounts (nanograms) of purified HMG-17 were used for quantitation. Sizes (kDa) of a low molecular mass marker are indicated at the left side. B, supercoiling assay. Single-stranded DNA was incubated for the indicated times in Xenopus egg extract in the presence of [alpha -32P]dATP. Incubation was in the absence (-) or presence (+) of HMG-17. Purified DNA was separated on a 0.8% agarose gel. The autoradiogram depicts the mobility of supercoiled (form I) and relaxed and open circular (form II) DNA. C, topoisomer analysis. Radiolabeled DNA was extracted from chromatin assembled in either the absence (-) or presence (+) of HMG-17 and separated on a 0.8% agarose gel in 1× TPE buffer (40 mM Tris-Cl (pH 8.0), 30 mM NaH2PO4, 1 mM EDTA; Ref. 27), containing chloroquine (form I, supercoiled DNA; form II, relaxed and open circular DNA). D, electron microscopy. Chromatin assembled in the absence (-) or presence (+) of HMG-17 was purified by sucrose gradients and analyzed by electron microscopy as described (44). bar = 50 nm.

The effect of HMG-17 on the efficiency of chromatin assembly was then examined by DNA supercoiling analysis (Fig. 1B). In a Xenopus egg extract, single-stranded DNA is replicated and assembled into regularly spaced chromatin in a time-dependent manner (33, 34). The kinetics of the assembly of the replicated DNA into chromatin can be monitored by the appearance of supercoiled DNA. The data show that the rate of chromatin assembly was not affected by the presence of HMG-17. After 30 min, some topoisomers are still visible, and after 120 min, the molecules were completely assembled into chromatin in both cases (Fig. 1B). For a further comparison of the number of nucleosomes assembled in the absence or presence of HMG-17, reconstituted chromatin was investigated by separation on chloroquine gels (Fig. 1C) and by electron microscopy (Fig. 1D). As revealed by both techniques, the presence of HMG-17 did not affect the number of nucleosomes assembled onto the plasmid. Analysis of the minichromosomes visualized by electron microscopy showed that the number of nucleosomes assembled in the absence and presence of HMG-17 was 34 ± 2 nucleosomes (Fig. 1D). Thus, HMG-17 did not affect the histone composition or the number of nucleosomes in the minichromosomes.

It has been suggested that incorporation of HMG-17 into nascent nucleosomes induces an extended chromatin conformation (27). Since our results clearly demonstrate that HMG-17 protein does not affect the number of nucleosomes in the minichromosomes and since we anticipate that changes in chromatin conformation may affect the rate of replication of a chromatin template, we tested whether HMG-17 also affects the structure of the minichromosomes under our assembly conditions. To investigate the effect of HMG-17 on the chromatin structure, minichromosomes assembled in the presence of [alpha -32P]dATP in either the absence or presence of HMG-17 were sedimented through sucrose gradients containing 70 or 400 mM KCl. Chromatin-containing fractions were identified by trichloroacetic acid precipitation of the individual fractions and agarose gel electrophoresis. On gradients containing 70 mM salt minichromosomes assembled in the presence of HMG-17 sedimented significantly slower than chromatin reconstituted in the absence of HMG-17 (Fig. 2A). Thus, in agreement with previous studies (27), minichromosomes assembled in the presence of HMG-17 seem to have a more open conformation than those lacking HMG-17. Purification on gradients containing 400 mM salt, which causes the removal of the HMG-17 protein from chromatin (compare Fig. 1A, 400 mM) sedimentation differences between chromatin, which was reconstituted in the absence or presence of HMG-17 protein, disappear (Fig. 2B). Both types of minichromosomes sediment slightly slower on gradients containing 400 mM salt compared with the sedimentation on gradients containing 70 mM salt, which is due to the removal of other non-histone proteins from the minichromosomes at 400 mM salt. These data demonstrate that the observed differences in chromatin structure are due to the presence of the HMG-17 protein.


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Fig. 2.   Sedimentation analysis of assembled chromatin. Minichromosomes assembled in the absence (-) or presence (+) of HMG-17 and [alpha -32P]dATP were purified in parallel by sucrose gradient sedimentation containing either 70 (A) or 400 mM KCl (B). An aliquot of each fraction was precipitated with trichloroacetic acid, and the incorporated radioactivity (cpm) was plotted as a function of the number of fractions.

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 [alpha -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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Fig. 3.   In vitro replication of reconstituted minichromosomes. Minichromosomes reconstituted in the absence (-) or presence (+) of HMG-17 were purified on sucrose gradients containing either 70 (A) or 400 mM KCl (C). Equal amounts of minichromosomes were incubated for 2 h under in vitro replication conditions. DNA was purified and investigated by agarose gel electrophoresis and autoradiography. RI, replicative intermediates; II, relaxed and open circular DNA; I, superhelical form I DNA. Replication efficiencies of minichromosomes purified on sucrose gradients containing 70 (B) or 400 mM KCl (D) as determined by trichloroacetic acid precipitation are standardized against the replication efficiency of minichromosomes reconstituted in the absence of HMG-17 and are shown as percent incorporation. The standard deviation is determined from three independent experiments.

The increase in the replication efficiency could be due to either a direct effect of HMG-17 on the replication apparatus or to changes in the chromatin structure leading to a more efficient production of replicated molecules. To distinguish between these two possibilities, increasing amounts of HMG-17 were added to the replication assay of minichromosomes that were assembled in the absence of HMG-17 (Fig. 4A). In a separate set of experiments, increasing amounts of HMG-17 were added to the replication assay of protein-free, double-stranded M13ori DNA (Fig. 4B). Purified replication products were analyzed by agarose gel electrophoresis and autoradiography. We found that the addition of HMG-17 up to a 10-fold excess had no effect on the replication efficiency of chromatin templates (Fig. 4A). Higher amounts of added HMG-17 inhibited replication efficiency, presumably because some of the protein incorrectly binds to the linker DNA and inhibits access of replication proteins to their target (data not shown). This result demonstrates that HMG-17 enhances the rate of replication of minichromosomes only when incorporated into chromatin during nucleosome assembly. Addition of HMG-17 to the in vitro replication assay of protein-fee DNA had no effect on replication efficiency (Fig. 4B), which demonstrates that the observed stimulation of replication is specific for chromatin and not due to a general stimulation of the replication efficiency by HMG-17.


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Fig. 4.   Addition of HMG-17 to pre-assembled chromatin or to protein-free DNA. Equal amounts of chromatin assembled in the absence of HMG-17 (A) or protein-free DNA (B) were incubated in parallel reaction mixtures with increasing amounts of HMG-17 under SV40 in vitro replication conditions. Purified DNA was analyzed by agarose gel electrophoresis and autoradiography. The mobility of replicative intermediates (RI), relaxed and open circular form II DNA (II), and superhelical form I DNA (I) are indicated.

The HMG-17-dependent stimulation of replication efficiency could be due to either a more efficient initiation of replication or to an increase in the rate at which the replicative fork moves along the replicated template. To address this point, replicating minichromosomal DNA was digested with restriction endonucleases to separate the SV40 origin-containing fragment from flanking DNA segments and the termination region (Fig. 5A). Minichromosomes assembled in either the absence or presence of HMG-17 were replicated for the indicated times (Fig. 5B). The deproteinized DNA was then digested with the restriction endonucleases AvaII, ClaI, and MscI and analyzed by agarose gel electrophoresis. Autoradiograms of restriction fragments (Fig. 5B) show that both in the absence and in the presence of HMG-17, nucleotides were incorporated first into the origin fragment (Fig. 5A, fragment a) and then, after a short delay, into both flanking DNA segments (fragments b and c) and finally into the termination region (fragment d). Thus, replication occurs in a bidirectional manner regardless of whether the minichromosomes contain HMG-17 protein or not. However, quantitative analysis indicates that HMG-17 protein significantly affects the kinetics of nucleotide incorporation into the various regions of the minichromosome. We determined the relative intensities of the autoradiographic signals from each DNA segment by laser scanning densitometry and plotted the values as a function of replication times (Fig. 5C). These data show that nucleotides were incorporated with higher efficiency into the origin fragment of HMG-17-containing chromatin compared with HMG-17-free chromatin. This result suggests that the presence of HMG-17 facilitates initiation of replication. To evaluate the elongation rates of the two different templates, the mean values of the intensities of fragments b, c, and fragment d, determined by laser densitometric scanning of the autoradiogram, were represented in a fitted line against the replication time. The slopes of the lines were different and indicate that the rate of elongation in minichromosomes containing HMG-17 was faster than in minichromosomes devoid of HMG-17. Thus we conclude, that the presence of HMG-17 stimulates both the initiation of replication and the rate of replication fork movement along the chromatin template.


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Fig. 5.   Restriction analysis of replicating chromatin. A, restriction map showing restriction sites on circular double-stranded M13ori-DNA. Numbers refer to the M13 nucleotide coordinates. The positions of the origin (ori) of bidirectional replication and the termination region (ter) are indicated. a, origin fragment; b and c, flanking fragments; d, termination fragment. B, chromatin assembled in the absence (-) or presence (+) of HMG-17 was replicated under standard conditions in parallel reactions. Aliquots were removed at the indicated times (min), and the extracted DNA was incubated with the restriction endonucleases ClaI, AvaII, and MscI. DNA fragments were analyzed by agarose gel electrophoresis and autoradiography. C, kinetics of nucleotide incorporation. The autoradiograms of three independent experiments were analyzed by laser scanning densitometry. The results are expressed in arbitrary units and plotted as a function of incubation times. Top graph, origin fragment (fragment a in panel B); middle graph, flanking fragments (mean value of fragments b and c in panel B); bottom graph, termination fragment (fragment d in panel B).

    DISCUSSION
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Abstract
Introduction
Procedures
Results
Discussion
References

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.

    ACKNOWLEDGEMENTS

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.

    FOOTNOTES

* 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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Top
Abstract
Introduction
Procedures
Results
Discussion
References

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