KtG'G
Molec. gen. Genet. 145, 215-218 (1976)
© by Springer-Vertag 1976
The Influence of DNA Binding Protein on the Substrate Affinities of DNA Polymerase from Ustilago maydis: One Polymerase Implicated in Both DNA Replication and Repair Geoffrey T. Yarranton, Peter D. Moore, and Adonis Spanos National Institute for Medical Research, Mill Hill, London NW7 1AA, England
Summary. The DNA polymerase of Ustilago maydis is stimulated by a DNA binding protein from the same organism. Analysis of this stimulation shows that there is an increase in affinity for both substrates of the reaction. The apparent Km for deoxynucleoside triphosphates is decreased 3 fold, and that for denatured DNA by 4 fold. In both cases the maximum velocity (Vmax) is increased 1.2 to 1.4 fold. It is suggested that the variability in the affinity of the enzyme for deoxynucleoside triphosphates mediated by the binding protein may provide the basis for the UV sensitivity of pyrimidine auxotrophs in this organism.
apparent Michaelis constant (Km) for deoxynucleoside triphosphates over a range of concentrations which would allow DNA replication to proceed in the absence of DNA repair synthesis in pyrimidine auxotrophs.
Materials and Methods 1. DNA and Enzymes DNA polymerase (Fraction V) was purified by the method of Banks et al. (1976), and the DNA binding protein by the method of Banks and Spanos (1975). Calf thymus DNA was obtained from Worthington Biochemicals, and either heat denatured or 'activated' using pancreatic DNase.
Introduction 2. DNA Polymerase Assays
The UV sensitivity found associated with pyrimidine auxotrophy in the fungus Ustilago maydis was originally suggested to be due to a difference in the affinity for pyrimidine nucleoside triphosphates between DNA polymerases required for repair and chromosomal replication (Holliday, 1965). It has since been shown that the pyr 1-1 mutant has extremely low levels of thymidine nucleotides, and the repair deficiency in this mutant is consistent with it being unable to successfully complete repair synthesis of DNA (Moore, 1975). A direct prediction from this hypothesis was that at least two DNA polymerases exist i n this organism and that they differ in their affinity for deoxynucleoside triphosphates. An alternative explanation is that a single enzyme performs both repair synthesis and chromosome replication and that its affinity for deoxynucleoside triphosphates is greater during replication than during repair synthesis. We present evidence here that an interaction of the D N A polymerase from U. maydis with a DNA binding protein from the same organism alters the
DNA polymerase assays were performed at 37° as described by Banks et al. (1976). Where assays contained DNA binding protein, the DNA was preincubated with the binding protein at 0° C, before addition to the assay mixture. Measurements of the apparent Km for dTTP were made in the presence of an excess of the three other deoxynucleoside triphosphates.
Results
Recently, a DNA binding protein which binds specifically to single stranded DNA has been isolated from U. maydis (Banks and Spanos, 1975). This protein stimulates the DNA polymerase activity from the same organism 3 to 5 fold when a denatured DNA template is used, but not when a native DNA template, nicked with pancreatic DNase I (activated DNA) is used. No stimulation is observed if the polymerase from U. maydis is replaced by those from: Escherichia coli (DNA polymerase I), Micrococcus luteus, T4 or T7 bacteriophages.
216
G.T. Yarranton et al. : DNA Binding Protein and DNA Polymerase
0.08 0.06
0.04
0.04
°/ ~ o~° , °
>-
0.02
0
-2
0 (1/[TTP]),uM-1
I
2
I
4
o
/
-+"~/
-0,03
~
-0,02
~
-0,01
0
0.0]
0,02
(1/[DNA])~M-1
Fig. 1. The effect of DNA binding protein on the kinetics of DNA polymerase activity on denatured DNA template in the presence of limiting concentrations of TTP (double reciprocal plot). V, the velocity of the reaction is measured as pmoles total nucleotide incorporated per 20 rain: o - - o in absence of binding protein; o - - o in presence of binding protein
Fig. 3. The effect of DNA binding protein on the kinetics of DNA polymerase activity in the presence of limiting concentrations of denatured DNA: o - - o in absence of binding protein; o - - o in presence of binding protein
Table 1. Kinetic constants measured for limiting concentrations of deoxynucleoside triphosphate concentrations Binding protein
Km (gM)
V~x pmoles incorporated/20 min
Denatured DNA (40 gM)
+
1.1 0.3
26.7 41.3
Activated DNA (30 ~tM)
+
1.2 1.2
62.5 62.5
0.08
0.04
Table 2. Kinetic constants measured for limiting concentrations of denatured DNA
0
•
I
-1
Y
I
0
2
I
/.,
Binding protein
Km (IaM)
Vm,x pmoles incorporated/20 min
+
125 31.3
40 50
(1/[TTP])~uNq Fig. 2. The effect of DNA binding protein on the kinetics of DNA polymerase activity on an activated DNA template in the presence of limiting concentrations of TTP: © - - o in absence of binding protein; o - - o in presence of binding protein
Denatured DNA
Stimulation of the U. maydis DNA polymerase activity by the binding protein seems to involve a decrease in the apparent Km values for both substrate molecules. The affinity for denatured DNA is increased 4 fold with a 1.2 fold increase in the Vmax, whilst the apparent affinity for dTTP is increased 3 to 4 fold and the Vmax 1.5 fold when denatured DNA is the template (Figs. 1 and 3, Tables 1 and 2). Similar results have been obtained with dATP. Synthesis on an activated DNA template, however, is not stimulated by binding protein; neither are the kinetic constants altered (Fig. 2, Table 1).
Discussion Higher eukaryotic organisms are reported to posses at least three D N A polymerases (c~,/3 and 7), as well as a discrete mitochondrial enzyme (Weissbach et al., 1975), and some authors have attempted to assign specific roles to these enzymes based on their affinity for deoxynucleoside triphosphates (Edenberg and Huberman, 1975; Laipis and Levine, 1973). The 7 polymerase, which has a high affinity for deoxynucleoside triphosphates, has been implicated in Okazaki fragment synthesis, whilst the ~ and/3 polymerases may
G.T. Yarranton et al. : DNA BindingProtein and DNA Polymerase be involved in repair and gap filling (Edenberg and Huberman, 1975). Simple eukaryotes may differ from higher organisms in the number of distinct DNA polymerases that they possess. The ascomycete fungus Saccharomyces cerevisiae is reported to posses two discrete DNA polymerases (A and B), as well as a discrete mitochondrial enzyme (Wintersberger, 1974), whilst Tetrahymena pyriformis seems to possess a single activity (Crerar and Pearlman, 1974). The basidiomycete fungus Ustilago maydis also seems to possess only a single DNA polymerase activity in mitotic cells (Jeggo and Banks, 1976; Banks et al., 1976). The DNA polymerase activity from U. maydis has been purified to homogeneity and its stimulation by a DNA binding protein from the same organism studied. Synthesis on a denatured calf thymus DNA template is stimulated 3 to 5 fold by the binding protein, and analysis of the nature of this stimulation has shown that the affinity of the polymerase for both substrate molecules is increased. The apparent Km for deoxynucleoside triphosphates is reduced from 1.1g M to 0.3g M and there is a 1.5 fold increase in the Vmax, and the apparent Km for denatured DNA is reduced from 125gM to 31.2gin with a 1.2 fold increase in the Vmax. If activated DNA is used as template, however, no stimulation by binding protein is observed. If we consider that the only difference between the two DNA templates is the length of single stranded regions, then several postulates can be made from these results. Firstly, since DNA binding protein probably binds cooperatively to DNA, most of the denatured DNA will be saturated with binding protein, whilst the short single stranded regions of activated DNA will not. Secondly, the increase in Vmax observed in the presence of binding protein probably reflects the increased strength of polymerase binding to DNA, and also the removal of secondary structure barriers to chain extension. Thirdly, since the apparent Km for deoxynucleoside triphosphates is altered by the binding protein, some physical interaction between DNA binding protein and polymerase is implied. However, since this alteration in Km is dependent upon the nature of the template, we suggest that the interaction is between DNA polymerase and binding protein complexed to DNA rather than between DNA polymerase and free binding protein. Fourthly, the increased affinity of the polymerase for denatured DNA in the presence of binding protein may cause the increased affinity for deoxynucleoside triphosphates. It has been shown for DNA polymerase I of Escherichia coli that the affinity for deoxynucleoside triphosphates is greater when a DNA template is present (McClure and Jovin, 1975). Hence,
217 by increasing the binding affinity of the DNA template, there might be a concomitant increase in the binding affinity of deoxynucleoside triphosphates. Since U. maydis seems to posses a single DNA polymerase activity, the ability of this enzyme to alter its affinity for deoxynucleoside triphosphates when synthesising on different types of templates may be of physiological importance. We suggest that repair and replicative DNA synthesis may be catalysed by the same DNA polymerase in U. maydis, and that the two modes of operation are distinguished from one another by their requirement for deoxynucleoside triphosphate concentration. Repair synthesis in vivo may occur on the type of template represented by activated DNA in vitro. Synthesis on this template probably consists of the addition of a few nucleotides without the displacement of preceeding DNA chains. The apparent Km for deoxynucleoside triphosphates in this type of synthesis is 1.1 ~ M. Chromosomal replication in vivo, however, may proceed on a template more nearly represented by denatured DNA in association with binding protein; this type of synthesis in vitro has a Km for deoxynucleoside triphosphates of 0.3pM. Hence, we suggest that DNA binding protein may mediate a change in the Km for deoxynucleoside triphosphate, and that this allows DNA repair synthesis and chromosomal replication to be distinguished from one another. Evidence for the physiological importance of nucleotide levels within the cell comes from the data of Moore (1975). The intracellular concentrations of dTTP can be estimated for wild-type and pyr 1-1 strains of U. maydis. In the wild-type it is 5p M, whilst in the pyrimidine mutant it is less than 0.75p M. (These calculations are based on the assumption that the dTTP is distributed evenly both within and between cells in a log phase population). From the values obtained, it can be seen that in the wild-type both DNA repair and replication can proceed normally, whilst in the repair defective pyr 1-1 mutant the level of dTTP is low, and repair synthesis severely restricted in comparison with chromosomal replication,
Acknowledgements. We are indebted to Dr. R. Holliday and Dr. G.R. Banksfor valuableadviceand discussion. References
Banks, G.R., Holloman,W.K., Kairis, M.V., Spanos, A., Yarranton, G.T. : A DNA polymerasefrom Ustilagomaydis. I. Purification and properties of the polymeraseactivity.Europ. J. Biochem. 62, 131-142 (1976) Banks, G.R., Spanos, A. : The isolationand properties of a DNA unwinding protein from Ustilago maydis. J. molec. Biol. 93, 63-77 (1975)
218 Crerar, M., Pearlman, R.E. : Deoxyribonucleic acid polymerase from Tetrahymena pyriformis. Purification and properties of the major activity in exponentially growing cells. J. biol, Chem. 249, 3123-3131 (1974) Edenberg, H.J., Huberman, J.A. : Eukaryotic chromosome replication. Ann. Rev. Gen. 9, 245-280 (1975) Holliday, R. : Radiation sensitive mutants of Ustilago maydis. Mutation Res. 2, 557-559 (1965) Jeggo, P.A., Banks, G,R. : DNA polymerase from Ustilago maydis: Partial characterization of the enzyme and a pol 1 mutation. Molec. gen. Gen. 142, 209-224 (1976) Laipis, P.J., Levine, A.J. : DNA replication in SV40 infected cells. IX. The inhibition of a gap-filling step during discontinuous synthesis of SV40 DNA. Virology 56, 580 -594 (1973) McClure, W.R., Jovin, T.M.: The steady state kinetic parameters and non-processivity of Escherichia coli DNA polymerase I. J. biol. Chem. 250, 40734080 (1975)
G.T. Yarranton et al. : DNA Binding Protein and DNA Polymerase Moore, P.D. : Radiation-sensitive pyrimidine auxotrophs of Ustilago maydis. II. A Study of repair mechanisms and UV recovery in pyr 1. Mutation Res. 28, 367-380 (1975) Weissbach, A., Baltimore, D., Bollum, F., Gallo, R., Korn, D. : Nomenclature of eukaryotic DNA polymerases. Europ. J. Biochem. 59, 1-2 (1975) Wintersberger, E. : Deoxyribonucleic acid polymerases from yeast. Further purification and characterization of DNA-dependent DNA polymerases A and B. Europ. J. Biochern. 50, 4147 (1974)
Communicated by B.A. Bridges Received January 12, 1976
Molec. gen. Genet. 145, 215-218 (1976)
© by Springer-Vertag 1976
The Influence of DNA Binding Protein on the Substrate Affinities of DNA Polymerase from Ustilago maydis: One Polymerase Implicated in Both DNA Replication and Repair Geoffrey T. Yarranton, Peter D. Moore, and Adonis Spanos National Institute for Medical Research, Mill Hill, London NW7 1AA, England
Summary. The DNA polymerase of Ustilago maydis is stimulated by a DNA binding protein from the same organism. Analysis of this stimulation shows that there is an increase in affinity for both substrates of the reaction. The apparent Km for deoxynucleoside triphosphates is decreased 3 fold, and that for denatured DNA by 4 fold. In both cases the maximum velocity (Vmax) is increased 1.2 to 1.4 fold. It is suggested that the variability in the affinity of the enzyme for deoxynucleoside triphosphates mediated by the binding protein may provide the basis for the UV sensitivity of pyrimidine auxotrophs in this organism.
apparent Michaelis constant (Km) for deoxynucleoside triphosphates over a range of concentrations which would allow DNA replication to proceed in the absence of DNA repair synthesis in pyrimidine auxotrophs.
Materials and Methods 1. DNA and Enzymes DNA polymerase (Fraction V) was purified by the method of Banks et al. (1976), and the DNA binding protein by the method of Banks and Spanos (1975). Calf thymus DNA was obtained from Worthington Biochemicals, and either heat denatured or 'activated' using pancreatic DNase.
Introduction 2. DNA Polymerase Assays
The UV sensitivity found associated with pyrimidine auxotrophy in the fungus Ustilago maydis was originally suggested to be due to a difference in the affinity for pyrimidine nucleoside triphosphates between DNA polymerases required for repair and chromosomal replication (Holliday, 1965). It has since been shown that the pyr 1-1 mutant has extremely low levels of thymidine nucleotides, and the repair deficiency in this mutant is consistent with it being unable to successfully complete repair synthesis of DNA (Moore, 1975). A direct prediction from this hypothesis was that at least two DNA polymerases exist i n this organism and that they differ in their affinity for deoxynucleoside triphosphates. An alternative explanation is that a single enzyme performs both repair synthesis and chromosome replication and that its affinity for deoxynucleoside triphosphates is greater during replication than during repair synthesis. We present evidence here that an interaction of the D N A polymerase from U. maydis with a DNA binding protein from the same organism alters the
DNA polymerase assays were performed at 37° as described by Banks et al. (1976). Where assays contained DNA binding protein, the DNA was preincubated with the binding protein at 0° C, before addition to the assay mixture. Measurements of the apparent Km for dTTP were made in the presence of an excess of the three other deoxynucleoside triphosphates.
Results
Recently, a DNA binding protein which binds specifically to single stranded DNA has been isolated from U. maydis (Banks and Spanos, 1975). This protein stimulates the DNA polymerase activity from the same organism 3 to 5 fold when a denatured DNA template is used, but not when a native DNA template, nicked with pancreatic DNase I (activated DNA) is used. No stimulation is observed if the polymerase from U. maydis is replaced by those from: Escherichia coli (DNA polymerase I), Micrococcus luteus, T4 or T7 bacteriophages.
216
G.T. Yarranton et al. : DNA Binding Protein and DNA Polymerase
0.08 0.06
0.04
0.04
°/ ~ o~° , °
>-
0.02
0
-2
0 (1/[TTP]),uM-1
I
2
I
4
o
/
-+"~/
-0,03
~
-0,02
~
-0,01
0
0.0]
0,02
(1/[DNA])~M-1
Fig. 1. The effect of DNA binding protein on the kinetics of DNA polymerase activity on denatured DNA template in the presence of limiting concentrations of TTP (double reciprocal plot). V, the velocity of the reaction is measured as pmoles total nucleotide incorporated per 20 rain: o - - o in absence of binding protein; o - - o in presence of binding protein
Fig. 3. The effect of DNA binding protein on the kinetics of DNA polymerase activity in the presence of limiting concentrations of denatured DNA: o - - o in absence of binding protein; o - - o in presence of binding protein
Table 1. Kinetic constants measured for limiting concentrations of deoxynucleoside triphosphate concentrations Binding protein
Km (gM)
V~x pmoles incorporated/20 min
Denatured DNA (40 gM)
+
1.1 0.3
26.7 41.3
Activated DNA (30 ~tM)
+
1.2 1.2
62.5 62.5
0.08
0.04
Table 2. Kinetic constants measured for limiting concentrations of denatured DNA
0
•
I
-1
Y
I
0
2
I
/.,
Binding protein
Km (IaM)
Vm,x pmoles incorporated/20 min
+
125 31.3
40 50
(1/[TTP])~uNq Fig. 2. The effect of DNA binding protein on the kinetics of DNA polymerase activity on an activated DNA template in the presence of limiting concentrations of TTP: © - - o in absence of binding protein; o - - o in presence of binding protein
Denatured DNA
Stimulation of the U. maydis DNA polymerase activity by the binding protein seems to involve a decrease in the apparent Km values for both substrate molecules. The affinity for denatured DNA is increased 4 fold with a 1.2 fold increase in the Vmax, whilst the apparent affinity for dTTP is increased 3 to 4 fold and the Vmax 1.5 fold when denatured DNA is the template (Figs. 1 and 3, Tables 1 and 2). Similar results have been obtained with dATP. Synthesis on an activated DNA template, however, is not stimulated by binding protein; neither are the kinetic constants altered (Fig. 2, Table 1).
Discussion Higher eukaryotic organisms are reported to posses at least three D N A polymerases (c~,/3 and 7), as well as a discrete mitochondrial enzyme (Weissbach et al., 1975), and some authors have attempted to assign specific roles to these enzymes based on their affinity for deoxynucleoside triphosphates (Edenberg and Huberman, 1975; Laipis and Levine, 1973). The 7 polymerase, which has a high affinity for deoxynucleoside triphosphates, has been implicated in Okazaki fragment synthesis, whilst the ~ and/3 polymerases may
G.T. Yarranton et al. : DNA BindingProtein and DNA Polymerase be involved in repair and gap filling (Edenberg and Huberman, 1975). Simple eukaryotes may differ from higher organisms in the number of distinct DNA polymerases that they possess. The ascomycete fungus Saccharomyces cerevisiae is reported to posses two discrete DNA polymerases (A and B), as well as a discrete mitochondrial enzyme (Wintersberger, 1974), whilst Tetrahymena pyriformis seems to possess a single activity (Crerar and Pearlman, 1974). The basidiomycete fungus Ustilago maydis also seems to possess only a single DNA polymerase activity in mitotic cells (Jeggo and Banks, 1976; Banks et al., 1976). The DNA polymerase activity from U. maydis has been purified to homogeneity and its stimulation by a DNA binding protein from the same organism studied. Synthesis on a denatured calf thymus DNA template is stimulated 3 to 5 fold by the binding protein, and analysis of the nature of this stimulation has shown that the affinity of the polymerase for both substrate molecules is increased. The apparent Km for deoxynucleoside triphosphates is reduced from 1.1g M to 0.3g M and there is a 1.5 fold increase in the Vmax, and the apparent Km for denatured DNA is reduced from 125gM to 31.2gin with a 1.2 fold increase in the Vmax. If activated DNA is used as template, however, no stimulation by binding protein is observed. If we consider that the only difference between the two DNA templates is the length of single stranded regions, then several postulates can be made from these results. Firstly, since DNA binding protein probably binds cooperatively to DNA, most of the denatured DNA will be saturated with binding protein, whilst the short single stranded regions of activated DNA will not. Secondly, the increase in Vmax observed in the presence of binding protein probably reflects the increased strength of polymerase binding to DNA, and also the removal of secondary structure barriers to chain extension. Thirdly, since the apparent Km for deoxynucleoside triphosphates is altered by the binding protein, some physical interaction between DNA binding protein and polymerase is implied. However, since this alteration in Km is dependent upon the nature of the template, we suggest that the interaction is between DNA polymerase and binding protein complexed to DNA rather than between DNA polymerase and free binding protein. Fourthly, the increased affinity of the polymerase for denatured DNA in the presence of binding protein may cause the increased affinity for deoxynucleoside triphosphates. It has been shown for DNA polymerase I of Escherichia coli that the affinity for deoxynucleoside triphosphates is greater when a DNA template is present (McClure and Jovin, 1975). Hence,
217 by increasing the binding affinity of the DNA template, there might be a concomitant increase in the binding affinity of deoxynucleoside triphosphates. Since U. maydis seems to posses a single DNA polymerase activity, the ability of this enzyme to alter its affinity for deoxynucleoside triphosphates when synthesising on different types of templates may be of physiological importance. We suggest that repair and replicative DNA synthesis may be catalysed by the same DNA polymerase in U. maydis, and that the two modes of operation are distinguished from one another by their requirement for deoxynucleoside triphosphate concentration. Repair synthesis in vivo may occur on the type of template represented by activated DNA in vitro. Synthesis on this template probably consists of the addition of a few nucleotides without the displacement of preceeding DNA chains. The apparent Km for deoxynucleoside triphosphates in this type of synthesis is 1.1 ~ M. Chromosomal replication in vivo, however, may proceed on a template more nearly represented by denatured DNA in association with binding protein; this type of synthesis in vitro has a Km for deoxynucleoside triphosphates of 0.3pM. Hence, we suggest that DNA binding protein may mediate a change in the Km for deoxynucleoside triphosphate, and that this allows DNA repair synthesis and chromosomal replication to be distinguished from one another. Evidence for the physiological importance of nucleotide levels within the cell comes from the data of Moore (1975). The intracellular concentrations of dTTP can be estimated for wild-type and pyr 1-1 strains of U. maydis. In the wild-type it is 5p M, whilst in the pyrimidine mutant it is less than 0.75p M. (These calculations are based on the assumption that the dTTP is distributed evenly both within and between cells in a log phase population). From the values obtained, it can be seen that in the wild-type both DNA repair and replication can proceed normally, whilst in the repair defective pyr 1-1 mutant the level of dTTP is low, and repair synthesis severely restricted in comparison with chromosomal replication,
Acknowledgements. We are indebted to Dr. R. Holliday and Dr. G.R. Banksfor valuableadviceand discussion. References
Banks, G.R., Holloman,W.K., Kairis, M.V., Spanos, A., Yarranton, G.T. : A DNA polymerasefrom Ustilagomaydis. I. Purification and properties of the polymeraseactivity.Europ. J. Biochem. 62, 131-142 (1976) Banks, G.R., Spanos, A. : The isolationand properties of a DNA unwinding protein from Ustilago maydis. J. molec. Biol. 93, 63-77 (1975)
218 Crerar, M., Pearlman, R.E. : Deoxyribonucleic acid polymerase from Tetrahymena pyriformis. Purification and properties of the major activity in exponentially growing cells. J. biol, Chem. 249, 3123-3131 (1974) Edenberg, H.J., Huberman, J.A. : Eukaryotic chromosome replication. Ann. Rev. Gen. 9, 245-280 (1975) Holliday, R. : Radiation sensitive mutants of Ustilago maydis. Mutation Res. 2, 557-559 (1965) Jeggo, P.A., Banks, G,R. : DNA polymerase from Ustilago maydis: Partial characterization of the enzyme and a pol 1 mutation. Molec. gen. Gen. 142, 209-224 (1976) Laipis, P.J., Levine, A.J. : DNA replication in SV40 infected cells. IX. The inhibition of a gap-filling step during discontinuous synthesis of SV40 DNA. Virology 56, 580 -594 (1973) McClure, W.R., Jovin, T.M.: The steady state kinetic parameters and non-processivity of Escherichia coli DNA polymerase I. J. biol. Chem. 250, 40734080 (1975)
G.T. Yarranton et al. : DNA Binding Protein and DNA Polymerase Moore, P.D. : Radiation-sensitive pyrimidine auxotrophs of Ustilago maydis. II. A Study of repair mechanisms and UV recovery in pyr 1. Mutation Res. 28, 367-380 (1975) Weissbach, A., Baltimore, D., Bollum, F., Gallo, R., Korn, D. : Nomenclature of eukaryotic DNA polymerases. Europ. J. Biochem. 59, 1-2 (1975) Wintersberger, E. : Deoxyribonucleic acid polymerases from yeast. Further purification and characterization of DNA-dependent DNA polymerases A and B. Europ. J. Biochern. 50, 4147 (1974)
Communicated by B.A. Bridges Received January 12, 1976
