Properties of the Escherichia coli DNA Binding (Unwinding) Protein: Interaction with DNAPolymerase and DNAAuthor(s): Ian J. Molineux and Malcolm L. GefterSource: Proceedings of the National Academy of Sciences of the United States of America,Vol. 71, No. 10 (Oct., 1974), pp. 3858-3862Published by: National Academy of SciencesStable URL: http://www.jstor.org/stable/64082 .

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Proc. Nat. Acad. Sci. USA Vol. 71, No. 10, pp. 3858-3862, October 1974

Properties of the Escherichia coli DNA B: Interaction with DNA Polymerase and DF

(single-strand-specific nuclease/protein. protein and DNA

IAN J. MOLINEUX AND MALCOLM L. GEFTER

Department of Biology, Massachusetts Institute of Technology, Cambric

Communicated by Boris Magasanik, July 12, 1974

ABSTRACT The E. coli DNA binding protein reduces the activity of the single-strand-specific nucleases associ- ated with all three DNA polymerases known in E. coli. A slight excess of binding protein over that required to saturate the DNA template leads to total inhibition of activity of the 3' - 5' nucleases associated with DNA polymerases I and III, but restores maximum activity of the DNA polymerase II-associated nuclease. The binding protein forms a specific complex with DNA polymerase II in the absence of DNA, and it is this complex that degrades a DNA binding protein complex. Binding protein also facilitates the binding of DNA polymerase II to single- stranded DNA, whereas the binding to DNA of DNA polym- erase I is inhibited. These data may explain the specificity with which the binding protein enhances the synthetic ability of DNA polymerase 1I.

The DNA binding protein of E. coli specifically stimulates the synthetic activity of E. coli DNA polymerase II and re- duces the synthetic activities of both E. coli DNA polymerases I and III (1, 2). Analogous specific stimulations of DNA

polymerase activity have been reported for the bacteriophage T7-induced DNA polymerase and the T7 DNA binding protein (3) and also for the bacteriophage T4-induced DNA

polymerase and the T4 gene-32 protein (4). In the latter case of the T4 proteins, it was also shown that the DNA polymerase and gene-32 protein interact in the absence of DNA to form a

specific protein-protein complex, and it was suggested that

this complex formation may be the reason for the observed

specificity of the stimulation in the rate of DNA synthesis in vitro. In contrast to the enhancement of the synthetic activity of T4-induced DNA polymerase, the activity of the

single-strand-specific nuclease associated with the poly- merase is reduced by the presence of the gene-32 protein (5).

The three DNA polymerases of E. coli each has an asso- ciated nuclease that catalyzes the exonucleolytic digestion of single-stranded DNA in the 3' -- 5' direction (6-8), and it was of interest to study the effects of the binding protein on nuclease activity. The results reported here show that the

single-strand-specific nucleases associated with DNA poly- merases I and III are reduced in activity by the presence of the binding protein. In contrast, the nuclease associated with DNA polymerase II is either inhibited or stimulated in

activity by the binding protein, depending on the concen- tration of protein present. These results were consistent with

the formation of a DNA polymerase II-DNA binding pro- tein complex that is capable of digesting single-strapded DNA that itself is covered with binding protein.

In this report we discuss the formation and stability of this complex between DNA polymerase II and binding

38

inding (Unwinding) Protein: [A L protein interactions)

ge, Mass. 02139

protein, and, in addition, show that DNA polymerase II will bind to single-stranded DNA in the presence of binding protein. Data will also be presented showing the effects of binding protein on the affinities of DNA polymerases I and III for single-stranded DNA.

The results presented here give further insight into the apparent specificity of the binding protein in stimulating the synthetic activity of DNA polymerase II.

MATERIALS AND METHODS

[3H]Polydeoxythymidylic acid [poly(dT)] was synthesized by terminal transferase with (dT)4 as primer; the incubation mixture contained 0.2 M potassium cacodylate (pH 7.0), 2 mM CoC12, 0.75 iML (dT)4, 2 mM [3H]dTTP (100 cpm/ pmole), 0.5 mg/ml of bovine serum albumin, and enzyme. The mixture was incubated at 37? for 24 hr, and the product was isolated by gel filtration through Sephadex G-100. DNA polymerase I (18,000 units/mg) was prepared by the method of Jovin et al. (9); DNA polymerase II (250 units/ mg) was prepared as described (8), followed by chromatog-

raphy on phosphocellulose (10); DNA polymerase III

(>10,000 units/mg) was prepared as described (8); DNA

binding protein was purified through DNA-cellulose and DEAE-Sephadex as described (2) and was free of detectable nucleolytic activity. Bacteriophage fd DNA was prepared by phenol-sodium dodecyl sulfate extraction of purified phage. fd [14C]DNA (40 cpm/pmole) was prepared by grow- ing the phage on a thymine-requiring host in the presence of [14C]thymine.

Glycerol gradient analysis of binary and ternary complexes was by sedimentation through glycerol (10-30%, v/v) in 67 mM Tris HCI (pH 7.6), 6.7 mM MIgCl2, 10 mM 2-mer- captoethanol. Centrifugation was at 49,000 rpm in a Spinco SW 50.1 rotor (Beckman Instruments) at 4?. Duration of centrifugation is given in the figure legends; 0.17-ml fractions were collected from the bottom of the tube and aliquots used for assay of enzyme activity.

Nuclease assays were performed in an incubation mixture (0.1 ml) containing 67 mM Tris HCl (pH 8.0), 6.7 mM Mg- C12, 10 mM 2-mercaptoethanol, 10 jM [3H]poly(dT), and DNA binding protein in varying concentrations. Incubation was for 5 min at 30?, and the reaction was terminated by precipitation with 5% perchloric acid, with denatured calf thymus DNA (250 jg/ml) as carrier. The acid-soluble radio- active nucleotides remaining in the supernatant after centrif- ugation were counted after neutralization of the acid in Bray's solution. The amount of DNA polymerase used was

58

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Proc. Nat. Acad. Sci. USA 71 (1974)

.0

- 2 4

x 1

o co

Er0.5 a_

r-I

0 2 4 Binding Protein (nug)

FIG. 1. Degradation of [3H]poly(dT) by DNA polymerase in the presence of the binding protein. 0, DNA polymerase I; O, DNA polymerase II; *, DNA polymerase III.

predetermined as that which gave a maximal rate of diges- tion in the absence of binding protein under assay conditions.

RESULTS AND DISCUSSION

The activity of the 3' -- 5' single-strand-specific nuclease of DNA polymerase I is reduced in the presence of binding protein, as is the 3' - 5' nuclease of DNA polymerase III (Fig. 1). At a ratio of one binding protein monomer (22,000 daltons), to 10 nucleotides, both of these enzyrmes are in- hibited completely. The activity of the 3' - 5' nuclease of DNA polymerase II, however, is reduced in the presence of low concentrations of binding protein, but at higher concen-

trations, nuclease activity is apparently fully active (Fig. 1). The ratio of binding protein: nucleic acid at which DNA

I I I _

30 0

o0-

cc b 20

E O II

Fraction

o d 0_30

10

Fraction I FIG. 2. Glycerol gradient analysis of DNA polymerase sedimentin

the presence of both 0.4 jig of fd [14C]DNA and 2.5 jig of binding proi tion is plotted from right to left. DNA polymerase was assayed on

polymerase; Panels a and d, DNA polymerase I; panels b and e, DIX

Interactions of the E. coli DNA Binding Protein 3859

polymerase II 3' -- 5' nuclease is least active in this reaction is about one protein monomer (22,000 molecular weight) to 20 nucleotides, and reaches maximum rate again at one protein monomer for 10 nucleotides. Sigal et al. (1) have de- termined, by sedimentation of DNA in the presence of bind- ing protein, that the stoichiometry of binding protein: DNA is 1 protein molecule (monomeric) to 8 nucleotides. There- fore, the minimum rate of digestion of DNA by the 3' -- 5' nuclease of DNA polymerase II appears to be when the DNA is slightly more than half covered with binding protein, while the maximum rate of nucleolytic digestion is re-established at a concentration of binding protein slightly more than is required to saturate the DNA (i.e., free binding protein is present).

The inhibition of DNA polymerase II 3' -- 5' nuclease ac- tivity at low binding protein concentrations can be explained in at least two ways: (i) DNA-binding protein complex is not a good substrate for the enzyme; (ii) some DNA poly- merase II is being sequestered in an inactive configuration by binding protein.

To determine whether DNA polymerase II is able to bind to single-stranded DNA in the presence of binding protein such that nucleolytic digestion is inhibited, we performed the following experiment. Bacteriophage fd [14C]DNA (circular) was mixed with DNA polymerase either in the pres- ence or absence of binding protein and sedimented through a 10-30% linear glycerol gradient. As is shown in Fig. 2b, DNA polymerase II polymerizing activity is only present at the top of the gradient, whereas no activity is seen sediment- ing with DNA when binding protein is absent (i.e., DNA polymerase II does not bind to internal regions of single- stranded DNA). However, when binding protein is present, more than 50% of the DNA polymerase II activity is seen

b c

0

A" I \ 2

0.

e f

20 30 0 10 20 30 Nlumber , in the presence of 0.4 jig of fd [14C] DNA (panels a, b, and c) and in ein (panels d, e, and f). Centrifugation was for 2 hr, and sedimenta- "gapped" calf thymus DNA (8) at 30?. 0, fd [14C]DNA; , I)NA A polymerase II; panels c and f, DNA polymerase III.

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3860 Biochemistry: Molineux and Gefter

a

x 2 E

O

C)

0

o 1 co

i

o 0

o 10 20 30

Fraction

FIG. 3. I)etection of DNA polymerase complexed to binding pr was for 20 hr. Binding protein was assayed by its ability to stimula'

plate (2). DNA polymerase was assayed on "gapped" calf thymus I

mentation of DNA polymerase I (60 units) in the presence (0) or

polymerase II (5 units) in the presence (O) or absence (*) of binding in the presence (0) or absence (0) of binding protein. Internal and e leukemia virus (70,000 daltons) and avian myeloblastosis virus (15

ability to catalyze the synthesis of poly(dG) using poly(rC)- (dG)i2-i1

in panels c and d.

sedimenting with DNA (Fig. 2e). It thus appears that bind- ing protein facilitates the binding between the internal sites on single-stranded DNA and DNA polymerase II. Since the DNA remains circular under these conditions (data not shown), the conclusion is warranted.

1.5 a b

0

E o 1.0

a) co co

n 0.5-

0 2 4 0 2 4 Binding Protein (og)

FIGc. 4. (a) Degradation of [3H]poly(dT) by DNA polymerase II in the presence of binding protein. The standard reaction mixture was supplemented by addition of KC1. *, No KC1; O, 0.02 AM KCl; o, 0.05 M KC1; ?, 0.1 M KC1. (b) Degradation of [3H]poly(dT) toy I)NA polymerase I (closed symbols) or DNA polymerase III (open symbols) in the presence of binding protein. The standard reaction mixture (0, 0) was supplemented by the addition of 0.1 M KC1 (?, m[).

Proc. Nat. Acad. Sci. USA 71 (1974)

: I

E E 0.

C

O 10 20 30 C d

5 E 0. V o

) 10 20 30 Number

?tein by sedimentation through glycerol gradients. Centrifugation ,e DNA polymerase II activity at 12? with poly(dA)- (dT)lo as tern- )NA at 30?. (a) Sedimentation of binding protein (37 jg). (b) Sedi- absence (0) of binding protein (37 jg). (c) Sedinmentation of I)NA protein (37 jig). (d) Sedimentation of D)NA polymerase III (5 units) xternal markers of the RNA-directed DNA polymerase from murine

3,000 daltons) were included in the gradients and assayed by their as template. Only the 70,000-dalton enzyme was included internally

Similar experiments of looking at the effects of binding protein on the binding of the other DNA polymerases of E. coli to single-stranded DNA are also shown in Fig. 2. DNA polymerase I has been shown to bind to internal regions of single-stranded DNA (6), and this indeed was observed under our conditions (Fig. 2a). However, in the presence of binding protein (Fig. 2d), much less enzyme activity remains bound, while the majority sediments slowly, characteristic of free DNA polymerase I. The residual DNA polymerase activity that sediments with the DNA under these conditions can, in fact, be completely eliminated (data not shown) by a higher concentration of binding protein (protein:nucleic acid equal to 10:1, w/v). DNA polymerase I, therefore, has a reduced

affinity for single-stranded DNA in the presence of binding

protein. DNA polymerase III, however, does not appear to bind to single-stranded DNA, as measured by activity either

in the absence (Fig. 2c) or the presence (Fig 2f) of binding

protein. The inhibition of DNA polymerase II 3' - 5' nuclease

activity by low c toncentrations of binding protein may therefore be explained either in terms of the formation of a ternary complex of DNA binding protein-DNA polymerase II, where te latter enzyme is bound to internal regions of the DNA , a is, therefore, unable to digest the DNA exo- nucleolytically, or by the formation of a DNA binding protein complex that is not a substrate for DNA polymerase II 3' -

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Proc. Nat. Acad. Sci. USA 71 (1974)

2

x 1

Q011

co

CD

I

0 10 Fraction

FIG. 5. (a) Glycerol gradient analysis of DNA polymerase II sedi binding protein. Centrifugation was for 2 hr, and sedimentation is

"gapped" calf thymus DNA. *, DNA polymerase II; 0, binding prot marker. (b) As in panel a, except that the glycerol gradient was mat

5' nuclease. The inhibition of nucleolytic activity of DNA

polymerases I and III by binding protein cannot, however, be explained by a similar ternary complex. These enzymes may simply not utilize DNA- binding protein as a substrate for exonucleolytic digestion or, with particular reference to DNA polymerase I, the ability of the enzyme to bind to DNA * binding protein may be reduced.

The re-attainment of maximum rate of nucleolytic activity of DNA polymerase II in the presence of higher concentrations of binding protein occurs at a ratio of protein to nucleic acid of 10:1 (w/w), or rather more protein than is required to saturate the DNA. It was considered possible, therefore, that free binding protein (i.e., that not complexed to DNA) may complex with DNA polymerase II and that the protein* -

protein complex is able to utilize DNA * binding protein com-

plex as a template for exonucleolytic digestion. To detect

the presence of a complex between binding protein and DNA

polymerase, we mixed the proteins at 0?, incubated them at that temperature for 5 min, and then sedimented them

through a glycerol gradient. Each set of gradients contained at least one internal reference marker and two external mark- ers in order to superimpose polymerase activity from two separate gradients and to help estimate molecular weights.

Interactions of the E. coli DNA Binding Protein 3861

I I

a 4

2o

0.-

0) 20 30 -

C

m b m

C) a)

c4 o

2

0 20 30

Number

nenting in the presence of 0.4 ,g of fd DNA and 3 jug of 35S-labeled

plotted from right to left. DNA polymerase II was assayed on min. The arrow marks the sedimentation of an external fd [14C] DNA [e 0.1 M in KC1.

Fig. 3a shows the sedimentation profile of the binding protein, assayed by its ability to stimulate polymerizing activity of DNA polymerase II (2). The bulk of the protein sediments at an apparent molecular weight of 90,000 and is presumed to be a tetramer of the 22,000 molecular weight protein (2). Binding protein activity was also detected at lower molecu- lar weight estimates, and these species approximate to dimeric and monomeric forms of the protein.

DNA polymerase I and DNA polymerase III sediment as

proteins with apparent molecular weights of 110,000 and

150,000, respectively (Fig. 2b and d, closed circles), and their sedimentation properties are not altered by the presence of the binding protein (open circles). Pure DNA polymerase II, however, sediments with an apparent molecular weight of 120,000, yet when sedimented in the presence of binding protein it has an apparent molecular weight of 140,000. This observation suggests an interaction of the two proteins has occurred, leading to a faster sedimenting species that has a molecular weight compatible with a complex between one DNA polymerase II molecule and one 22,000-dalton binding protein monomer. The DNA polymerase II binding protein was further characterized by isolating the polymerase activity from the DNA polymerase II and binding protein

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3862 Biochemistry: Molineux and Gefter

gradient, iodinating the protein with 125I, and showing, by sodium dodecyl sulfate-acrylamide gel electrophoresis, the

presence of both DNA polymerase II and binding protein (unpublished results of I.J.M.). By the same technique of

glycerol gradient centrifugation followed by sodium dodecyl sulfate-acrylamide gel analysis, no binding protein aggregate larger than the tetrameric form has been detected. Therefore, the presence of binding protein sedimenting with DNA

polymerase II activity implies that an interaction between

binding protein and DNA polymerase II has occurred. There are, thus, at least two competing phenomena pos-

sible in a reaction mixture containing DNA, DNA polymerase II, and binding protein. The DNA polymerase may form a

ternary complex consisting of all three components or it may complex with binding protein alone. To discern the relation-

ships of these complexes to enzyme activity, we examined the sensitivity of the nuclease reaction to the presence of salt.

Fig. 4a shows the effect of various concentrations of KC1 (between 0 and 0.1 M) on the rate of degradation of single- stranded DNA by DNA polymerase II. In the absence of

binding protein, the rate of degradation is slightly stimulated

by the presence of up to 50 mM KC1, in agreement with the stimulation of polymerase activity by salt (10). The reduction in rate of digestion in the presence of low concentrations of

binding protein appears to be independent of the presence of

salt; however, the re-establishment of maximum nuclease

activity in the presence of higher binding protein concentra- tions is salt-sensitive above 0.02 M KC1 and is not observed at all at 0.1 M KC1. The effect of salt on the 3' -- 5' nucleases of DNA polymerases I and III is to reduce the rate of nucleo-

lytic digestion (Fig. 4b). Addition of binding protein to those reactions that contain 0.1 M KC1 is qualitatively the same as in the absence of salt; i.e., that binding protein is inhibitory to the nucleolytic activity of those enzymes.

As would be predicted from the salt sensitivity of the re- establishment of DNA polymerase II 3' -+ 5' nuclease ac-

tivity at higher concentrations of binding protein, the forma- tion of a DNA polymerase II-binding protein binary com-

plex is also salt-sensitive. Sedimentation of DNA polymerase II through glycerol gradients in the presence of 0.1 M KC1 is unaltered by the presence of binding protein (unpublished observation of I.J.M.). The formation of the ternary complex between DNA binding protein and DNA polymerase II, however, is also salt-sensitive. Fig. 5a shows the sedimenta- tion of fd DNA, 35S-labeled binding protein, and DNA poly- merase II activity. In the absence of salt, all the binding protein and most of the DNA polymerase activity sediments

slightly faster than an external marker of fd DNA, and rep- resents the ternary complex. Sedimentation of the same mix- ture through glycerol gradients in the presence of 0.1 M KC1, however, shows all the DNA polymerase II activity remain-

ing at the top of the gradient, whereas all the binding protein remains bound to the DNA (Fig. 5b).

The kinetics of degradation of single-stranded DNA cata- lyzed by DNA polymerase II in the presence of binding

Proc. Nat. Acad. Sci. USA 71 (1974)

protein are qualitatively independent of salt concentration, whereas the formation of the ternary complex is salt-sensitive. The implication of these results is that ternary complex for- mation is not the reason for the inhibition of DNA polymerase II nucleolytic activity by low concentrations of binding protein, as salt would then relieve the initial decrease in activity. It must be concluded, therefore, that DNA poly- merase II, like DNA polymerases I and III, cannot digest a complex between DNA and binding protein. Stimulation of DNA polymerase II nucleolytic activity by higher con- centrations of binding protein is probably a result of the formation of a DNA polymerase II * binding protein complex that is able both to bind to and to digest DNA-binding protein complex.

These data are further evidence for the specificity of the binding protein in its ability to stimulate DNA synthesis catalyzed by DNA polymerase II (1, 2). They further imply that the specificity may be not only in the binding protein- catalyzed conformational change of the DNA substrate, as was suggested by Sigal et al. (1), but also (or even com- pletely) in the DNA polymerase II . binding protein complex.

Two novel features of the DNA binding protein are evident from this study. The protein appears to have two distinct sites, each capable of binding to a macromolecule. One site is specific for single-stranded DNA regardless of its sequence, and the other is specific for DNA polymerase II. Further- more, this feature implies that the protein may serve as a "discriminator" substance with regard to those enzymes capable of interacting with DNA. For example, a DNA coated with unwinding protein makes that DNA a substrate for DNA polymerase II but not of DNA polymerase I. It also prevents RNA polymerase from transcribing single-stranded DNA (2). Thus, this protein may serve a key role in the cell in reactions involving repair, replication, recombination, and transcription.

This work was supported in part by a grant from the National Institutes of Health, 5-ROI-GM20363-02, and in part by a grant from the National Science Foundation, B-36649.

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2. Molineux, I. J., Friedman, S. & Gefter, M. L. (1974) J. Biol. Chem., in press.

3. Reuben, R. C. & Gefter, M. L. (1973) Proc. Nat. Acad. Sci. USA 70, 1846-1850.

4. Huberman, J. A., Kornberg, A. & Alberts, B. M. (1971) J. Mol. Biol. 62, 39-52.

5. Huang, W. M. & Lehman, I. R. (1972) J. Biol. Chem. 247, 3139-3146.

6. Kornberg, A. (1969) Science 163, 1410-1418. 7. Gefter, M. L., Molineux, I. J., Kornberg, T. & Khorana, H.

G. (1972) J. Biol. Chem. 247, 3321-3326. 8. Kornberg, T. & Gefter, M. L. (1972) J. Biol. Chem. 247,

5369-5375. 9. Jovin, T. M., Englund, P. T. & Bertsch, L. L. (1969) J. Biol.

Chem. 244, 2996-3008. 10. Kornberg, T. & Gefter, M. L. (1971) Proc. Nat. Acad. Sci.

USA 68, 761-764.

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