Nucleic Acids Research, 2000, Vol. 28, No. 2 620-625
© 2000 Oxford University Press
The N-terminal region of DNA polymerase 
catalytic subunit is necessary for holoenzyme function
Institute of Veterinary Biochemistry, University of Zürich, Winterthurerstrasse 190, CH-8057 Zürich, Switzerland
Received August 9, 1999; Revised October 25, 1999; Accepted November 12, 1999.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Genetic and biochemical studies have shown that DNA polymerase
(Pol) is the major replicative Pol in the eukaryotic cell. Its functional form is the holoenzyme composed of Pol, proliferating cell nuclear antigen (PCNA) and replication factor C (RF-C). In this paper, we describe an N-terminal truncated form of DNA polymerase (
N Pol) from calf thymus. The N Pol was stimulated as the full-length Pol by PCNA in a RF-C-independent Pol assay. However, when tested for holoenzyme function in a RF-C-dependent Pol assay in the presence of RF-C, ATP and replication protein A (RP-A), the N Pol behaved differently. First, the N Pol lacked holoenzyme functions to a great extent. Second, product size analysis and kinetic experiments showed that the holoenzyme containing N Pol was much less efficient and synthesized DNA at a much slower rate than the holoenzyme containing full-length Pol. The present study provides the first evidence that the N-terminal part of the large subunit of Pol is involved in holoenzyme function. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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DNA replication requires the finely tuned action of many enzymes, proteins and cofactors. In eukaryotic cells, eight DNA polymerases called
, ß, 
, , 
, 
, 
and 
have been identified (1). Three of them [namely polymerase (Pol), Pol and Pol] are essential for DNA replication. Pol contains primase activity and is responsible for initiation of DNA synthesis on both the leading and lagging strands; Pol and Pol seem to be involved in elongation of the DNA primers synthesized by Pol on the leading and lagging strand, respectively (2,3). Genetic and biochemical studies have shown that Pol is the major replicative Pol in the eukaryotic cell. In addition to DNA replication, Pol has been implicated in many other DNA transactions, such as nucleotide excision repair, mismatch repair and recombinational repair (reviewed in 4). More recently, Pol has also been identified in base excision repair as a back-up enzyme (5).
Pol has been purified from mammalian cells, including fetal calf thymus tissue, as a heterodimer with a catalytic subunit of 125 kDa and a second subunit of 48 kDa (6). Both the polymerase and the 3'
5' exonuclease activities are located on the large p125 subunit. The precise function of the small subunit is still unknown. It has been shown that the 48 kDa subunit is required for efficient stimulation of Pol by the sliding clamp proliferating cell nuclear antigen (PCNA) (7–9). In addition to PCNA, at least two other accessory proteins are required at the replication fork: replication factor C (RF-C) and replication protein A (RP-A). RF-C, also called the clamp loader, recognizes DNA and utilizes ATP hydrolysis to assemble the PCNA clamp around the DNA. The two auxiliary proteins RF-C and PCNA form a complex that is able to track along the DNA strand as a moving platform until it encounters a 3'-OH primer template junction (10). This moving platform then recruits and tethers Pol to the DNA. The complex between Pol, PCNA and RF-C forms the Pol holoenzyme (11).
How these proteins interact and whether RF-C remains a part of this complex in the elongation process is still not known. Deletion mutants of Pol that selectively abolish interactions of Pol with PCNA or RF-C would help to clarify this issue. Unfortunately, these mutants are not presently available due to the apparent difficulty in obtaining recombinant fully active Pol overexpressed from either bacteria (7) or baculovirus-infected insect cells (12). A major problem might be that Pol appears to be more complex than anticipated so far, since a third subunit has recently been identified in human cells (13).
In this paper we describe an N-terminal truncated form of Pol (N Pol) that does not share the same characteristics as full-length Pol. N Pol is as much stimulated as the full-length Pol by PCNA in a RF-C-independent Pol assay but not in a RF-C-dependent holoenzyme assay. The product size analysis and kinetic experiments with the holoenzyme complex showed that the holoenzyme containing N Pol was much less efficient and synthesized DNA at a much slower rate than the holoenzyme containing full-length Pol. Our data indicate that the N-terminal part of Pol large subunit is important for holoenzyme function.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Enzymes and proteins
RF-C was isolated as described (14). Human PCNA was purified to homogeneity as described (15). Recombinant human RP-A was purified according to Henricksen et al. (16) and Escherichia coli single-strand DNA-binding protein (SSB) according to Lohman et al. (17). Polyclonal antibodies against the exonuclease box and against the C-terminal domain of mouse Pol
were prepared as described in Cullmann et al. (18) and Hindges and Hübscher (7), respectively. Rabbit antisera against two different peptides in the N-terminal and C-terminal regions of the large subunit were gifts from P. Fischer and K. Downey (19).
Buffers
The following buffers were used: buffer A, [50 mM Tris–HCl pH 8, 100 mM NaCl, 250 mM D-(+)-sucrose, 1 mM DTT, 1 mM EDTA, 10 mM NaHSO3, 1 mM phenylmethylsulfonylfluoride (PMSF), 1 µg/ml aprotinin, 1 µg/ml leupeptin, 1 µg/ml antipain, 1 µg/ml chymostatin]; buffer B, [50 mM Tris–HCl pH 8, 10% (v/v) glycerol, 1 mM DTT, 1 mM EDTA, 10 mM NaHSO3, 1 mM PMSF]; buffer C-50, [50 mM KPO4 pH 7.5, 10% (v/v) glycerol, 1 mM DTT, 1 mM EDTA, 0.1 mM PMSF]; buffer C-500, [500 mM KPO4 pH 7.5, 10% (v/v) glycerol, 1 mM DTT, 1 mM EDTA, 0.1 mM PMSF]; buffer D, [50 mM Tris–HCl pH 7.5, 20% (v/v) glycerol, 1 mM DTT, 1 mM EDTA, 0.1 mM PMSF, 1 mg/l leupeptin]. Salt concentrations (as NaCl) are indicated by a suffix, e.g. buffer B100 = buffer B + 100 mM NaCl.
Pol purification
All steps were carried out at 0–4°C.
Crude extract. To 880 g of fetal calf thymus was added 1.5 l of buffer A. The tissue was thawed and homogenized in a Sorvall Omnimixer. After centrifugation of the homogenate at 10 000 g for 20 min, the supernatant was filtered through four layers of cheesecloth.
Phosphocellulose P11 chromatography. The crude extract was adsorbed in batch for 1 h on P11 resin (100 ml column) equilibrated in B100 buffer. The resin was packed onto a column washed with 3 volumes of B100. The polymerase activity was eluted with 3 volumes of a linear gradient from B100 to B800. Under these conditions, the PCNA-dependent polymerase activity was identified in the wash.
Q-Sepharose chromatography. The P11 wash was loaded on a 40 ml Q-Sepharose resin equilibrated in B100 buffer. The column was washed with 5 volumes of B100. Elution was performed with 10 volumes of a linear gradient from B100 to B800. The Pol activity eluted at the very beginning of the gradient, at around B110 (Q-Seph. pool).
Ceramic Hydroxyapatite cHAP chromatography. The Q-Seph. pool was loaded onto a ceramic hydroxyapatite (12 ml column) pre-equilibrated with B100 buffer. The column was washed with 6 volumes of C-50 and proteins were eluted with a 10 column volumes gradient from C-50 to C-500. Fractions with an activity strongly dependent on PCNA were pooled (HAP pool) and dialyzed against buffer D100.
MonoQ FPLC column. For concentration, the HAP pool was injected onto a 1 ml MonoQ column (Pharmacia), which was then washed with 4 volumes of buffer B100 and eluted with a 5 column volumes gradient from 100 to 800 mM NaCl. The active Pol fractions were pooled and stored in 20 µl aliquots in liquid nitrogen until use.
Enzymatic assays
RF-C-independent Pol assay. The stimulation of Pol by PCNA was assayed in a final volume of 25 µl containing 50 mM Bis–Tris pH 6.5, 1 mM DTT, 0.25 mg/ml BSA, 6 mM MgCl2, 20 µM [3H]dTTP (300 c.p.m./pmol), 0.5 µg poly(dA)/oligo(dT)16 base ratio 10:1, and variable amounts of Pol and PCNA. The reactions were incubated at 37°C for 30 min and then stopped with 10% trichloracetic acid. Acid-insoluble radioactivity was quantified as described (20). One unit of polymerase activity corresponds to the incorporation of 1 nmol dTMP into acid-precipitable material in 60 min at 37°C in the assay described above.
Km determination for the 3'-OH terminus. The reactions were carried out with the RF-C-independent Pol assay as described above using 100 ng PCNA, 0.1 U Pol (either full-length or N Pol) and varying amounts of template poly(dA)/oligo(dT) (0.06, 0.1, 0.2 and 0.4 µM).
RF-C-dependent Pol holoenzyme assay. The assays were carried out as described (21) in the presence of 500 ng RP-A, 100 ng PCNA, 0.017 U RF-C and variable amounts of full-length or N Pol. One unit of RF-C allows the incorporation of 1 nmol dNMP into acid-precipitable material in 60 min at 37°C in standard holoenzyme assay conditions.
Product analysis in the RF-C-dependent holoenzyme assay. The assays were carried out as described (22). A mix of 800 ng RP-A, 100 ng PCNA, 0.017 U RF-C and variable amounts of full-length or N Pol were incubated at 37°C for 30 min.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Purification of a truncated form of Pol
from fetal calf thymusPol
from calf thymus was purified through four consecutive chromatographic steps (see Materials and Methods). During purification, a form of Pol was observed that bound unusually weakly to columns, e.g. eluting in the wash or at the very beginning of a gradient. Nevertheless, after the three chromatographic steps, Phosphocellulose, Q-Sepharose and Hydroxyapatite, this Pol was separated from Pol and from Pol (data not shown). At this stage, Pol required the addition of PCNA whereas Pol was PCNA-independent (23). Since PCNA can stimulate Pol on a linear poly(dA)/oligo(dT) template in the absence of RF-C, the activity detected by this assay is referred to as RF-C-independent polymerase activity. After concentration on a MonoQ column, the active fractions containing only Pol were pooled. These fractions were characterized further by immunoblotting with monoclonal antibodies against the small p50 subunit (Fig. 1C) and two different polyclonal antibodies against the large subunit of Pol: one against the C-terminal region (Fig. 1A) and the other against a region comprising the exo box (7,18). As a positive control, we used a full-length Pol purified from calf thymus (23). The data in Figure 1A show that >90% of the large subunit of the unusual Pol has an apparent molecular weight of 116 kDa, compared with 125 kDa for full-length Pol. In order to see which part, N- or C-terminal, was missing, immunoblots were performed with rabbit antisera against two different peptides: KRRPGPGGVPPKRARC in the N-terminal region and CDQEQLLRRFGPPGP in the C-terminal region of the large subunit (19). The data in Figure 1B show that the antibody specific for the C-terminal part recognized both full-length and truncated Pol and the antibody specific for the N-terminal part recognized only full-length Pol. We concluded then that the N-terminal part (~80 amino acids) of the large subunit was missing in this Pol preparation, which was designated N Pol.
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Full-length and
N Pol are stimulated by PCNA in a RF-C-independent Pol assay and have the same Km for 3'-OH primer terminiDuring purification, the activity of
N Pol was monitored, both in the presence and absence of PCNA, using poly(dA)/oligo(dT) as a linear DNA template. This assay does not require RF-C since under low pH conditions (e.g. pH 6.5) PCNA can slide on DNA by itself. The incorporation of [3H]dTMP by both full-length Pol and N Pol was stimulated over 100 times by PCNA. Moreover, titration of PCNA clearly indicated that both forms of Pol could be stimulated by PCNA in a very similar manner (Fig. 2A). Next we tested whether the missing N-terminal region was precluding the binding of N Pol to DNA. For this purpose, the affinity of both polymerases for the 3'-OH terminus was determined by measuring the kinetic constants Km and Vmax with the RF-C-independent Pol assay mentioned above. As shown in Figure 2B, the Km and Vmax of full-length Pol were identical to those of N Pol, being ~0.1 µM for Km and 2 pmol/min for Vmax, respectively. Thus, both Pol forms have the same affinity for the 3'-OH primer terminus in the presence of PCNA.
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Full-length but not
N Pol is active in a RF-C-dependent Pol holoenzyme assayWhen a circular singly primed template M13 DNA is used as a model template, at least two other auxiliary proteins are required: first RF-C and ATP to load PCNA onto the DNA and second RP-A, which covers the single-stranded DNA, thus preventing non-specific binding of RF-C. Interestingly, under conditions where loading of PCNA onto the DNA by RF-C is required, the activity of
N Pol was only very modestly stimulated by PCNA whereas under these conditions full-length Pol was, as expected, stimulated >10-fold (Fig. 3A). When increasing amounts of PCNA or RF-C were added, identical results were obtained (data not shown). These experiments might suggest two conclusions: there is either a factor inhibiting the activity of Pol or the missing part of Pol plays an important role in the interaction with RP-A and/or RF-C. The presence of a contaminating factor inhibiting the activity of N Pol was first tested by mixing increasing amounts (0–0.3 U) of N Pol to 0.15 U of full-length Pol. No decrease in the full-length Pol holoenzyme activity was observed even with the highest amount of N Pol added, clearly showing that there was no factor inhibiting Pol (data not shown). A specific interaction between the N-terminal part of Pol and RP-A was subsequently investigated by replacing RP-A with E.coli SSB. As shown in Figure 3B, the activities in the RF-C-dependent Pol assay of both full-length and N Pol were the same when SSB was used instead of RP-A (compare Fig. 3A and B).
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The holoenzyme containing
N Pol is significantly less efficient and slower than that containing full-length PolTo analyze in more detail the effects of
N Pol on DNA synthesis, a radioactive 5'-32P-labeled primer (40mer) was annealed to single-strand M13 DNA and replication was monitored after 30 min in the RF-C-dependent holoenzyme assay using increasing amounts of full-length and N Pol. The products were separated on a denaturing polyacrylamide gel. As shown in Figure 4A, the amount of full-length products was proportional to the amount of full-length Pol added. The holoenzyme containing N Pol, in contrast, is much less efficient in synthesizing full-length products (compare lanes 2–4 with 5–7 in Fig. 4A). Next, kinetic experiments were performed in order to compare the rate of the reaction catalyzed by N Pol with full-length Pol (Fig. 4B). The products of DNA synthesis catalyzed by 0.2 U Pol were analyzed at different incubation times (0, 1, 3, 10, 20 and 30 min) on a denaturing polyacrylamide gel. With full-length Pol, full-length products were synthesized within the first minute of the reaction, while N Pol synthesized full-length products only after 10 min.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In this paper we present the characteristics of a truncated form of Pol
from calf thymus. Immunoblot analysis with polyclonal antibodies suggested that 9 kDa (~80 amino acids) of the N-terminal part of the large subunit of Pol were missing (Fig. 1A and B). Several cleavage sites for proteases are present around this position. For example, trypsin cleaves at position W78 and chymotrypsin at W78 and R80. A multiple sequence alignment (20 sequences) produced by the program MAXHOM (24) predicted a solvent-exposed loop conformation for the 100 amino acids at the N-terminal end of the catalytic subunit (25,26), making it accessible to proteases. Interestingly, this N-terminal part of the large subunit is less conserved among species and is completely absent from the polymerases from Chlorella virus and from Archaea, which start at position 99 and 123, respectively. We took advantage of this N Pol to investigate its properties compared with those of the full-length Pol previously purified from the same tissue, using two different assays: the RF-C-independent and the RF-C-dependent Pol assays.
When DNA synthesis was measured with the RF-C-independent Pol assay (Fig. 2), both forms of Pol were equally active, suggesting that the 80 amino acids at the N-terminus do not function in catalysis per se. These results are in agreement with other studies showing that deletion mutants at the N-terminus (amino acids 2–249) retain polymerase activity, but deletion of the C-terminal part of the large subunit abolished all polymerase activity (27). This C-terminal domain is thought to be responsible for DNA interaction, as shown by photocrosslinking performed together with immunoblot analysis (28). The truncated Pol, which contains the intact C-terminal region of the catalytic subunit, has the same affinity for DNA as full-length Pol (Fig. 2B). When N Pol was tested for stimulation by PCNA, a similar behavior to the full-length Pol was observed (Fig. 2A), suggesting that the N-terminal domain is not involved in the interaction with PCNA. Studies with model peptides derived from the catalytic subunit of Pol indicated the presence of a variant of the PCNA-binding motif (residues 142 to 149) still present in N Pol (29–31). However, this domain does not per se appear to be functional, since DNA synthesis by the catalytic subunit of mammalian Pol is not stimulated by PCNA. The two-subunit form of Pol (p125·p48) is required for stimulation by PCNA (7,12,32–34). Confirming these results, immunoblot analysis with monoclonal antibodies showed that the small p50 subunit in N Pol was intact and present at expected levels (Fig. 1C). At present, it is not clear whether PCNA directly interacts with the small p50 subunit or whether the binding of p50 to the p125 subunit leads to a conformational change that increases the interaction of the catalytic subunit with PCNA. Recently, a more complex form of Pol has been isolated from Schizosaccharomyces pombe, with at least four, and possibly even five, subunits (35), and from Saccharomyces cerevisiae (36,37) and human (13), with three subunits. Although the sequence similarity between the three subunits Pol32p (S.cerevisiae), Cdc27 (S.pombe) and p66 (human) is very low, the presence of a PCNA-binding motif (22) in each of these subunits suggests that these three proteins are functional homologs involved in the interaction with PCNA.
When the two Pol forms were tested under conditions where PCNA cannot assemble around DNA by itself, but must be loaded onto DNA by the clamp loader RF-C, N Pol and full-length Pol behaved differently (Figs 3 and 4). The mechanism by which RF-C enables Pol to use primed circular DNA as a template in vitro has been studied in detail (11,38–41). Our results showed that when RF-C is required, the activity of N Pol is only moderately stimulated by PCNA, in contrast to full-length Pol (Fig. 3). This moderate stimulation could be due to the residual amounts of full-length large subunit (see Fig. 1A). Furthermore, we showed that the holoenzyme containing N Pol was significantly less efficient and slower than that containing full-length Pol (Fig. 4), suggesting that the N-terminal part of Pol is involved in the interaction with another protein. Three proteins could be candidates for such an interaction: PCNA, RP-A and RF-C. First, an interaction between the N-terminal part of Pol and PCNA is very unlikely, since both forms of Pol are similarly stimulated by PCNA on a poly(dA)/oligo(dT) template. Second, a specific interaction between the N-terminal part of Pol and RP-A was investigated using E.coli SSB: the polymerase activities in the RF-C-dependent Pol assay of both full-length and N Pol were the same when SSB was used instead of RP-A (compare Fig. 3A and B). Consequently, the fact that N Pol could be only moderately stimulated by PCNA in the RF-C-dependent holoenzyme assay was not due to loss of a specific interaction between the N-terminal part of Pol and RP-A. However, it does not eliminate the possibility that N Pol is no longer able to interact with both E.coli and human SSB. The fact that Pol has been shown to interact directly with the p70 subunit of RP-A favors this hypothesis (42). The third possibility is that the N-terminal part of the large subunit of Pol might interact with RF-C. This conclusion is supported by the previous isolation of a multiprotein complex active in DNA replication containing at least Pol, Pol and RF-C from calf thymus (21) and human cells (43), suggesting a direct interaction between Pol and RF-C. Furthermore, Pol has been shown to interact directly with RF-C (42), consistent with interaction between Pol and the p40 subunit of RF-C (13,44). The role of RF-C in the holoenzyme complex (beyond its role of loading PCNA) seems to be dependent on the presence of RP-A, via interactions between three RF-C subunits (p140, p40 and p38) and the p70 subunit of RP-A, as RF-C does not remain in the holoenzyme if E.coli SSB is substituted for RP-A (45) but is not released from RP-A-coated DNA (42,46). This report shows that the N-terminal part of the large subunit of Pol is important for holoenzyme function, probably through its interaction with RF-C and/or RP-A. Such an interaction could play a role in stabilization of the Pol holoenzyme (Pol, RF-C and PCNA) at the mammalian DNA replication fork. Recently, evidence that Pol can be mutated in human colon cancer cells, particularly those having no mismatch repair defects, has been presented (47). Six of 19 mutations were localized in the first 119 amino acids of the N-terminal part of the Pol large subunit, underlining the important role played by this region that we propose interacts with RF-C and/or RP-A.
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ACKNOWLEDGEMENTS |
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We thank Aengus Mac Sweeney and Zophonías O. Jónsson for critical reading of the manuscript. We thank Paul Fischer and Kathleen Downey for providing the antisera against the the N- and C-terminal parts of Pol
and Alexander Yuzhakov for a copy of his manuscript in press. This work was supported by the UBS, ‘im Aufrag eines Kunden’ to S.B., by the Swiss Cancer League (grant KFS 164-9-1995) to M.S. and by the Kanton of Zürich.
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FOOTNOTES |
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* To whom correspondence should be addressed. Tel: +41 1 635 5472; Fax: +41 1 635 6840; Email: hubscher@vetbio.unizh.ch
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  S. C. Alley, M. A. Trakselis, M. U. Mayer, F. T. Ishmael, A. D. Jones, and S. J. Benkovic Building a Replisome Solution Structure by Elucidation of Protein-Protein Interactions in the Bacteriophage T4 DNA Polymerase Holoenzyme J. Biol. Chem., October 12, 2001; 276(42): 39340 - 39349. [Abstract] [Full Text] [PDF]
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 J. Huang and D. B. Levin Expression, purification and characterization of the Spodoptera littoralis nucleopolyhedrovirus (SpliNPV) DNA polymerase and interaction with the SpliNPV non-hr origin of DNA replication J. Gen. Virol., July 1, 2001; 82(7): 1767 - 1776. [Abstract] [Full Text] [PDF]
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V. L. Gerlach, W. J. Feaver, P. L. Fischhaber, and E. C. Friedberg Purification and Characterization of polkappa , a DNA Polymerase Encoded by the Human DINB1 Gene J. Biol. Chem., January 5, 2001; 276(1): 92 - 98. [Abstract] [Full Text] [PDF]
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H. J. Einolf and F. P. Guengerich Fidelity of Nucleotide Insertion at 8-Oxo-7,8-dihydroguanine by Mammalian DNA Polymerase delta . STEADY-STATE AND PRE-STEADY-STATE KINETIC ANALYSIS J. Biol. Chem., February 2, 2001; 276(6): 3764 - 3771. [Abstract] [Full Text] [PDF]
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