kinetics and mechanism of bal 31 nuclease action on small substrates and single-stranded dna

ELSEVIER Biochimica et Biophysica Acta 1251 (1995) 125-138

BB Biochi~ic~a et Biophysica A~ta

Kinetics and mechanism of BAL 31 nuclease action on small substrates and single-stranded DNA

Tao Lu l, Horace B. Gray Jr. * Department of Biochemical and Biophysical Sciences, University of Houston, Houston, TX 77204-5934, USA

Received 5 April 1995; accepted 27 April 1995

Abstract

Kinetic and mechanistic aspects of the action of two forms of the BAL 31 nuclease (EC 3.1.11) from Alteromonas espejiana on model substrates, small oligonucleotides, larger oligonucleotides and poly[d(A)] have been examined. The minimal oligonucleotide substrate is a 5'-phosphorylated dinucleotide and a phosphodiester not containing a nucleotide residue is not cleaved. Both forms act predominantly in an exonucleolytic fashion on single-stranded DNA polymers in a highly processive manner; however, the mechanism becomes distributive for small oligomers (3-4 nucleotide residues). The direction of attack is from the 5' end, in contrast to the mode of digestion of duplex DNA which involves attack at the 3' termini. An endonucleolytic mode of attack also exists, but at a level 2-3% or less of that of the terminally directed cleavage. Apparent values for the catalytic efficiency of the action on long DNA polymers are too large to fit a simple kinetic scheme involving a direct enzyme-substrate encounter and lead to an interpretation in which nuclease molecules are non-productively bound away from the 5' ends and undergo facilitated diffusion to yield productive (terminally bound) enzyme-substrate complexes.

Keywords: Exonuclease; Endonuclease; Processivity; Facilitated diffusion

1. Introduction

The extracellular nucleases from Alteromonas espe-

j iana consist mostly of two enzyme species derived proteo- lytically from a common precursor [1]. These have been designated the ' s low' (S) and 'fast ' (F) forms because of their relative rates of degradation of linear duplex DNA [2] and are collectively known as BAL 31 nuclease. Studies on these enzymes have focused on the kinetic [2,3] and mechanistic [4] properties of the reaction to shorten duplex DNA from both ends and the ability of the nucleases to cleave endonucleolytically in response to a variety of covalent [5-8] and non-covalent [6,9,10] alterations in duplex DNA. Characterization of their action on single- stranded DNA has been limited to the demonstration that 5'-mononucleotides, but not short oligonucleotides, are found in partial digests [6] and to the determination of apparent kinetic parameters [2]. An endonuclease activity against single-stranded DNA was also evidenced [2].

* Corresponding author. E-mail: [email protected]. Fax: +1 (713) 7438351.

Present address: Department of Molecular Genetics, The University of Texas System Cancer Center, M.D. Anderson Hospital and Tumor Institute, Houston, TX 77030, USA.

0167-4838/95/$09.50 © 1995 Elsevier Science B.V. All rights reserved SSDI 01 67-4838(95)0009 1-7

In this study, the actions of the S and F forms against dinucleotide analogs, oligonucleotides and single-stranded DNA polymers were examined. A 5' ~ 3', highly processive mode of exonuclease action on single-stranded substrates was evidenced which contrasts with the 3'--+ 5', quasi-processive attack on duplex DNA [4]. The kinetic data, analyzed according to a model developed in this work, suggest that the nucleases can bind to DNA segments away from the ends and form productive (terminally bound) complexes without intervening dissociation; this appears to be the first instance in which such an interpretation has been made for an enzymatic reaction based on steady-state kinetic parameters. The results have implica- tions for the overall mechanism of degradation of linear duplex substrates.

2. Materials and methods

2.1. Oligonucleotides

(Bis)-p-nitrophenyl phosphate (~bNO2-p-~bNO 2) and thymidine 3 ' - o r 5'-monophosphate p-nitrophenyl ester (dTp-~bNO 2 or ~bNO2-pdT), d(A) 2, d(T) 2, pd(A) 2 , pd(T) 2 ,

126 T. Lu, ll.B. Gray Jr./Biochimica et Biophysica Acta 1251 (1995) 125-138

pd(A) 3 were from Sigma Chemicals. pd(A)16 and d(GTT- TTCCCAGTCACGAC) (MI 3 ' - 40' sequencing primer, referred to below as 17-mer) were products of Pharmacia LKB and United States Biochemicals, respectively. d(TTA), d(AATT) and d(A) 6 were synthesized on a Biosearch 8700 DNA synthesizer and deblocked by heat- ing at 65°C for 18 h in 14.5 M NH4OH solution. The purity of the synthesized oligomers was more than 95% as estimated from the relative areas of the peaks corresponding to desired compounds and contaminating ultraviolet- absorbing peaks from chromatographic analysis (below).

Determination of concentrations of stock solutions of the ~hNO2-containing compounds, and of the extinction coefficient of p-nitrophenyl phosphate in the nuclease reaction buffer (16.0 X 103 M l c m - t) were as described Ill].

2.2. Enzymes and reaction conditions

BAL 31 nucleases (F and S forms) were purified as described [1,2,11]. Purity was verified by sodium dodecyl suifate-polyacrylamide gel electrophoresis and from the much faster degradation of linear duplex DNA by the F form [12] at the same nuclease concentration. All BAL 31 nuclease reactions and assays were done in standard assay buffer (SAB) (600 mM NaCI, 12.5 mM CaCI z, 12.5 mM MgC1 z, 20 mM Tris-HCl, 1 mM EDTA (pH 8.0)) at 30°C. The ionic strength of SAB (0.69 M) is in the range where optimal nuclease activity occurs [6] and is comparable to that of the enzyme's natural sea water milieu (0.61 M). The divalent metal ion concentrations are sufficient to ensure maximum activity [4]. The nuclease assay, based on the hydrolysis of single-stranded DNA, and the definition of unit nuclease activity are as described [6,13]. The unit definitions of all other enzymes were as stated by the suppliers.

2.3. 3H-labeled poly[d(A)]

3H-labeled d(A),, was synthesized using terminal de- oxynucleotidyl transferase using modifications [1 I] of ex- isting procedures [14,15]. The d(A),, was purified elec- trophoretically [14], extracted with phenol/chloroform and precipitated with ethanol. 32p end-labeled aliquots displayed detectable material in the range from 450 to 510 nucleotides (number-average molecular size was 480 nucleotides) upon polyacrylamide gel electrophoresis with denatured 32p-labeled molecular weight markers ('1 kb' DNA 'ladder' from Gibco BRL). The extinction coefficient for d(A),, at 260 nm was taken to be 9.45 X 10 3 M-~cm -1 [16]. The molar activity of the d(A)450_510, denoted d(A)4~ in the following, was 36.2 Ci/mol of nucleotide.

2.4. Terminal labeling

The d(A)4~ 6 polymer, pd(A)j6 and 17-mer were 5' end-labeled with [y-3zp]ATP (4500 Ci/mmol) and T4

polynucleotide kinase and purified as described [11]. The samples were dried after purification and resuspended in TE buffer (20 mM Tris-HC1, 1 mM EDTA (pH 8.0)) and non-labeled oligonucleotides were added to obtain a desired specific activity.

The pd(A)j6 and 17-mer were labeled with 32p at the 3' end with [a-3: P]dATP using the terminal transferase reaction and an approximately four-fold molar excess of the oligonucleotide over dATP, thus creating pd(A)~7 and an 18-mer, respectively. An aliquot of the 3'-labeled 17-mer was 5'-phosphorylated in the presence of a 5-fold molar excess of non-labeled ATP.

2.5. Quantitation of radioactir, ity in polyacrylamide gels

Polyacrylamide gel electrophoresis analysis of nuclease digestion products was done using DNA sequencing tech- niques according to Ausubel et al. [17]. Gels contained 6% and 20% acrylamide for the analysis of pd(A)4- ~ and the two oligomers, respectively. Autoradiography of the partial digestion products for localization of 32p radioactivity was followed by fractionation of the gels [11]. 3H and 32p radioactivity were assayed for digestion products of pd(A)4~ and the two oligomers, respectively [11].

2.6. High-performance liquid chromatography

For the HPLC analysis of the digestion products of the small oligonucleotides, alkaline salt gradient elution with a Mono-Q HR 5 / 5 (Pharmacia LKB) anion exchange column was done as described [11]. Dilution and mixing with the alkaline eluent were used to stop the reactions instead of EDTA due to its interference in the chromatographic profiles. All peaks identified were well-separated and confirmed by chromatography of the individual compounds.

For the assays to obtain kinetic parameters for the F and S nucleases with pd(A) 2 as substrate, a reverse-phase column (Bio-Sil ODS-10, Bio-Rad Laboratories) was used [11]. The EDTA peak from the use of this compound to stop the reactions did not interfere with this analysis.

2.7. Determination of kinetic parameters

Initial tests for nuclease activity on the ~bNO2-containing compounds were done at 5 mM substrate with either nuclease at 20 U/ml . Determination of kinetic parameters for the ~bNO2-pdT substrate, which was the only one readily cleaved to release p-nitrophenol, was done by following the increase in A4°°mnm using a Beckman DU-7 spectrophotometer with a thermostatted cuvette. F and S nucleases were used at 15.0 U/ml and substrate concentrations were varied from 2 to 40 mM which proved to cover the range of 0.02 to 0.8 × Km. Plots of A4°°mnm vs. time were linear. At most, 1% of the starting substrate was converted.

The kinetic assay of the nucleases with pd(A) z as substrate was by analysis of peak areas corresponding to

T. Lu, H.B. Gray Jr. / Biochimica et Biophysica Acta 1251 (I 995) 125-138 127

pdA and pd(A) 2 after separation via reverse phase HPLC. The extinction coefficients and correction for hyper- chromicity due to hydrolysis of pd(A) 2 were the values quoted [11]. The non-treated control sample showed two small peaks in addition to the pd(A) 2 peak which were unaffected by nuclease treatment. Contamination by pdA was not detectable in the range of substrate concentrations used, which was from 0.4 to about 6 X K m. The concentration for both nucleases was 0.3 U/ml . Reactions were carried out as noted [11] under which conditions less than 15% of the starting substrate was digested.

Apparent kinetic parameters K m and Vmax~,~ for hy- • ap

drolysls of 3H-d(A)4-~ by both BAL 31 nucleases were determined by measuring the acid-soluble 3H radioactivity released. The concentration of both nucleases was 3.13 X

10 - 3 U/ml and substrate concentrations ranged from 0.1 to 10.8 nM in DNA molecules (0.2 to 20X Km,~p) (13 concentrations) and from 0.8 to 20 nM (0.4 to 9 X Km ~ ) (11 concentrations) for the F and S enzymes, respectively. Reactions, including controls, were carried out as noted [11]. In no case was more than 15% of the starting substrate hydrolyzed. Replicate determinations of the velocity at each substrate concentration were made and the average velocity was used in all calculations.

For calculation of forward reaction rate c o n s t a n t s (kcat) , the molecular weights for the F and S form were taken as 109 X 10 3 and 85 X 10 3, respectively, and their respective specific activities as 25.3 and 29.8 U / ~ g [2]. Calculations using least-squares analysis of data plotted according to the Lineweaver-Burk method [18] and the statistically

1 2 3 4 5 6 7 8

17-mer Q I ID 18-mer

pdG O

pdA

Fig. 1. Autoradiogram of 5'- or 3'-terminally 32p-labeled 17-mer or 18-mer samples (approximately 2 .7/xM in DNA molecules) treated with SVPD or BAL 31 nucleases and electrophoresed in 20% polyacrylamide gel. Incubations with SVPD were at 37°C in 0.11 M Tris-HC1 (pH 8.9), 0.11 M NaCI and 15 mM MgC1 z. Lanes 1-4, 5'-labeled 17-mer; lanes 5-8, 3'-labeled 18-mer. Lane 1, not treated; lanes 2 and 3, digested with SVPD (8.7 × 10 -2 U / m l ) for 3 and 15 rain, respectively; lane 4, digested with BAL 31 F nuclease (0.5 U / m l (0.18 nM)) for 15 min; lane 5, not treated; lanes 6 and 7, digested with SVPD as for lanes 2 and 3; lane 8, digested with BAL 31 F nuclease as in lane 4. Fastest-migrating bands are pdG (lanes 1-4) and pdA (lanes 5-8). Contaminating species shorter than 17-mer (lane 1) are relatively minor as exposures were long enough to reveal relatively faint (quantitated in Fig. 4a) intermediate bands from BAL 31 nuclease digests.

128 T. Lu, H.B. Gray Jr./Biochimica et Biophysica Acta 1251 (1995) 125 138

weighted Wilkinson method [19] agreed within a maximum of 20% and 7% of the average values of the kinetic parameters for the ~bNO2-pdT and d(A)4- ~, substrates, respectively. The values presented were those calculated according to Wilkinson as this method weights the reaction velocities assuming a constant absolute error, which is reasonable in photometric analysis.

3. Results

3.1. Activity o f BAL 31 nucleases on p-nitrophenyl phosphate substrate analogs and small oligonucleotides

Three compounds containing the p-nitrophenol moiety (~bNO2-p-thNO 2, dTp-~bNO 2 and ~bNO2-pdT) were examined. Only the pdT-containing dinucleotide analog was cleaved at a significant rate so as to release p-nitrophenol. At the same enzyme and substrate concentrations, dTp- ~bNO 2 was cleaved at about 1% of the rate for ~bNO2-pdT and cleavage of ~bNO2-p-&NO 2 was not detectable.

DNA dinucleotides with 5'-terminal phosphates (pd(A) 2 and pd(T) 2 were tested) were readily digested to nucleoside monophosphates but dimers lacking 5'-terminal Pi (d(A) 2 and (d(T) 2) were not attacked by either form of the nuclease. At enzyme and substrate concentrations of 0.4 U / m l and 25 /zM, respectively, about 30% of each of the dimers with 5'-phosphates were converted to nucleoside monophosphates in 3 - 4 min. With longer incubation (20 min), these dimers were completely converted. However, no digestion of d(A) 2 or d(T) 2 was observed at nuclease concentrations as high as 60 U / m l and incubation for 2 days.

Two trinucleotides, with and without a terminal 5'-phosphate, and a tetranucleotide were tested with both forms of the nuclease. The only digestion products observed for d(TTA) were d(T) 2 and pdA. pd(A) 2 and pdA were observed in partial digests of pd(A) 3 but extended incubation yielded only pdA. The products of exhaustive digestion of d(AATT) were d(A) 2 and pdT which species were also observed in partial digests. Attempts to detect other intermediates from this substrate were not successful, appar-

C 1 2 3 4 5 6 7 8

C A G A A C T

G

A

C C C T T

T

T

G

Fig. 2. 32p autoradiogram after electrophoresis in 20% polyacrylamide gel of 5'-labeled 17-mer partially digested to various extents with BAL 31 nucleases. Lanes 1-4 represent incubations with F nuclease (0.5 U/ml) at 3, 8, 15 and 25 rain, respectively, and lanes 5-8 are for S nuclease (0.5 U/ml (0.20 riM)) at the same respective times of incubation. Lane C, not treated. Sequence corresponding to each band is shown to the right.

T. Lu, ll.B. Gray Jr. / Biochimica et Biophysica Acta 1251 (1995) 125-138 129

Table 1 BAL 31 nuclease terminal digestion products of small oligonucleotides and analogs a

Oligonucleotide or analog Digestion products

~bNO2-p-$NO 2 N.R. dTp-~bNO 2 N.R. b $NO2-pdT HOq~NO 2, pdT d(A)2 N.R. d(T)2 N.R. pd(A) 2 pdA pd(T) 2 pdT d(A)3 d(A) 2 , pdA d(TTA) d(T) 2, pdA pd(A)3 pdA d(AATT) d(A) 2 , pdT

a N.R., no reaction detected. Results were identical for F and S nucleases. b No reaction to produce p-nitrophenol (HOthNO2).

ently due to overlap of elution times for the starting substrate and pd(T) 2. The result for pd(A) 3 indicates that removal of nucleotides from very short oligomers is not necessarily processive. For longer oligomers, however, digestion is processive until only short oligomers remain (below). Both forms of the nuclease produced the same cleavage products in each case. The digestion products for the above substrates are summarized in Table 1.

3.2. Mode of digestion of oligonucleotides

The 5'- 32 P-labeled 17-mer and the 3'- 32 P-labeled 18-mer (derived from the non-labeled 17-mer by addition of a labeled pdA residue), respectively, behaved as expected when incubated with snake venom phosphodiesterase (SVPD), which digests DNA non-processively from the 3'-end [20]. Lanes 2 and 3 of Fig. 1 show the expected radioactive digestion intermediates for the 5'-labeled oligomer. Lane 3 displayed all 17 possible species, provid- ing for identification of the intermediates of partial digestion with BAL 31 nuclease (lane 4). The fastest-migrating spot corresponds to the 5'-terminal nucleotide pdG. The 3'-labeled oligomer yields only pdA and remaining intact 18-mer as labeled species in partial SVPD digests (lanes 6 and 7). The slower rate of migration for pdG than for pdA in such gels has been noted [21].

The products of progressive BAL 31 digestion of 5'- 32 p and 3'-32p end-labeled 17-mer and 18-mer, respectively, are shown in Figs. 2 and 3. In all these reactions, a high molar ratio of oligomer to enzyme ( > 10 4) was used so that single-hit kinetics apply at low (e.g., less than 50% of molecules attacked) extents of digestion. The profiles of radioactivity vs. fraction number from the gels of Figs. 2 and 3 are shown for the F nuclease in Fig. 4a and Fig. 4b. The profiles for the S nuclease (not shown) are very similar to those of the F enzyme for each substrate. In each case, however, they are very different for oligomers labeled at the 3'- or 5'-end.

For the oligomer labeled at the 5'-end, the bulk of the

radioactivity appears at the position corresponding to the intact oligomer or at the monomer position (quantitated below). This is consistent with exonucleolytic attack from the 5' end. The low level of less than full-length oligomers bearing the original 5'-termini rules out endonucleolytic attack followed by dissociation of enzyme from substrate, as the major initial cleavage event as well as such cleavage followed, without intervening dissociation, by 5' ~ 3' exonuclease action. For the initial cleavage to be endonucleolytic, it would have to be followed, without dissociation, by highly processive 3' ~ 5' exonuclease attack in order to produce pdA as the predominant digestion product. The fainter bands between the full-length and mononucleotide positions ( Figs. 2 and 4a) would then represent relatively small amounts of species attacked via an initial endonucleolytic event that is not immediately followed by processive 3' ~ 5' exonuclease activity.

In the case of the 3'-labeled 18-mer, the partial digestion products contained substantial amounts of labeled

1 2 3 4 5 6 7 8 C

18.mer

pdA

Fig. 3. 32p autoradiogram after electrophoresis in 20% polyacrylamide gel of 3'-labeled 18-mer partially digested to various extents with BAL 31 nucleases as in legend to Fig. 2. Lane assignments are as in Fig. 2.

130 T. Lu. H.B. Gray Jr./Biochimica et Biophysica Acta 1251 (1995) 125-138

100

"~. 80

~ 20 a_

I00

"~ 80

~ 60

"~ 20

a

6 11 16 Fraction Number

17 16 15 14 13 12 U 10 9 8 7 6 5 4 3 2 1 Oligomer Length (nucleotldes)

b

18-16 15 7

3 v ~ ~ 7 9 Fraction Number

6 5 4 3 2 1 Oligomer Length (nucleotides)

Fig. 4. Profile of percent of total 32p radioactivity vs. fraction number/oligonucleotide position from gels of Figs. 2 and 3. (a) 5'-labeled 17-mer, digestion times (min): C), 0; [2, 3; II, 8; (b) 3'-labeled 18-mer, digestion times (min): C), 0; r-q, 3; II, 8; 0, 15.

short oligomer, mostly dimers and trimers ( Figs. 3 and 4b). The molar ratios among these did not depend strongly on the extent of digestion. This pattern, taken together with that for the 5'-labeled oligomer, still allows for the con- certed endonuclease-processive 3' ---> 5' exonuclease mechanism above, with the stipulation that the initial cleavage takes place at or near the 3'-end. It is also consistent with highly processive initial digestion of longer single-stranded DNAs from the 5'-end, with the mechanism becoming progressively more distributive as the substrate is reduced to only several nucleotides in length.

A purely endonucleolytic (independent of free ends) initial event would have to be base composition- or sequence-dependent in order to preferentially release oligonucleotides from near one end. This possibility was examined by using pd(A)~6 as substrate after labeling either the 3' or 5' terminus with 32p as above. Treatment with SVPD as in Fig. 1 was done to verify the labeling and oligomer sizes. Patterns of products were seen in intermediate digests very similar to those for the 17-mer, although there were no 'missing' intermediate oligomers in the patterns for the 5'-labeled homopolymer (cf. missing 10-mer in Fig. 2). The 5' end-labeled pd(A)16 displayed the majority of radioactivity at either the full-length position or the monomer position. When pd(A)16 was 3'-end-labeled (to make pd(A)j7) and partially digested, short oligomers were

generated as before with mass ratios among the monomer and oligomers similar to those seen when the 18-mer was used as substrate. The similarity of the patterns observed for the two kinds of oligomers shows that the mode of digestion is not dependent on base composition or sequence-specific effects.

Taken together with the finding that pd(A) 3 is not digested processively, these results strongly suggest that BAL 31 nuclease digests single-stranded DNA predominantly from the 5'-end. The nuclease will release 32 P-pdN from DNA labeled at the 5'-end and digest single-stranded DNA highly processively when the substrate is long enough (longer than the heptamer as seen in Fig. 3). The high processivity of BAL 31 nuclease action on much longer single-stranded DNA is evidenced below. As the substrate becomes shorter than a heptanucleotide, the probability of dissociation from the enzyme between catalytic events progressively increases and small oligomers bearing the original 3'-terminus appear in partial digests. The alterna- tive explanation, fully processive 3' ---> 5' digestion in con- cert with endonucleolytic attack near the 3' end, is incon- sistent with the non-processive attack on pd(A) 3 (5'-labeled oligomers should be produced).

The 3'-end labeled pd(A)~7 has a phosphate group at its 5'-end while the 18-mer thus labeled does not have the 5'-terminal phosphate. The similar patterns for partial digests of these two substrates suggests that the 5'-phosphate group is not important in BAL 31 nuclease attack on oligomers despite the fact that the nuclease does not digest dinucleotides without 5'-phosphates. Also, the patterns from such digests were unchanged from those for non-phosphorylated T-labeled 18-mer when this substrate was subjected to 5'-phosphorylation using non-labeled ATP. This agrees with the finding that tri- or tetranucleotides without 5'-phosphates can be partly digested by BAL 31 nuclease, leaving the d(N) 2 at the 5'-end intact, and indicates that the nucleases will produce d(N) 2 as the 5'-terminal digestion product instead of pN if the 5'-end of DNA substrate is not phosphorylated. None of these experiments would have revealed 5'-terminal d(N) 2 from the oligomers as a ra- dioactively labeled species.

The low level of 5'-labeled oligomers in partial digests involving a 5'-directed attack requires that nucleotides are removed one at a time from oligomers or polymers bearing 5'-phosphoryl termini. This is because significant fractions of any dinucleotides or higher oligomers produced in an initial cleavage event occurring at a rate comparable to that of removal of the terminal residue would persist in partial digests, where there is not a high probability of attack on the fragments from initial cleavage of a given molecule. The cleavage of pd(A) 2 is consistent with this finding.

3.3. Digestion o f a single-stranded DNA polymer

pd(A)4~i, Y-terminally labeled with 32p and uniformly labeled with 3H, was partially digested with the BAL 31

T. Lu, H.B. Gray Jr./Biochimica et Biophysica Acta 1251 (1995) 125-138 131

M 1 2 3 4 5 6 7 8 9

a a

5171506 ~ ~l~ 9 ~ O g 344

298 In,

220 Q 201

154 Q 134 m

75a75 b

rected attack by SVPD causes the progressively shortened polymer to appear as a smear migrating faster than the non-degraded material (lanes 6 and 7). T7 gene 6 exonuclease removes mononucleotides from the 5' end [22,23] and produced only full-length and very fast-migrating material in partial digests (lanes 8 and 9) as expected. When aliquots of these same samples were electrophoresed in 20% acrylamide gels, a single fast-moving species was seen (not shown); there were no significant bands of 32p_labeled dimers or larger small oligomers so that the fast-migrating spot from T7 and BAL 31 nuclease treat- ments is confirmed as pdA.

The 32p autoradiogram of partial BAL 31 digests (Fig. 5, lanes 2-5) is consistent with processive digestion. The profile of 3H radioactivity from fractionation of gel lanes representing progressive partial digests (shown for F nuclease in Fig. 6) better illustrates this as it shows that most of the DNA mass is distributed between the full-length and monomer positions. A high degree of processivity in a predominantly exonucleolytic mode of attack is thus demonstrated since: (1) all digestion products appear in this profile (not just terminally labeled molecules), (2) endonucleolytic attack alone could not produce such high fractions of the radioactivity as a monomer while non-degraded polymer still remained.

3.4. Endonuclease actiL, i~ on single-stranded DNA

pdA • ~ 8 db dlb

Fig. 5. Autoradiograrn after electrophoresis in 6% polyacrylamide gel of 5'-terminally 32P-labeled pd(A)4~ ~ (0.22 /xM in DNA molecules) partially digested to various extents with BAL 31 F nuclease (0.4 U/ml ) (lanes 2, 3; 15 and 25 min, respectively), BAL 31 S nuclease (0.4 U/ml ) (lanes 4, 5; 15 and 25 min, respectively), SVPD (8.7× 10 -2 U/ml ) (lane 6,7; 10 and 25 min, respectively) and phage T7 gene 6 exonuclease (160 U/ml ) in 50 mM Tris-HCI (pH 8.1), 5 mM MgCI2, 20 mM KCI, 5 mM /3-mercaptoethanol at 37°C (lanes 8,9; 10 and 25 min, respectively. Lane 1, not treated; lane M, 5 '-32P-labeled single-stranded molecular weight standards (oligonucleotide lengths to left). Fastest-migrating band is pdA; most intense band near top of gel is intact polymer distributed about weight-average length of near 480 nucleotides. Due apparently to the presence of glycine in the SVPD buffer used here, much of the pd(A)4~ 6 was retained at the top of the gel. No such retardation was observed for the oligomers (Fig. 1), where Tris-HC1 buffer was used. Numbers to left indicate marker lengths in nucleotide residues.

nucleases at a molar ratio of DNA to enzyme of > l0 3. This substrate was also treated with other nucleases and aliquots subjected to electrophoresis under DNA sequencing gel conditions. A 32 p autoradiogram of this gel (Fig. 5) shows that the digestion products of 5' 3 2 p _ pd(A)4 ~ by snake venom phosphodiesterase and T7 gene 6 exonuclease were as expected. The non-processive 3 ' ~ 5' di-

The reported endonuclease activity against circular single-stranded DNA [2] is a minor mode of degradation as seen from the relatively low fraction of 32p radioactivity appearing between the starting substrate and monomer positions for the 5'-end-labeled oligomers (quantitated for the 17-mer in Fig. 4a) and for d(A)4-~- 6 (Fig. 5). The intermediate 3H cpm for d(A)4- ~ (Fig. 6) can also be related to the endonuclease activity except that products of

I00,

"~ 80

~ 60

~" 40

~ 2o

o 1 6 11 16 21 26

Fraction Number I I I I I I i I I ] I

N ~ Oligomer Length (nucleotides)

Fig. 6. Profile of percent of total ~H radioactivity vs. fraction number/polynucleotide position after electrophoresis in 6% polyacrylamide gel of products of partial digestion of 3H-labeled pd(A)4~ 6 with BAL 3n F nuclease (0.4 U/ml). Digestion times (rain): O, 0; [:3, 4; II, 8; 0 , 15; zx, 25.

132 T. Lu, H.B. Gray Jr./Biochimica et Biophysica Acta 1251 (1995) 125 138

Table 2

Kinetic parameters for the BAL 31 nucleases with ~bNO2-pdT as substrate ~

Nuclease Vma x K m kca t kcat/K m ( n m o l / U min) (mM) (rain-1 × 10-3) (1 m o l - i m i n - i × 10-3)

F 0.14 + 0.01 49 +__ 5 0.39 + 0.03 8.0 + 1.0 S 0.16 ___ 0.01 53 + 7 0.41 ___ 0.03 7.8 ___ 1.2

a Errors are standard deviations calculated from the Wilkinson treatment [19] or derived from these error estimates by propagation of error formulas.

incompletely processive digestion (larger than small oligomers, which would not appear separated from the monomer in the fractionation scheme used) would con- tribute artifactually to the mass at intermediate positions, while endonucleolytic cuts followed, without dissociation, by processive 5 ' ~ 3' exonucleolytic degradation would reduce the apparent endonuclease activity. These factors would not affect the profiles for 5'-labeled material.

Estimates of the ratio of the frequency of endonuclease to exonuclease cleavages were made from the profiles for the 5'-labeled oligomers with correction for the presence of dimers, trimers, etc. (Fig. 4b) as described [11]. The values ranged from 0.016 + 0.003 to 0.034 __+ 0.002 (over two extents of partial digestion, at each of which over 50% of intact oligomer remained) with the S enzyme showing slightly higher ratios than the F enzyme for both oligomers and with pd(A)16 having the higher ratio for the respective enzymes than the 17-mer.

Such estimates were also made for d(A)4-~- 6 from the 3H profiles (Fig. 6 for F nuclease), assuming that there is no 3 ' ~ 5' exonuclease activity and that oligomers large enough to appear outside the fraction nominally representing the monomer do not result from incompletely processive 5' ~ 3' attack. A correction was made which assumes that the failure of the fully intact polymer (fractions 2 and 3 of Fig. 6 and the corresponding fractions for the S nuclease) to represent 100% of the radioactivity in the absence of nuclease action is the result of the presence of some of this material as smaller than full-length polymer and that this smaller DNA is not converted to monomer. For the F and S enzymes, the endo/exo ratios are 5.6 × 10 -3 and 6.5 × 10 -3, respectively, at less than 25% loss of the initial d(A)4~ 6 so that single-hit kinetics apply. If a processive endonuclease ~ exonuclease activity is present, the above ratios would increase by a factor of two. These are significantly lower than the averages for the oligomers (above), confirm that the endonuclease activity is relatively

minor and indicate that the ratio can depend on substrate chain length and/or substrate concentration.

A sequence and/or base composition dependence of the relative rates of endonuclease and exonuclease activities was suggested by the results for the oligomers. A striking example of an apparent sequence effect is the absence of the decamer in partial digests of the 5'-labeled 17-mer (Fig. 2), which corresponds to the failure of the sequence 5'-d(GT) to undergo endonucleolytic attack. This cannot arise from a 5' ~ 3' exonuclease digest as such digestion products will not be labeled. Whether this dependence is at the dinucleotide level or requires a longer sequence con- text cannot be ascertained using this substrate.

3.5. Kinetic parameters

The kinetic parameters for both S and F nucleases acting on ~bNO2-pdT and pd(A)e are in Tables 2 and 3. kca t represents the tumover number or forward reaction rate constant as per the usual definition kca t = Vmax/[E] , and refers to the rate of phosphodiester bond hydrolysis; the catalytic efficiencies are given by k c a t / K m. The simplest Michaelis-Menten model with a single enzyme-substrate intermediate appears applicable for these small substrates and the measured kinetic parameters are here assumed to be the actual ones. None of the parameters differ markedly between the two nucleases for a given substrate and are the same within experimental error for ~bNO2-pdT. The large differences between the two substrates are considered in Section 4. Proportionality between initial reaction velocity and enzyme concentration was observed (determined for pd(A) 2 only, data not shown) over the concentration range examined (0.1-1.0 U/ml) .

The apparent kinetic parameters for the F and S forms acting on 3H-d(A)4--g-6 are in Table 4. In order for the units of Vma × 00' kcat (also an apparent value) and K m to be consistent, the values of Vmax~00 correspond t~ p moles

Table 3

Kinetic parameters for the BAL 31 nucleases with d(pA) 2 as substrate

Nuclease Vma x K m kca t ' kcat/K m ( n m o l / U min) ( /xM) ( m i n - i X 10- 3) (1 m o l - 1 min I × 10- 9)

F 8.17 + 0.05 16.5 ___ 3.1 22.5 + 0.14 1.4 + 0.3 S 7.47 + 0.02 10.5 _+ 1.2 18.9 + 0.05 1.8 _+ 0.2

T. Lu, ll.B. Gray Jr./Biochimica et Biophysica Acta 1251 (1995) 125-138

Table 4 Kinetic parameters for the BAL 31 nuclease with d(A)48 o as substrate

133

N u c l e a s e Vma x pp K m p k c a t kcat/Km.pp. (pmo[/U min) (nM~ (min-i ) (1 mol- i mm- 1 X 10- to)

F 8.1 + 0.4 0.55 + 0.07 22.5 -1- 0.8 4.1 + 0.6 S 7.7 + 0.4 2.2 _ 0.4 19.6 + 1.0 0.9 5:0.2

d(A)4--~6 degraded per unit of enzyme per minute and were obtained by dividing the values based on rate of bond breakage by 480; K m is then properly expressed as

. a pp

molar concentration of polymer. It is noted that the values of Vmaxa and kcat, o n the basis of the rates of intemu-

• P , . .

cleotlde ~ond hydrolysis, are similar for pd(A) 2 and the polymer with those for the dinucleotide higher by a factor of roughly two.

The K m of the F form for single-stranded DNA was , app • •

not determined m earher work because it was too low for photometric measurement [2]. With the labeled compound, measurements at the low total nucleotide concentrations required were feasible. The two nuclease species clearly are not distinguishable with respect to Vmax,pp and kca t

given the experimental errors. The apparent catalytic efficiencies exceed the largest experimental value for a simple Michaelis-Menten model (Section 4).

4. Discussion

4.1. Specificity and kinetics f o r small substrates

p-Nitrophenol-containing phosphodiesters have long been used to determine the cleavage specificities of nucleases. The BAL 31 enzymes are not 'general' phosphodi- esterases because they did not digest the canonical phosphodiesterase substrate ~bNO2-p-~bNO 2. The minimum 'DNA' substrate is pd(N) 2 (d(N) 2 is not a substrate) independently of whether purine or pyrimidine bases are involved. The degree of base stacking does not strongly affect the kinetics as comparable extents of cleavage of pd(T) 2 and pd(A) 2 were observed under the same set of incubation conditions but the analogous RNA dimers (U) 2 and (A) 2 exhibit the most and the least base stacking, respectively, of all 16 (N) 2 compounds [24].

The activity against ~bNO2-pdT to release pdT is consistent with the production of 5'-dNMPs from both single- stranded and duplex substrates [6] and suggests that at least one nucleotide moiety is essential for substrate recognition. In view of the failure to cleave d(N) 2, this relatively slow cleavage is interpreted as the result of an ability of the NO 2 and phenyl groups to mimic very weakly the 5'-phosphate and nucleoside moieties of a 5'-phosphoryl terminal nucleotide. ~bNO2-pdT is indeed a poor substrate as seen from the K m values, which are (3 -5) - 103 times greater for the p-nitrophenol compound than for pd(A) 2 and in a range (50 mM) at least an order of magnitude higher than

for the natural substrates of enzymes in general. Also, the value of kca t for pd(A) 2 is some 50-fold higher than for ~bNO2-pdT. These data may be contrasted with those for the general phosphodiesterase SVPD, which cleaves d(T) 2 as well as ~bNO2-p-~bNO 2 and has comparable, 'enzyme- like' K m values for these two substrates as well as for ~NO2-pdT and pd(T) 2 (range 0.2-0.8 mM) [25].

The 5'-directed mode of attack evidenced above, the lack of activity against d(N) 2 and the production of d(T) 2 and d(A) 2 as terminal digestion products of d(TTA) and d(AATT), respectively, suggest that the absence of the 5' phosphate causes the oligonucleotide to bind as if the phosphate of the first internucleotide bond starting from the 5' end is the 'terminal' one; this bond is not attacked because its phosphate replaces a terminal phosphate. The oligomer can then be digested at subsequent phosphodiester bonds in a 5' ~ 3' direction• Here, the enzyme treats the substrate lacking a 5'-phosphate as if the terminal nucleoside residue were not present.

The endonuclease activity, though minor, is significant because the endonucleolytic introduction of cleavages into one strand of closed circular duplex DNA in response to covalent or non-covalent alterations [5-10] is likely a manifestation of the endonuclease activity against single- stranded DNA. The low level of this activity makes separate catalytic sites for exonucleolytic and endonucleolytic cleavage appear unlikely. Since binding of DNA molecules away from the termini is argued below to be a significant part of the mechanism, it is reasonable to postulate that internucleotide bonds in or near the cleavage site have a relatively low probability of being attacked. The trivial argument that the endonuclease activity is due to a contam- inant is difficult to promote because the S and F forms are well-separated by gel filtration [2] and yet display similar levels of such activity, requiring that similar endonuclease activities are the result of different-sized contaminating enzymes, each of which happens to co-elute with a species of BAL 31. Also, the endonucleolytic activity on non-su- percoiled closed DNA containing apurinic sites paralleled the peaks of S and F nuclease activity on single-stranded DNA [81.

4.2. Processivity o f action on single-stranded DNA substrates

A DNA polymer close to 500 nucleotide residues in length was evidenced to be completely degraded in a majority of productive enzyme-substrate binding events,

134 T. Lu, H.B. Gray Jr./Biochimica et Biophysic~ Acta 1251 (1995) 125-138

demonstrating a high processivity of the nuclease action on a single-stranded substrate. This is compared with the quasi-processive nature of the attack on duplex DNA, where about 18 and 28 residues are removed from 3' termini per event by the S and F species, respectively [4].

High processivity in exonucleases has been postulated to be the result of a second binding site other than the catalytic site [14,20,23,26]. This two-position interaction of a catalytic and an 'anchor' site: (1) results in strong overall binding, without necessitating such strong binding to the catalytic site alone so as to place the enzyme-substrate complex in a deep free energy 'well ' which requires the overcoming of a large free energy barrier to reach the 'activated' form of the enzyme-substrate complex [27]; (2) provides for the interesting mechanochemical reaction of translocation of the DNA chain into the catalytic site as the terminal nucleotides are removed. Brody et al. [14] apparently were the first to evidence such two-site interactions when they showed that exonuclease I from E. coli digests single-stranded DNA highly processively from the 3'-end when the substrate is longer than an l l-mer, but non- processively for substrates shorter than the ll-mer. For oligomers too short to be bound effectively to both the anchor site and the catalytic site, the mechanism goes over to one of removal of one residue per binding event because the catalytic site cannot perform the translocation step and the substrate dissociates after each cleavage. The increase of K m rr for the degradation with decreasing chain length and th~ failure of this enzyme to digest oligomers shorter than the tetramer [14] are consistent with this model.

The processivity demonstrated for their action on single-stranded substrates makes a structure with anchor and catalytic sites, with loss of processive attack as the oligomers become too short to bind effectively to the anchor site, a very attractive one for the BAL 31 nucleases. However, the mode of action is distinct from that of exonuclease I in that the shortest oligomer, the dinucleotide, is digested if it bears a 5'-phosphate. A two-site binding model for the BAL 31 nucleases must allow for the independent function of the catalytic site from the anchor site, else it is difficult to rationalize the attack on the dinucleotide when attack on the trinucleotide is not processive. The independence of the two sites is a novel aspect of the BAL 31 enzymes.

Since pd(A) 2 represents the minimal DNA substrate, t h e Km,rp values for this dinucleotide could be inter- pretable according to the simple Michaelis-Menten model and thus well approximate those for the catalytic sites of the two forms independently of interaction with the anchor site. These K m values are (3 -5 ) . 1 0 3 times greater than those for d(A)a-g- 6. Inasmuch a s K m values can be regarded as measures of enzyme-substrate affinity, lower values are reasonable for the polymer where the anchor site is involved. However, such an interpretation requires that the forward reaction rate constant be smaller than the dissociation constant of the enzyme • substrate complex where only

a single such intermediate is required and needs additional assumptions in the case of d(A)4-~6 if a catalytically non- productive nuclease • DNA complex is postulated (below).

4.3. Directionality of attack

While the data of this study best support a 5 ' 4 3' exonuclease mechanism as the predominant mode of attack on single-stranded substrates, double-stranded DNA is digested predominantly from the 3'-end and 5'-terminated single-stranded tails of very significant length relative to the number of nucleotides removed can be generated in partial digests [4]. There was no evidence for 3'-terminated single strands [4]. This indicated that a 5 ' 4 3' directed activity, if present, was far slower than the 3' 4 5' one with respect to the degradation of duplex DNA. Since a 5' ~ 3' mode of attack apparently exists on single-stranded substrates, it is concluded that the nuclease dissociates at the junction between single- and double-stranded regions. Such a 'dissociation signal' has been proposed for E coli exonuclease I [23].

The average single-stranded tail lengths can be over 50 residues per 100 removed at low nuclease concentrations but level off at a value near 7 residues per 100 removed for both forms above certain enzyme concentration ranges [4]. The presence of a processive 5 ' 4 3' exonuclease activity that cannot enter duplex regions suggests an explanation for the finding that at higher nuclease concentrations, where relatively short tails are observed, a high fraction (up to 50%) of the ends are fully base-paired [4] - a result of the termination of the processive exonucleolytic attack at the first base pair of duplex DNA. The limiting average size of the tails reflects the fact that the 5' 4 3' exonuclease attack cannot take place until the 3 ' 4 5' event does, so that the molecular population always con- tains some molecules with tails of average length dictated by the quasi-processivity of the 3' 4 5' activity. This is the case even though the kinetic parameters (this work; Lu, T. and Gray, H.B., Jr., unpublished) suggest that the rate- limiting factor is the 3 ' 4 5' exonuclease activity. The above explanation is much more plausible than removal of single-stranded tails by a relatively infrequent endonucleolytic event as proposed [4], since such events would have to be concentrated near the duplex-single strand junction. The dependence of tail length on the rate at which a given number of nucleotides are removed from the 3' ends (nuclease concentration dependence [4]) remains unex- plained.

Both modes of exonucleolytic attack on DNA substrates have been observed in a single enzyme, but only one other species, an extracellular nuclease from Bacillus subtilis, has been reported to digest single-stranded DNA from the 5' end but duplex DNA from the 3' end [28]. The action of this enzyme is distinct from that of the BAL 31 nucleases, however, in that 3'-dNMPs are produced and there was no

T. Lu, H.B. Gray Jr./Biochimica et Biophysica Acta 1251 (1995) 125-138 135

evidence for endonuclease activity on single-stranded DNA.

BAL 31 S nuclease extensively treated with an endopro- tease can undergo a dramatic loss in its exonuclease activity on duplex DNA while maintaining most of its activity on single-stranded DNA and its ability to cleave duplex DNA endonucleolytically in response to strand breaks [1]. In light of the present study, this suggests that the 3' ~ 5' activity can be inactivated readily by proteoly- sis while the 5' ~ 3' activity is resistant. The process is not accompanied by any apparent molecular weight reduction in the absence of extreme denaturing conditions [1] so that the question of separate catalytic domains has not been answered.

4.4. Kinetic parameters and their interpretation

The turnover numbers for the exonuclease activity on single-stranded substrates are not distinguishable within experimental error (Table 4), in agreement with the older data [2]. These values are also in reasonable agreement with the earlier work (maximum difference of 26% of the average value) even though a naturally occurring initially circular substrate was used while a homopolymer was employed in this study. The similarity in the Vmax,~, and kca t values for the two forms might appear to suggest that the catalytic activity is due to sites that are similar in their inherent capacity to cause the hydrolysis of internucleotide bonds• However, this interpretation can be erroneous (below).

The value of the apparent Michaelis c o n s t a n t s Km~ p for the S nuclease from the present study, converted to a nucleotide concentration basis so that it may be compared to that of Wei et al. [2], is 3-fold less than that from the earlier work• A value for the F enzyme was not obtainable in that work. The estimates and uncertainties for K m for

• ~ P P

single-stranded DNA polymers of this study (Table 4) indicate that the values for F and S nucleases are distinguishable•

The catalytic efficiency must be less than the second- order rate constant for the formation of an enzyme-substrate complex in a unisubstrate reaction that can be mod- eled by a single such complex 2. The high apparent catalytic efficiencies for the action of the nucleases on single-stranded DNA (Table 4) are even more significant with respect to the upper limit for a diffusion-controlled enzyme-substrate encounter (usually taken to be near 10 ~ 1/mol per min) when the presence of a relatively slowly diffusing macromolecular substrate is taken into account. An upper limit was estimated using the Debye-

2 Although the nuclease catalyzes a bisubstrate reaction with H20 as the second substrate, the concentration of this second 'substrate' is at all times saturating and the kinetics may be treated as if a unisubstrate reaction were being catalyzed.

Smoluchowski equation by Winter et al. [29] as approx• 108 1/mol per s (6 × 109 l / m o l / m i n ) for the interaction of the lac repressor and a 50 000 base pair duplex DNA. Using diffusion coefficients estimated for the BAL 31 nucleases and d(A)4- ~ and the other parameters as per Winter et al., calculation in this equation gives an upper limit for k 1 of 2.4 × 10 ~° 1/mol per min (details on request)• The value from Table 4 for the F enzyme is 1.7-times this limit• More significantly, it is 2.5-times the largest experimental value tabulated [30], which is for an enzyme that does not have the restriction of acting on a macromolecular substrate. It is emphasized that k~ corresponds to the constant for the direct binding of the protein to a specific site (e.g., an end) and not to the overall rate of non-specific binding to a DNA chain.

It is not necessary to ascribe unlikely catalytic efficiencies to the nucleases if a reaction scheme is used that allows for the existence of non-hydrolyzed enzyme • DNA complexes in which the nuclease is bound internally and forms productive complexes (enzyme bound at a 5'- terminus) only by 'search' processes that do not involve an intervening macroscopic (release of protein so as to diffuse out of the domain of polymer segments of the DNA 3) dissociation; formation of a productive complex directly by random nuclease-DNA interaction occurs at a negligibly low frequency. If macroscopic dissociation occurs, the internally bound complex must be reformed. The relatively infrequent endonuclease attack is ignored•

The kinetics of such 'search' processes have been described theoretically for non-enzymatic reactions such as the binding of lac repressor to its operator in a duplex DNA molecule much larger than the operator sequence, where non-specific binding of the repressor occurs at any sequence which is not the operator itself [34]. This analysis shows that processes labeled 'facilitated diffusion' can greatly enhance the rate of formation of repressor • operator complexes, starting from non-specific repressor. DNA complexes, through such mechanisms as 'sliding' (the non-specifically bound repressor can undergo one-dimensional diffusion along the contour of the chain without dissociation to the extent that it could reassociate at a

3 The DNA polymer of these studies is estimated (below) to contain at least 12 statistical segment lengths and thus can be considered to have a coil conformation. The statistical segment length (Kuhn statistical length) was estimated as 14 nm from Studier's [31] empirical correlation between s20.~ , and molecular weight for single-stranded DNA in alkaline solution and the following substitutions in Eq. (10) of Gray et al. [32]: the molecular weight-independent term is set equal to zero, the molecular weight per unit length is one-half that of duplex DNA, the excluded volume parameter e = 0.20 (from the exponent in Studier's equation), r/c , = 0.01 poise, p = 1.0 g / c m 3 and the partial specific volume = 0.445 cm3/g [33]. This estimate should be high because the DNA is likely more flexible in neutral electrolyte solution, where the additional charge repulsions of titrated dG and dT residues are not present. To estimate the total chain length of d(A) z~ and mass per unit length, the internucleotide spacing of B-DNA was assumed.

136 T. Lu, H.B. Gray Jr./Biochimica et Biophysica Acta 1251 (1995) 125-138

distant site along the chain contour) and/or intersegment transfer (non-specifically bound protein is transferred from one segment to another of the same molecule without an intervening macroscopic dissociation). Thus, the apparently competing process of non-specific binding becomes essential in order to achieve overall association rate constants consistent with those experimentally observed. In the present case, the search for an end is held to be similarly facilitated.

The reaction scheme is

k2 k3 k p / n E + n N t E + D R # ED. # ES,, ...-)

k 2 k-3 kl $ $ k_ 1

E + D,,

where EDn and ES, represent, respectively, the non-hydrolyzed complex with enzyme bound away from the 5' end and the hydrolyzable complex with enzyme at this terminus. The step from ED, to ES n formally corresponds to an 'isomerization' in kinetic schemes involving multiple enzyme-containing complexes. The hydrolysis step is clearly irreversible. Although shown above for comparison with the situation in which degradation is not processive, full processivity removes the steps corresponding to k_ ~ and k_B: productive complexes, once formed, neither dissoci- ate nor re-form ED, but are digested to n nucleotides Nt. This means that the rate of the step to release free enzyme is the rate of digestion of an entire molecule, which is kp/n i f kp is the forward rate constant for removal of a single nucleotide. The negligibly small rate for direct formation of ES, from E + D, implies that k I << k 2 so that this path for formation of ES~ is effectively removed 4 k 2 and k 3 should be length-dependent [34], but both would be constants for a given value of n as is the case here.

The amount of substrate released as acid-soluble material in each velocity determination was such that each nuclease molecule would have had to degrade more than one DNA molecule. This is required in order for the scheme above to hold; if less than one DNA molecule were degraded in the time of measurement, the apparent rate of release of E would not be kp/n . kp/n is repre- sented as k' in the following. It is not equal to kca t (Table 4) unless certain relationships among rate constants exist (below)

For a steady-state situation, the treatment of King and Altman [35] for kinetic schemes involving multiple enzyme species, as described by Segel [36], may be used. This treatment allows expressions for the concentration of each enzyme species, divided by the total enzyme concen-

4 If k I is not set equal to zero, the further inequality k2k 3 >> k_2kl, which is consistent with k 2 >> kj, is formally required to obtain Eq. (1).

tration [ E],, to be easily obtained. Details of the use of this method in the present work are available [11].

For ED,, the result is

[EDn] k2[O.]k'

[E]---7 - + + k2( 3 + t,')[D°] (1)

The velocity of the overall reaction (where it is under- stood that k' must be the rate constant for degradation of a whole molecule) is just the net velocity of any step. For the step from ED. to ES., for example

P = k 3 [ E D n ] ( 2 )

since the reverse velocity of this step is zero. Substituting for [ED.] from Eq. (1) and assuming that

the velocity is the initial value

k3k2[Dn]k'[E],

v°= k ' (k 3 + k 2) +k2(k 3 +k')[D.] (3)

This is compared to the mathematical form of the standard Henri-Michaelis-Menten relationship, which is used in obtaining the kinetic parameters of Table 4 with the substrate concentration taken to be that of 5' ends which is equal to [D,]

Vmax ~po [ D n ] ~'0 = (4)

Km .r + [D.]

If all terms of Eq. (3) are divided by k2(k 3 + k'), Eq. (3) has the Michaelis-Menten form and expressions for Vmax, and K m are obtained in terms of the rate con-

r P app stants of Eq. (3)

k3k'[E], Vmaxapv (k 3 q- k') (5)

and

kt(k3 ~- k_2) Kmooo ~2 ( k3 + k')

k2 ) (6)

Eq. (5) provides for the usual interpretation of the maximum velocity as being a constant multiplied by [E],, but the constant is not the forward reaction rate constant k' (as in the simplest Michaelis-Menten scheme) except in the limit that k 3 >> k'. Only in this case can the observed kca t be taken to be k'. The behavior in this limit is reasonable because it would mean that the 'isomerization' (search process) from ED, to ES, is relatively very rapid and the rate-limiting step then becomes that corresponding to k'. In such a case, interpretation of the similar maximum velocities for the two species would imply a similar capacity to cause bond hydrolysis.

T. Lu, ll.B. Gray Jr. / Biochimica et Biophysica Acta 1251 (1995) 125-138 137

If, however, k' >> k 3, the maximum velocity is k3[E] t and the rate of 'isomerization' of ED n to ES, controls the rate at saturating concentrations of D n. In that case, the measured kca t appears to be a measure of the rate of facilitated diffusion. Here, it is seen that the similarity in Vmaxapp values for the two forms could reflect nothing more than a similar overall rate constant for 'search' for a 5' end; the actual forward rate constants could be quite different.

The apparent Michaelis constant (Eq. (6)) has a different form in terms of rate constants than in the simple unisubstrate Michaelis-Menten kinetic scheme. When k' >> k 3, the second equality of Eq. (6) becomes equal to the second factor in brackets and is analogous to that of the simple scheme with the significant differences that k3, k_ 2 and k 2 replace, respectively, the forward reaction rate constant and the rate constants for dissociation from and binding to substrate in the simple scheme. There is only a 4.0 ___ 0.9-fold difference between the two Kmapp values.

The analog of the catalytic efficiency

k' kz( k3 + k ') - - - ( 7 )

Km,pp k 3 + k_ 2

no longer has the interpretation requiring it to be less than a diffusion-controlled second-order rate constant for direct formation of the initial enzyme-substrate complex (kl in this treatment) as k 2, not k l, appears in the denominator above, k 2 is the total rate constant for enzyme-chain association and can be much larger than kl; the latter constant was neglected in this scheme by that rationale. The diffusion-imposed limits for catalytic efficiencies where facilitated diffusion is not involved can be well exceeded even if the multiplier of k 2 in Eq. (7) were significantly less than one. Thus, facilitated diffusion involving non-specific binding, which has been able to account for measured apparent second-order binding constants between lac repressor and its operator (contained in a long DNA molecule) that are 2-3 orders of magnitude greater than possible for a single-step, diffusion-controlled process [29,34], is shown in this work to be incorporated easily into a plausible model for an enzyme-catalyzed reaction and to provide a rationale for apparent catalytic efficiencies that, in the case of the F nuclease, exceed the limitations imposed by diffusion-mediated encounters as well as the largest experimental value and are larger for both species, relative to the estimated maxima, than for any other enzyme. It is noted that the catalytic efficiencies for the dinucleotide (Table 3) are more than an order of magnitude below the maximum for the simple Michaelis- Menten scheme; efficiencies below the maximum must be observed if the simple model, which implies that any 'isomerization' reaction(s) to form the productive complex are relatively very rapid, is adequate.

Another proposed mechanism (reviewed by von Hippel and Berg [37]) by which macromolecular reaction rates can

exceed diffusional limits is the existence of large electrostatic fields about macromolecules that are 'shaped' so as to guide a substrate into an active site. This seems very unlikely to be the explanation for the BAL 31 nuclease observations, however, because the DNA backbone is uniformly negatively charged so that such an effect would not seem likely to aid in directing the nuclease toward a 5' end of a long DNA chain. Moreover, these effects are abol- ished in other systems by increasing the ionic strength of the solution but all studies here were done at a high ionic strength (0.69 M).

4.5. Role o f facilitated diffusion fo r other enzymes acting on DNA

The are a number of other enzymatic reactions on DNA for which facilitated diffusion has been suggested as significant in the rate of location of specific sites (see [38] for a list of references). Also, eukaryotic enhancer sequences may exert their action on promoters distant along the contour of the DNA chain by a mechanism involving sliding of regulatory protein(s) between the two specific sites [39]. It is generally considered that the facilitated diffusion process is electrostatic in nature and thus sensi- tive to the salt concentration, so that at high concentrations of monovalent salt the mechanism goes over into a three- dimensional search (distributive search) as a result of shorter 'on' times of random DNA. protein complexes [40].

Most of the above studies were done in vitro and there had been some question as to the significance of facilitated diffusion in vivo because the search mechanisms become distributive at ionic strengths well below those thought to prevail inside the cell. Moreover, in the case of several site-specific restriction enzymes, the presence of a non- saturating amount of a bacterial histone-like protein also apparently rendered the mechanism distributive [41 ]. How- ever, in the case of the ultraviolet damaged repair enzyme T4 endonuclease V, there is in vivo evidence of facilitated diffusion to incise processively at multiple ultraviolet-induced lesions in a single DNA molecule after a single initial binding event [42]. Moreover, it has been shown that mutants of this enzyme, that maintain in vitro catalytic abilities similar to the wild-type enzyme but are unable to act processively, confer reduced levels of survival of ultraviolet light-induced damage, compared to that for the wild-type endonuclease, to E. coli cells that otherwise lack an ultraviolet repair function [38].

The BAL 31 nucleases display high catalytic efficiencies, which are readily explained by a facilitated diffusion mechanism according to a kinetic analysis developed for the first time in this study, for the cleavage of single- stranded DNA. This is apparently the first example in which catalytic efficiencies greater than the maximum expected for direct diffusion-limited formation of enzyme. substrate complexes have been demonstrated. In marked

138 T. Lu, H.B. Gray Jr. / Biochimica et Biophysica Acre 1251 (1995) 125 138

contrast to expectations based on other studies, this occurs in vitro in a medium of high ionic strength chosen to mimic that of the natural marine environment of these extracellular proteins. Because they normally degrade DNA outside of cells (presumably to provide nutrients for the Alteromonas organism), it is much less likely that com- plexing of DNA with proteins would be a factor in the natural activity of these enzymes. Thus, even if further work showed that bound proteins obviated the role of facilitated diffusion in this reaction, it would not follow that this mechanism was unimportant for the BAL 31 nucleases in their natural role.

Acknowledgements

This work was supported in part by Grant GM-21839 from the National Institutes of Health and in part by Biomedical Research Support Grant S07RR-07147 (Uni- versity of Houston).

References

[1] Hauser, C.R. and Gray, H.B., Jr. (1990) Arch. Biochem. Biophys. 276, 451-459.

[2] Wei, C.-F., Alianell, G.A., Bencen, G.H. and Gray, H.B., Jr. (1983) J. Biol. Chem. 258, 13506-13512.

[3] Bencen, G.H., Wei, C.-F., Robberson, D.L. and Gray, H.B., Jr. (1984) J. Biol. Chem. 259, 13584-13589.

[4] Zhou, X.-g. and Gray, H.B., Jr. (1990) Biochim. Biophys. Acta 1049, 83-91.

[5] Legerski, R.J., Gray, H.B., Jr. and Robberson, D.L. (1977) J. Biol. Chem. 252, 8740-8746.

[6] Gray, H.B., Jr., Winston, T.P., Hodnett, J.L., Legerski, R.J., Nees, D.W., Wei, C.-F. and Robberson, D.L. (1981) in Gene Amplifica- tion and Analysis. II. Structural Analysis of Nucleic Acids (Chirikjian, J.G. and Papas, T.S., eds.), pp. 169-203, Elsevier, New York.

[7] Wei, C.-F., Legerski, R.J., Robberson, D.L., Alianell, G.A. and Gray, H.B., Jr. (1983) in DNA Repair: A Laboratory Manual of Research Procedures (Friedberg, E.C. and Hanawalt, P.C., eds.), Vol. 2, pp. 13-40, Marcel Dekker, New York.

[8] Wei, C.-F., Legerski, R.J., Alianell, G.A., Robberson, D.L. and Gray, H.B., Jr. (1984) Biochim. Biophys. Acta 782, 404-414.

[9] Lau, P.P. and Gray, H.B., Jr. (1979) Nucleic Acids Res. 6, 331-357. [10] Kilpatrick, M.W., Wei, C.-F., Gray, H.B., Jr. and Wells, R.D.

(1983) Nucleic Acids Res. 16, 3811-3822.

[11] Lu, T. (1992) Ph.D. dissertation, University of Houston, Houston, TX.

[12] Hauser, C.R. (1989) Ph.D. dissertation, University of Houston, Houston, TX.

[13] Gray, H.B., Jr. and Lu, T. (1993) in Methods in Molecular Biology. Vol. 16. Enzymes of Molecular Biology (Burrell, M.M., ed.), pp. 231-251, Humana Press, Totowa, NJ.

[14] Brody, R.S., Doherty, K.G. and Zimmerman, P.D. (1986) J. Biol. Chem. 261, 7136-7143.

[15] Chang, L.M.S. and Bollum, F.J. (1971) Biochemistry 10, 536-542. [16] Cassani, G.R. and Bollum, F.J. (1969) Biochemistry 8, 3928-3936. [17] Ausubel, F.M., Brent, R., Kingston R.E., Moore, D.D., Seidman,

J.G., Smith, J.A. and Struhl, K. (1991) Current Protocols in Molecu- lar Biology, Vol. 1, pp. 7.6.1-7.6.7, Wiley, New York.

[18] Segel, I.H. (1975) Enzyme Kinetics, pp. 46-49, Wiley, New York. [19] Wilkinson, G.N. (1961) Biochem. J. 80, 324-332. [20] Nossal, N.G. and Singer, M.F. (1968)J. Biol. Chem. 243, 913-922. [21] Frank, R. and Koster, H. (1979) Nucleic Acids Res. 6, 2069-2087. [22] Ken, C. and Sadowski, P.D. (1972)J. Biol. Chem. 247, 311-318. [23] Thomas, K.R. and Olivera, B.M. (1978) J. Biol. Chem. 253, 424-

429. [24] Warshaw, M.M. and Tinoco, I., Jr. (1966) J. Mol. Biol. 20, 29-38. [25] Razzell, W.E. and Khorana, H.G. (1959) J. Biol. Chem. 234,

2105-2113. [26] Klee, C.B. and Singer, M.F. (1968) J. Biol. Chem. 243, 923-927. [27] Fersht, A.R. (1985) Enzyme Structure and Mechanism, 2nd ed., pp.

324-327, W.H. Freeman, New York. [28] Okazaki, R., Okazaki, T. and Sakabe, K. (1966) Biochem. Biophys.

Res. Comm. 22, 6! 1-619. [29] Winter, R.B., Berg, O.G. and von Hippel, P.H. (1981) Biochemistry

20, 6961-6977. [30] Fersht, A.R. (1985) Enzyme Structure and Mechanism, 2nd Ed., p.

152, W.H. Freeman, New York. [31] Studier, F.W. (1965)J. Mol. Biol. 11,373-390. [32] Gray, H.B., Jr., Bloomfield, V. and Hearst, J.E. (1967) J. Chem.

Phys. 46, 1493-1498. [33] Hearst, J.E. (1962) J. Mol. Biol. 4, 415-417. [34] Berg, O.G., Winter, R.B. and yon Hippel P.H. (1981) Biochemistry

20, 6929-6948. [35] King, E.L. and Altman, C. (1956) J. Phys. Chem. 60, 1375-1378 [36] Segel, I.H. (1975) Enzyme Kinetics, pp. 505-515, Wiley, New

York. [37] yon Hippel, P.H. and Berg, O.G. (1989) J. Biol. Chem. 264,

675-678. [38] Dowd, D.R. and Lloyd, R.S. (1990) J. Biol. Chem. 265, 3424-4331. [39] Wasylyk, B., Wasylyk, C., Augereau, P. and Chambon, P. (1983)

Cell 32, 503-514. [40] Leirmo, S., Harrison, C., Cayley, D.S., Burgess, R.R. and Record,

M.T., Jr. (1987) Biochemistry 26, 2095-2101. [41] Ehbrecht, H.-J., Pingoud, A., Urbanke, C., Maass, G. and Gualerzi,

C. (1985) J. Biol. Chem. 260, 6160-6166. [42] Gruskin, E.A. and Lloyd, R.S. (1988) J. Biol. Chem. 263, 12728-

12737.

kinetics and mechanism of bal 31 nuclease action on small substrates and single-stranded dna

Documents