,J. Mol. Bid. (1976) 103, 61-76

Studies on the Mechanism of Enzymatic DNA Elongation by Escherichia coli DNA Polymerase II LINDA A. SHERMAN AND IULCOLM

L. GEFCER

Department of Biology Massachusetts Institute of Technology Cambridge, Mass. 02139, U.S.A. (Received 27 October 1975, and in revised form 23 January 1976) The mechanism of enzymatic elongation by Escherichiu coli DNA polymerase II of a DNA primer, which is annealed to a unique position on the bacteriophage fd viral DNA, has been studied. The enzyme is found to dissociate from the substrate at specific positions on the genome which act as “barriers” to further primer extension. It is believed these are sites of secondary structure in the DNA. When the template is complexed with E. coli DNA binding protein many of these barriers are eliminated and the enzyme remains associated with the same primertemplate molecule during extensive intervals of DNA synthesis. Despite the presence of E. co.5 DNA binding protein, at least one barrier on the fd genome remains rate-limiting to chain extension and disturbs the otherwise processive mechanism of DNA synthesis. This barrier is overcome by increasing the concentration of enzyme. In contrast, it is found that DNA polymerase I is not rate-limited by structural barriers in the template, however, it exhibits a non-processive mechanism of elongation. These tidings provide a framework for understanding the necessity for participation of proteins other than a DNA polymerase in chain extension during chromosomal replication.

1. Introduction Since the initial discoveries of Escherichia coli DNA polymerases II and III it has been clear that the basic catalytic activities of the three E. coli DNA polymerases are quite similar (for a review, see Gefter, 1975). This is in contrast to their physiological roles which appear to be quite different (Gefter, 1975). Thus, many investigations have been conducted in order to determine the chemical basis for the diverse biological functions of these enzymes. On careful examination of their catalytic activities, it has been noted that both DNA polymerase II (Sigal et al., 1972; Wickner et aZ., 1972) and DNA polymerase III (Gefter, 1974; Livingston et al., 1975) are capable of efficiently extending a DNA primer only when the template strand is less than 100 to 300 nucleotides in length. When this length is exceeded, both the observed initial rate of primer extension and the final extent are reduced. DNA polymerase I activity on the other hand, is relatively unaffected by the length of the template. Thus it is expected that there is a fundamental difference in the mechanism of catalysis of these enzymes and that 61

L.

62

A.

SHERMAN

AND

M.

L.

GEFTER

additional proteins might be found that effect the elongation reaction catalyzed by DNA polymerases II and III. In searching for such “factors”, it was found that a DNA binding protein isolated the enzymatic activity of DNA polymerase from E. coli was capable of stimulating II by 10 to 50-fold (Sigal et al., 1972; Molineux et al., 1976). This protein has the property of binding tightly and co-operatively to single-stranded DNA (Sigal et al., 1972; Molineux et al., 1976) as well as binding to DNA polymerase II (Molineux & Gefter, 1974). In addition, it has also been observed that the DNA binding protein stimulates DNA polymerase III-catalyzed primer extension provided that additional factors are included in the reaction (Geider & Kornberg, 1974: Hurwitz & Wickner, 1974). In order to elucidate the parameters in enzymatic DNA chain elongation which necessitate the participation of proteins other than a DNA polymerase, we have undertaken a study of the mechanism by which DNA primers are elongated in vitro. We have begun this study utilizing DNA polymerase II since this enzyme, as well as its stimulatory co-factor, the E. coli DNA binding protein, have been purified to homogeneity (Sherman, 1976; Molineux & Gefter, 1974). Previous studies on the nuclease activity of DNA polymerase II have suggested that one way in which binding protein may stimulate polymerase activity would be the induction of a processive mechanism of enzymatic synthesis (Molineux & Gefter, 1976). More explicitly, this hypothesis predicts that in the ab-,ence of DNA binding protein the polymerase dissociates from the DNA substrate after each of several nucleotides have been added. Thus overall chain growth occurs by a repeated series of associations and dissociations of the enzyme from the DNA substrate. However, in the presence of binding protein, the enzyme molecule remains associated with the same primer-template molecule until it is completely copied. To test such a possibility, as well as to facilitate determination of potentially ratelimiting steps in elongation, a defined substrate has been constructed. It consists of bacteriophage fd DNA SST to which is annealed a specific primer molecule obtained as a restriction nuclease fragment. Elongation can be monitored by size analysis of the primer using gel electrophoresis under denaturing conditions. It has been found that secondary structure in the template inhibits ongoing synthesis by polymerase II. The effect of the E. coli DNA binding protein (Sigal et al., 1972) which has the capacity to remove such regions of template secondary structure is studied. In addition, the details of the reaction mechanism of DNA polymerase II are contrasted to that of E. coli DNA polymerase 1.

2. Materials and Methods Unlabeled deoxyribonucleoside triphosphates and “gapped” calf-thymus DNA are as previously described by Sherman (1976). All four tritium-labeled deoxyribonucleoside triphosphates were purchased from New England Nuclear Corp. Bacteriophage fd strand (SS) DNA was prepared by hot phenol extraction of purified pha,ge (Yamamoto & Alberts, 1970) in the presence of 2?/, sodium dodecyl sulfate, followed by repeated precipitation with ethanol. Unlabeled replicative form I fd (RFI) DNA was prepared by the method of Model & Zinder (1974) with the following alterations: cell lysis and the preparation of a viral DNAt Abbreviations

used: SS, virion

positive

strand DNA;

RF, replicative

form DNA.

DNS

POLYMEHASE

II:

MECHANISM

OF

ELONGATION

63

containing supernatant, solution was carried out by the method of Reuben et al. (1974). Deproteinization was achieved by extraction with an equal volume of chloroform/isoa,myl alcohol (25: I, v/v). The aqueous layer was then adjusted to 70°& ethanol and the DNA collected by centrifugation, was then air-dried and rcprocipit)ated. The precipitate, suspended in 50 ml of 10 mM-Tris.HCl (pH 7.5), 10 mM-NaCl, 5 mM-EDTA (Tris/EDTA/Na hllffer). The solut,ion was extracted with phenol and the RF1 was purified as described b> Model & Zinder (1974). The final yield of RF1 was 0.5 mg/l of infected cells. from cells grown in 200 ml of til R.F DNA uniformly labeled with 32P was prepared low-phosphate medium (Pieczmik et al., 1972) supplemented with 10 pg of K2HP0,/ml. The [32P]ort,hophosphate was added 10 min aft’er infection. The viral DNA-containing supcrnat,ant was prepared as doscribed above for a non-radioactive sample. After phenol (\xtract ion and ethanol precipitation, the pellet was resuspended in 1 ml of Tris/EDTA/Na I)llffer. 50 i~g of pancreatic RNAase were added and the solution was incubat’ed for 30 min cont’aining at 37°C. It, wa,s then layered onto a 59;) to 20°b (w/v) sucrose gradient 10 mM-Tris.HCi (pH 7.5), 1 mM-NaCl, and 1 m&I-EDTA. Cent’rifugation was for 4 h ai, 40,01)0 rcavsjmin in an SW41 rot,or (Beckman Instrumants Co.). 0.3-ml fractions \VCIY collect,cd as the gradient was pumped through a syringe needle inserted to t,he bott’om of the: tube. The peak of acid-precipit)able 32P-labeled material, which migrates to a position c*tlaract8cGtic of RF as determined by an external marker, was collected and used in t,h(x l)rc>paration of primer fragment’s after concentration t,o 1 ml by dialysis against, 30”,, polyothylenc~ glycol in 10 mM-Tris.HCl (pH 7.4), 1 miw-NaCl, and 1 mM-EDTA. Such prc’Ijarations yield 0.1 m of DNA which contains 0.3”; of the input radioactivit,y. Both unlabeled and 32P-labeled HpaII (see Materials and Methods, section (h)) frapIIICY~~,S\vcxrt> 1~1~~par~~i in an incubation mixture which contained 10 miw-Tris.HCl (pH 7.4). 10 mM-Mg(.‘l,, 6 tn.M-KCI, 10 mM-2-merca,ptoet,hanol, .50 to 100 pg fd RF/ml, and sufficient cl,lz,vmcb to allow for ctompleto digestion after 2 h of incubation at 37°C. “2P-labclt,d f&g:ruvnts were! separated by directly layering the 1 ml of incubation mixture adjusted t,o 15”,, surros~ onto a soA polyacrylamide slab gel (20 cm x 20 cm x 1.5 cm) (Loening, 1969). Fragm~~nt posit’ion was determined by aut,oradiography. In t,h(x case of ur~labelrd DNA, sllfficient 32P-labcletl fragment’s were added to allow for aut80radiographic detection. Thll incubation mixture was then extracted wit,h phenol and precipitat,ed with ethanol. ‘I’h~~ fr;rgmt,nt,s wercl dissolved in 10 mM-Tris.HCl (pH 7.4), 1 mm-NaCl, 1 mM-EDT>%, 15”,, slIcros(‘ at, a concentration of 0.25 mg/ml. Up to 2 ml was applied per slab gel. Hpall fi.agment’s 1, J - K, L and M (Seeburg & Schaller, 1975) were cut out, and eluted into ‘l’l~isjEDTd/Na buffor by gel homogenization. Gel part,iclos were rcamoved by ccnt,rifugatio1l :jt 12,000 g for 20 min. The supernatant, was further clarified and contaminating lo\\ molccu1a.r weight 32P-labpled material removed by adsorption to a column of DEAEc~c~llulose (DE23) (0.5 cm x 0.4 cm) previously equilibrated with Tris/EDTA/Na buffer. The column was t’hen washed with 5 ml of 0.25 M-Na.Cl, 10 mM-TriseHCl (pH 7.5), 5 mMlI-Tris. 1 InM--\J:tCl, 1 mM-EDTil, and then further desalted by filtration on Sepbadex G25 (I’harn~ncia Fine Chemical). 32P-labeled substrate was prepared as described above excc>pt that. t,hc, molar rat,io of fragments TaoSS w&s 0.2 to allow for 80”;, ntilization of th(l raciioi1c.t i\,(a rrc~yat~i\~~~st ranll of’ cxach fragmcant,. .i- .TO”,, of fd SS rnolcc~1tlcs :lrc l)rimc(l.

64

L. A. SHERMAN

AND

M.

L.

GEFTER

(b) Enzymes E. coli DNA polymerase I (fraction ‘7; Jovin et al., 1969) was a gift from Dr David Baltimore of this institution. E. coli DNA polymerase II (2500 unitsjmg) was prepared as previously described (Sherman, 1976). Hemophilua puruinjkenzae endonuclease II (HpaII) was prepared by the method of Sharp et al. (1973). E. coli DNA binding protein was purified by the method of Molineux & Gefter (1974). It was concentrated by adsorption to DEAE-cellulose equilibrated with 20 m&r-Tris.HCl (pH 7~4)~ 50 mM-NaCl, followed by elution with 1 M-N&~ in this same buffer. It was then dialyzed against 50% glycerol in 20 m?d-TrisHCl, 50 mm-N&l, 1 mM-EDTA, 1 mM-2-mercaptoethanol. Contaminating DNA polymerase II activity was eliminated by heating the binding protein for 10 min at 70°C. Assays for DNA polymerases I and II on “gapped” DNA were done as previously described (Sherman, 1976). Defining a unit as that amount of enzyme necessary for the incorporation of 1 nmol of thymidine 5’-phosphate in 5 min at 3O”C, the specific activity of DNA polymerases I and II was 7000 and 2500, respectively, on gapped calf-thymus DNA?. Incubation mixtures for fragment-primed synthesis of fd SS (30 ~1) contained 67 InMTris-acetate (pH 7*4), 6.7 mM-MgCls, 3.3 mM 2-mercaptoethanol, 10 mM-ammonium sulfate, 15% (v/v) glycerol, O-1 mg bovine serum albumin/ml, and 33 PM of each of 4[3H]dNTPs (2000 cts/min per mol); 0.25 pmol of fragment-primed molecules, and enzyme. When binding protein complexed with DNA was used as substrate, only the thymidine 5’-triphosphate contained tritium label. For synthesis by polymerase I, only the TTP contained tritium label and 0.2 ~-Kc1 replaced the 10 mM-ammonium sulfate. Reactions were stopped by the addition of 210 ~1 of 0.1 M-N&~, 20 mM-EDTA, and 10 mmTrisHCl (pH 7.4). Unless otherwise stated, incubations were at 30°C. (c) Size anaZy8i.s Samples were prepared for polyacrylamide gel electrophoresis under denaturing conditions as follows: E. coli tRNA was added to a concentration of 100 pg/ml. Samples were then phenol-extracted, and ethanol-precipitated. After collecting the precipitate in siliconized tubes, the samples were desiccated, and dissolved in 20 pl of formamide which was previously deionized with Bio-Rad Mixed Bed Resin and buffered with 20 mbil-sodium barbital (pH 9.0). Solid sucrose and bromophenol blue were added. The sample was heated at 45°C for 20 min and then electrophoresis was carried out on polyacrylamide slab gels which contained either 98% formamide (Pinder et al., 1974) or 8 M-urea (Loening, 1969). For alkaline sucrose gradient analysis, reaction mixtures were either directly ethanolprecipitated, or deproteinized as described above and then precipitated. The pellet was resuspended in 0.2 M-NaOH, O-1 M-NaCl, 1 mm-EDTA, and layered on a 5% to 20% sucrose gradient made in 0.1 M-NaOH, O-9 M-NaCl, 1 mM-EDTA; it was centrifuged at 45,000 revs/min in a Beckman SW50.1 rotor for 4.5 h at 20°C.

3. Results (a) DNA synthesis by DNA polymerases I and II on fragment-primed bacteriophage fd single strands Previous studies of the catalytic properties of DNA polymerase II have demonstrated that its rate of polymerization is sensitive to the length of the single-stranded template being copied. The enzyme is unable to utilize efficiently DNA substrates containing single-stranded regions which are more than 100 nucleotides long (Sigal t The follows:

number

of pm01

of DNA

polymerase

pm01 -=unit The The

mol. mol.

w of DNA w of DNA

polymerase polymerwe

per unit

of enzymatic

1X109

mol

I is 109,000 II is 86,000

w x spec. act. units w (Jovin et al., 19691. (Sherman, 1976).

activity

was

calculated

as

DNA

POLYMERASE

II:

MECHANISM

OF

65

ELONGATION

TABLE 1 Synthesis

by E. coli DNA

polymerases Incorporation

Units

rate(pmol/5

Gapped DNA

.-. Polymrrase I

0.035

Polymerase

II

0.0 I

40

Polymerase

II 1. binding

0.01

60

protein

min) Primed fd SS

~__.-

140

36