Department of Biochemistry and Immunology, St Georges Hospital Medical School, Cranmer Terrace, London, SW17 0RE, UK
These authors contributed equally to this paper
*Author for correspondence (e-mail: s.cotterill{at}sghms.ac.uk)
Accepted March 10, 2001
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SUMMARY |
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The interaction with the DNA polymerase is not related to the special situation in early embryos where there are large amounts of maternal protein stockpiles of the polymerase, as it occurs to the same level in early and late embryos and also in proliferating cell culture. However, it does not occur in quiescent cells, making it likely that the protein is related to proliferation. This is also consistent with Dpit47 expression being higher in proliferating cells. The interaction between the Dpit47 and the polymerase takes place predominantly in the nucleoplasm, and seems to involve several subunits of the polymerase in comparable amounts, making it unlikely that it is solely required for the assembly of the polymerase complex. The polymerase can also be seen to interact with Hsp90, and the interaction between Dpit47 and the polymerase is increased by the specific Hsp90 inhibitor geldanamycin. This suggests that a complex of the Dpit47, Hsp90 and DNA polymerase exists in the cell. The interaction between DNA polymerase
and Dpit47 completely inhibits the activity of the polymerase.
These results suggest that Hsp90 acts as a chaperone for DNA polymerase and that this interaction is mediated through the novel co-chaperone Dpit47. This provides the first suggestion of a role for chaperones in DNA replication in higher eukaryotes.
Key words: Replication, Heat shock proteins, DNA polymerase, Drosophila, Chaperone
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INTRODUCTION |
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DNA polymerases are essential enzymes involved in DNA replication and in repair. Although at least nine different polymerases have now been isolated from eukaryotes (Hubscher et al., 2000) only one of these the DNA polymerase , contains a DNA primase activity. Therefore, DNA polymerase
is the only known DNA polymerase able to initiate de novo DNA synthesis (Foiani et al., 1997) and is required for both initiation at chromosomal origins and for the synthesis of Okasaki fragments on the lagging strand. In all species so far studied, the DNA polymerase
is composed of four subunits. The polymerase activity is associated with the largest (
180 kDa) subunit, whereas the primase activity is associated with the smallest (
50 kDa) (Bakkenist and Cotterill, 1994; Copeland and Wang, 1993; Nasheuer and Grosse, 1988; Santocanale et al., 1993). Little is known about the activities associated with the other subunits (
60 and
80 kDa). For efficient initiation of replication, DNA polymerase
has to be loaded and active at specific points during the cell cycle; it also has to be prevented from inappropriate DNA synthesis. How this regulation is achieved is not yet clear, but extensive study in a number of organisms has indicated that it is liable to be at a number of different levels. Although the rate of transcription is cell-cycle controlled in budding yeast, protein levels remain constant throughout the cell cycle (Falconi et al., 1993). Differential association of the polymerase with the chromatin has been observed in budding yeast (Desdouets et al., 1998), human cell culture (Stokke et al., 1991) and Drosophila embryos (Melov et al., 1992). Phosphorylation may also be involved as phosphorylation of various subunits has been observed in vivo for budding yeast (Foiani et al., 1995), fission yeast (Bouvier et al., 1993), humans (Nasheuer and Grosse, 1988) and Drosophila (Kuroda and Ueda, 1999), and in vitro a number of kinases have been shown to phosphorylate the enzyme, in some cases having effects on the polymerase activity of the enzyme (Voitenleitner et al., 1999) (Weinreich and Stillman, 1999).
In an effort to understand more about the mechanisms controlling the DNA polymerase from Drosophila melanogaster, we have cloned and characterised all four subunits of the complex (Bakkenist and Cotterill, 1994; Cotterill et al., 1992; Huikeshoven and Cotterill, 1999; Melov et al., 1992) and carried out screens to identify interacting proteins. In this paper we present our data on one of the proteins that we isolated by two-hybrid interaction with the p180 subunit. This protein, which we have called Dpit47, appears to be an Hsp90 co-chaperone protein. We present our analysis of the characterisation of Dpit47 and its interaction with Hsp90. We also present data on the interaction of DNA polymerase
with Dpit47 and Hsp90. Complexes of DNA polymerase
with Dpit47 show severely suppressed polymerase activity in vitro. This interaction with Hsp90 could therefore provide an additional level of control for the polymerase. An effect of heat shock proteins is already well documented in a number of prokaryotic DNA replication systems (Konieczny and Zylicz, 1999), and is also thought to be involved in the control of replication of some eukaryotic viruses, e.g. SV40 (Campbell et al., 1997) and papilloma (Liu et al., 1998), but we believe this constitutes the first evidence that Hsp90 may have a function in DNA replication in higher eukaryotes.
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MATERIALS AND METHODS |
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DNA sequencing was carried out using the Sequenase kit (US Biochemicals, Cleveland, OH, USA) or was sent to Advanced Biotechnology Centre, Charing Cross and Westminster Medical School, for ABI sequencing. Nucleotide sequence analysis and amino acid comparisons were performed using Geneworks (IntelliGenetics Inc.) or the Blast facility at NIH.
Production of Dpit47 antibodies
Dpit47 missing the first 20 amino acids was cloned into pQE31 (Qiagen) and the His-tagged protein expressed and purified according to the manufacturers instructions. Purified protein was sent to Neosystem laboratoire (Strasbourg) for antibody production in two rabbits. The polyclonal antisera of these rabbits were affinity purified using purified Dpit47 cross-linked onto CNBr activated sepharose 4B (Amersham-Pharmacia) using standard procedures (Harlow and Lane, 1988).
Immunoprecipitations
Antibodies were cross-linked to protein A-sepharose beads using dimethylpimelimidate. 50 µl of packed beads were incubated for 1 hour at 4°C with 300 µl of extract. Beads were then washed ten times with 20 volumes of IP buffer: 10 mM Tris, pH 7.5, 100 mM NaCl, 0.05% triton X100 and 5% glycerol buffer (1 ml per wash). The beads were then incubated with 100 µl of IP buffer plus 250 mM NaCl. This eluate was kept and the beads washed ten times with IP buffer plus 250 mM NaCl. This treatment was repeated with 500 mM NaCl, the beads were incubated with 2% SDS and the pellet was resuspended in 100 µl SDS-PAGE loading buffer. For the experiment with the addition of geldanamycin or ATP, MgCl2 was added to a final concentration of 1 mM and geldanamycin added at 3 µg/ml or ATP at 1 mM, final concentration.
Calculation of Dpit47 concentration in extracts
Dpit47 was immunoprecipitated from Drosophila embryo crude extract, as indicated above, so that it could be visualised on PAGE-SDS upon staining with sensitive Coomassie (Brilliant blue G colloidal) (e.g. Fig. 3; Fig. 4; Fig. 6b). The concentration of Dpit47 in the immunoprecipitate was estimated by comparison with known amounts of molecular weight markers. The immunoprecipitated Dpit47 was then used as a standard to estimate the amount of the protein in crude extracts by western blotting. In all cases, serial dilutions were loaded on the gels and the amounts estamated using the Alpha Innotech Corporation quantitation program.
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Embryos were counterstained with monoclonal anti-histones (1/1000, Chemicon) and horse anti-mouse Texas red monoclonal (1/500, Vectorlabs) following a similar procedure.
Drosophilaembryos, cell extracts and cellular fractionation
For whole cell extract, Drosophila embryos of the ages indicated in the text were homogenised in 1 volume of IP buffer using a Dounce homogeniser with a tight pestle in order to break open the nuclei. These were filtered through Miracloth and then centrifuged for 5 minutes at 6.5 K rpm in a microfuge.
For cellular fractionation the homogenisation was carried out using a loose pestle in order to maintain the integrity of the nuclei. The filtered homogenate was centrifuged for 10 minutes at 13 K rpm. The supernatant is the cytoplasmic fraction. The nuclei pellet was washed several times using the same buffer and subjected to further fractionation, first using 1% Triton X100 and then increasing the NaCl concentration as indicated in the text. In each case the first supernatant was kept and the pellet washed several times with buffer supplemented with Triton and/or NaCl. All buffers were supplemented with protease inhibitors (Boehringer Mannheim, CompleteTM, EDTA free).
Cell culture S2 cells were propagated in 1X Schneiders Drosophila media (GIBCO) supplemented with 10% FBS, 50 units/ml penicillin and 50 µg/ml streptomycin. Cells either in exponentially growing phase or in stationary phase were harvested and treated as above for whole cell extracts or cellular fractionation.
DNA polymerase assay
A 35 bp oligonucleotide (TGAGTCGTATTACAATTCACTGGCCGTCGTTTTAC) was annealed to a complementary 17 bp oligonucleotide (GTAAAACGACGGCCAGT). For each reaction 2.5 pmol of annealed primers were used. The reaction was carried out at 25°C in a final volume of 50 µl of 20 mM Tris-HCl, pH 8, 2 mM DTT, 200 µg/ml bovine serum albumin, 10 mM MgCl2, 50 µM dATP, 50 µM dGTP, 50 µM dTTP and 32PdCTP. The reaction was stopped by the addition of 10 µl EDTA (0.5 M) and 5 µl SDS 20%. The free nucleotides were removed on a G50 spin column. The products of the reaction were ethanol precipitated and analysed on a 15% polyacrylamide gel. The bands were visualised either by autoradiography or using a phosphoimager screen.
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RESULTS |
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Database searching also revealed several other Drosophila proteins related to the Dpit47 protein. It therefore seems likely that Dpit47 forms part of a protein family.
Analysis of the region upstream of the Dpit47 gene did not reveal any sites for the E2F (Duronio et al., 1995) or DREF (Yamaguchi et al., 1995) transcription factors both of which have previously been implicated in the control of genes involved in cell cycle and replication in Drosophila.
Dpit47 interacts with polymerase in crude extracts
To analyse Dpit47 further, an antibody was raised against an overexpressed hexa-his tagged version of the protein. In crude extract from Drosophila embryos, affinity purified antibody recognises a single band of approximately 50 kDa (data not shown), which corresponds well to the size predicted from the sequence of 46.5 kDa. Calculation of the amount of Dpit47 in the cell suggests that it is an abundant protein and is present at approximately 2x109 molecules per cell in stage 14 embryos. (see Materials and methods).
To obtain confirmation of the interaction between DNA polymerase and Dpit47, we carried out immunoprecipitations from crude 0-5 hour Drosophila embryo extract using the anti Dpit47 antibody. As shown in Fig. 2, Dpit47 is efficiently immunoprecipitated from embryo extract. The analysis of the eluted fractions by western blot using antibodies directed against DNA polymerase
reveals the presence of the 180 kDa subunit. The interaction is of moderate strength as all of the 180 kDa subunit has been eluted by 250 mM salt, and is further confirmed by the reverse immunoprecipitation of Dpit47 by anti-polymerase
antibodies (data not shown). This interaction between Dpit47 and the 180 kDa subunit of the DNA polymerase
is specific, as it is not detected when pre-immune or unrelated sera are used for the immunoprecipitation. In addition, the precipitates do not contain PCNA, RPA, cdk2, topoisomerase 2 or lamin (data not shown). Calculation of the amount of the DNA polymerase
associated with Dpit47 suggests that it is about 10% of total cellular polymerase.
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From these results we conclude that Dpit47 forms a tight complex with Hsp90 and Hsp70, and by analogy to the function of the related budding yeast protein CNS1 is likely to function as an Hsp90 co-chaperone.
Geldanamycin stabilises the interaction between Dpit47 and DNA polymerase
Because Dpit47 is likely to be an Hsp90 co-chaperone we were interested to know whether we could detect an interaction of Hsp90 with DNA polymerase . Analysis of proteins immunoprecipitated with a monoclonal antibody against the DNA polymerase
did show a band that cross reacted with Hsp90 antibodies (data not shown). However, in our hands the Hsp90 antibodies produced a high background; we therefore sought another way to confirm that the DNA polymerase
interacted with Hsp90.
Geldanamycin is a specific inhibitor of Hsp90 activity and acts by binding to its ATP binding site (Pearl and Prodromou, 2000). The presence of geldanamycin therefore interferes with the Hsp90 pathway and has been shown to alter the interactions between the component proteins of the pathway. For DNA polymerase , direct measurement of the interaction with Hsp90 under various conditions was limited by the quality of the Hsp90 antibodies. We therefore decided to look for an effect of geldanamycin on the interaction between Dpit47 and DNA polymerase
. An observed effect should allow us to argue for an involvement of Hsp90 with the DNA polymerase
, mediated through Dpit47. DNA polymerase
complexed with Dpit47 was therefore isolated by immunoprecipitation in the presence or absence of geldanamycin. As can be seen in Fig. 4A,a, immunoprecipitations incubated with geldanamycin consistently contain more DNA polymerase
(about twofold) than is detected in the control.
Although geldanamycin blocks the action of Hsp90, the addition of ATP to Hsp90 complexes has been shown to catalyse release of the substrate. Fig. 4A,b shows that the replacement of geldanamycin with ATP reduced the DNA polymerase level below detection levels. This is also consistent with what we would expect for the interaction of an Hsp90 complex with DNA polymerase
.
We therefore conclude that the appearance of Hsp90 in DNA polymerase immunoprecipitates, plus the observed effects of geldanamycin and ATP on the interaction of DNA polymerase
with Dpit47, are consistent with what would be expected if Hsp90 acted as a chaperone for DNA polymerase
and if Dpit47 was a co-chaperone that mediated this interaction.
Geldanamycin affects the interaction of Dpit47 with Hsp70
The specific interaction with DNA polymerase only represents a small percentage of the Dpit47 in the cell. We were therefore interested to determine the effect of geldanamycin in general on the interaction of Dpit47 with other components of the Hsp90 pathway.
We looked at the relative amounts of Hsp90 and Hsp70 immunoprecipitated in the pellets under the various conditions (Fig. 4B). In the control immunoprecipitation Hsp90 and Hsp70 can clearly be identified. When the immunoprecipitation is performed in the presence of geldanamycin, Hsp90 and Hsp 70 are pulled down in similar amounts as in the control. In the presence of ATP, similar amounts of Hsp90 are detected but the level of Hsp70 is much reduced. These results further support the involvement of Dpit47 in the Hsp90 pathway.
Dpit47 developmental profile
To determine the cellular function of the interaction between Dpit47 and DNA polymerase we needed to get more information about the behaviour of the Dpit47 protein in Drosophila. Total crude extracts from all stages of Drosophila life cycle were analysed on western blot with antibody directed against Dpit47. Fig. 5 shows the profile consistently obtained with Dpit47 antibodies. The protein is more abundant in young embryos, in pupae and in females and lower in late embryos, larvae and males. This pattern is characteristic of a protein having a role in proliferation.
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From these results we can conclude that the interaction between DNA polymerase and Dpit47 is not limited to young embryos but also takes place in older embryos and in cell culture.
The interaction between DNA polymerase and Dpit47 is not detected in non-proliferating cells
We next investigated whether the Dpit47:DNA polymerase interaction also occurred in non-proliferating cells. We made whole cell extracts from quiescent S2 cells and carried out immunoprecipitation using the anti-Dpit47 antibody. As a positive control we used proliferating S2 cells as above. Fig. 6c compares the level of DNA polymerase
in equivalent amounts of Dpit47 immunoprecipitates from exponentially growing and quiescent cells and shows that in quiescent cells the amount of DNA polymerase
is very much reduced. This is not due to the absence of DNA polymerase
as it is also detected in quiescent cells (Fig. 6c, loading control).
From this result we can conclude that the interaction between Dpit47 and DNA polymerase is confined to proliferating cells and therefore, as was originally suggested by the expression profile of the Dpit47, the interaction is likely to have some role in proliferation.
Dpit47 is found in the nucleus but is only loosely associated with the chromatin
Much of the activity of Hsp90 co-chaperones takes place in the cytoplasm. We therefore determined the cellular location of Dpit47 as a guide to where it was likely to carry out its function.
In embryos prior to cellularisation where the protein was most abundant, Dpit47 protein was visible throughout the embryo in the nucleus and cytoplasm (data not shown). After cellularisation, however, the protein was seen predominantly in the nucleus. Fig. 7a shows the staining pattern using affinity purified Dpit47 antibodies and counterstained with anti-tubulin antibodies to clearly visualise the stages of the cell cycle. Throughout the cell cycle even during mitosis when the nuclear envelope has partially broken down Dpit47 staining appears to be confined within the nuclear region. In Fig. 7b where the embryos are counterstained with histone it is apparent that the Dpit47 staining only partially overlaps that of the DNA in interphase and telophase and in the mitotic stages forms a cloud in the vicinity of, but not directly associated with, the DNA. In both Fig. 7a and Fig. 7b it is possible to see that two different patterns are obtained for Dpit47 staining in mitosis. In the upper of the two metaphase panels in each case the protein appears more proximal to the DNA than the other. Although both these patterns are visible regularly we have not been able to determine definitively which of them preceeds the other in the cell cycle.
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The interaction between Dpit47 and DNA polymerase is detected both in cytoplasmic and nucleoplasmic fractions
Because Dpit47 is detected in the cytoplasm and the nucleoplasm we were interested to determine whether the interaction that we had observed with DNA polymerase was also detected in both compartments. Immunoprecipitations were therefore performed on cytoplasmic and their corresponding nucleoplasmic fractions from embryos 0-18 hours old and analysed as described above. As shown in Fig. 9 the interaction between DNA polymerase
and Dpit47 is detected in both fractions. By comparison of the amount of DNA polymerase
present in the precipitate and in the crude cytoplasmic and nucleoplasmic fractions, it is possible to see that significantly more p180 is found associated with Dpit47 in the nucleoplasmic than in the cytoplasmic fraction.
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The DNA polymerase associated with Dpit47 is inactive
We also wished to determine whether the association between Dpit47 and DNA polymerase had any effect on its enzymatic activity. We therefore looked at the ability of the DNA polymerase
co-immunoprecipitated with Dpit47 to perform an end filling reaction on a 35 mer substrate. As a control we immunoprecipitated DNA polymerase
with a monoclonal antibody known not to affect its activity. The levels of polymerase were adjusted to be equivalent in both cases (Fig. 11, loading control). Fig. 11 clearly demonstrates that the DNA polymerase associated with Dpit47 has no detectable polymerase activity. This is not due to an inhibitory protein present in the Dpit47 immunoprecipitates as addition of Dpit47 precipitates has no effect on the level of polymerase activity detected in the DNA polymerase immunoprecipitates (data not shown).
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DISCUSSION |
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The recent release of the Drosophila genome sequence (Adams et al., 2000) has further allowed us to determine that Dpit47 is a member of a family of proteins in Drosophila. Seven other homologues can be identified from their genome sequence, although in most cases the homology is largely confined to the TPR region. One of these proteins has already been defined as the Drosophila Hop homologue; the others are as yet uncharacterised.
Database searching has also allowed us to identify Dpit47 homologues in other species from budding yeast to mammals. The closest of these is a human protein TTC4 (accession number NM_004623.1), which was first identified as a putative tumour suppressor gene frequently deleted in breast cancer (Su et al., 1999). Since this is closely related to Dpit47 it would be interesting to determine if TTC4 has retained functional characteristics of the Dpit47 protein.
Dpit47 is likely to function as a DNA polymerase co-chaperone
Most of the work presented here centres around the study of the interaction of the Dpit47 protein with the DNA polymerase . Although the interaction was first seen in the two hybrid system, we have also confirmed that the interaction can be detected in extracts from various types of cells. We have detected the interaction in both cytoplasmic and nucleoplasmic fractions, suggesting the interaction may occur in both compartments. However, we cannot completely rule out the possibility that Dpit47 associates with DNA polymerase
predominantly in the nucleoplasmic fraction, and that the interactions observed in cytoplasmic fractions are caused by leakage from the nucleus during the nuclear preparation, particularly from cells in mitosis.
Although both proteins are quite abundant, the interaction only involves about 10-20% of the polymerase and 1-2% of the Dpit47. This is consistent with the Dpit47 having other substrates (see later). For the DNA polymerase this could represent an instability of the complex to the isolation conditions, alternatively only a subpopulation of the DNA polymerase
molecules may bind to Dpit47.
In addition to showing the interaction between Dpit47 and DNA polymerase , we have also been able to immunoprecipitate Hsp90 directly with DNA polymerase
antibodies. Although we have not formally isolated the trimeric complex, this observation, taken together with the observed effect of geldanamycin on the Dpit47:polymerase interaction, strongly suggests that the DNA polymerase
:Dpit47 complex is likely to also include Hsp90.
Cellular function of Dpit47: DNA polymerase interaction
Our data is suggestive of a role for the Hsp90 pathway in DNA polymerase function. Interestingly, it is also consistent with a much earlier report in which geldanamycin was shown to inhibit DNA polymerase
activity (Yamaki et al., 1982). However, an important question is the function of this interaction in a cellular context. Among Hsp90 client proteins the purpose of the interaction with Hsp90 varies depending on the protein concerned. For a large group of proteins, including the widely studied glucocorticoid receptor (Buchner, 1999) and telomerase, it seems that the interaction is involved in the conversion of the protein from the inactive to the active state. For other proteins it is involved in holding the protein in a particular configuration so that it can interact with protein partners or be modified in some way (e.g. phosphorylated). The interaction with the DNA polymerase that we have studied here causes a severe inhibition of the polymerase activity in the complex (the specific activity is at least 50x less than that normally seen). This suggests that the most likely role of the interaction in this case is to sequester polymerase in an inactive form. What still remains to be determined is the role this plays in the normal functioning of the cell. It is likely to be involved in proliferation, as the interaction does not occur in quiescent cells. The presence of the interaction in late embryos/larvae and cell culture also suggests that it is not just related to the unusual situation in early embryos in Drosophila where excessive amounts of maternal proteins are present.
In the normal progression of the cell cycle there are a number of places where such an activity could be useful. Inactive polymerase must be maintained prior to initiation of DNA replication, or after the completion of one complete round, therefore Dpit47 could function at either of these stages. Because the observed location of Dpit47 is predominantly nucleoplasmic but not chromatin bound, any observed effect must take place prior to or subsequent to the association of the polymerase with the replication complex, rather than actually in the complex itself. Therefore, Dpit47 could function by facilitating rapid binding of the polymerase to the chromatin during initiation of DNA replication by concentrating the polymerase in an inactive form close to its potential substrate. Alternatively, it could function by allowing rapid sequestration of the polymerase after it has finished synthesis.
A sequestration role for the Dpit47 DNA polymerase interaction does not preclude the possibility that the interaction may have other additional effects on the polymerase that are more in line with the effects that the Hsp90 pathway has been seen to have on other client proteins. We cannot rule out completely that passage through the Hsp90 complex is required for activation of all polymerase molecules (as for the glucocorticoid receptor). However, the high level of activity of the polymerase isolated from early embryos when only small amounts need to have been activated owing to the small number of genomes to replicate, and the observation that polymerases from other organisms can be overproduced and are active, makes it less likely.
Equally, the observation that the Dpit47 immunoprecipitates contain all four subunits in equal amounts makes it unlikely that interaction is required for the assembly of the complex. However, it is still possible that the Dpit47 interaction is required for modification of the polymerase, or for allowing its interaction with other components of the replication complex.
Cellular function of Dpit47
Hsp90 is an essential abundant molecular chaperone involved in the folding, assembly and activation of number of proteins involved in signal transduction, cell cycle control, telomerase activity Hsp90 (Holt et al., 1999) or transcriptional regulation (Pratt, 1998). Here we have presented evidence that links the Hsp90 pathway with a mainstream replication protein, thereby providing the first report of a possible role for chaperones in the process of DNA replication in higher eukaryotes. However, the amount of Dpit47 is far greater than that of DNA polymerase , making it very likely that Dpit47 has other client partners. Sensitive Coomassie analysis of the fractions where DNA polymerase
is detected eluting from the anti-Dpit47 antibody reveal the presence of about a dozen bands of comparable intensity to DNA polymerase
. It would therefore be interesting to identify these proteins to determine if any others are involved in DNA replication and to see what other cellular processes might involve Dpit47.
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ACKNOWLEDGMENTS |
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