Proc Nut1 Ad Sei USA
Vol 77. No 2, pp 799-803, February 1980
Biochemistry

An Escherichia coli replication protein that recognizes a unique
sequence within a hairpin region in 6x174 DNA

(DNAdependent ATPase/sequence specificity/protein n'/origin of &X DNA complementary strand replication)

JOSEPH SHLOMAI AND ARTHUR KORNBERG
Department of Biochemistry. Stanford University School of Medicine, Stanford, California 94305

Contributed by Arthur Kornberg, Nouember 5,1979

ABSTRACT  Protein n', a prepriming DNA re lication en-
zyme of Escherichia coli, is a 4x174 DNAdepencfent ATPase.
Restriction endonuclease fragmentation and exonuclease VI1
digestion of 9x174 DNA have led to the identification of a
Snucleotide fra  ent that carries the protein n' recognition
sequence. Molecg hybridization and sequence analysis have
located this sequence within the untranslated region between
genes F and G, a map location analogous to that of the unique
complementa  strand origin of phage G4 DNA. Within the
55nucleotidexagment is a sequence of 44 nucleotides that
forms a stable haiqiin structure. This duplex ma be the signal
for protein n' to initiate the prepriming events tiat lead to the
start of 6x174 complementary DNA strand replication.

Conversion of the single-stranded chromosomes of phages M13,
G4, and 4x174 (4X) to their duplex replicative forms are model
systems for studies of the mechanisms of initiation of DNA
synthesis in Escherichia colt. The more complex $X system
appears to be particularly pertinent to the discontinuous phase
of E. colt chromosomal replication (1,2).
The M13, G4, and @X templates differ primarily in their
enzymatic requirements for the initiation of primer synthesis.
Coated with single-stranded-DNA binding protein (SB), M13
DNA can be primed directly by RNA polymerase (3) and G4
DNA, by primase (4,5). However, SSB-coated #JX DNA, al-
though primed by the same E. colt primase, must first be acti-
vated in a prepriming stage. In this prepriming reaction, the
E. coli proteins n', n and n", i, dnaB, and dnaC form an acti-
vated complex with 4X DNA (2.6-8).
The distinctions in primer synthesis on these three phage
templates are probably due to structural differences in "pro-
moter"-like sites recognized by the distinctive priming systems.
Although the origin of complementary DNA strand replication
is unique and well characterized for M13 (9) and G4 (10-14),
it does not appear to be at a unique site for 4X. as judged by in
oioo and tn oitro studies (7,9, 11, 15).
We will describe elsewhere the purification of protein n' to
near homogeneity, its 4X DNA-dependent ATPase activity,
and its capacity to destabilize an SWX DNA complex. In this
paper we report the recognition by protein n' of a specific se-
quence in 4X DNA located at an intergenic region and suggest
that it may be the signal that leads to the initiation of 4X
complementary DNA strand replication.

MATERIALS AND METHODS

Nucleic Acids, Enzymes, Resins, and Nucleotides. @X, qiX
replicative form (RF)I, G4, and M13 DNAs were prepared as
described (16). E. coli SSB was 4 X l(r units/mg (17); Hae 111
and HinfI restriction endonucleases were from Bethesda Re-
search Laboratories, Rockville, MD, and Mbo XI was from New
England BioLabs; exonuclease VI1 preparations were gifts from
S. P. Goff (Massachusetts Institute of Technology) and from J.
W. Chase (Albert Einstein Medical School); TCpolynucleotide
kinase was from P-L Biochemicals. Hydroxyapatite was ob
tained from Bio-Rad, agarose was from Bethwja Research

Table 1.

Localization of the n' recognition site to a particular

nuclease restriction fragment

ATP hydrolyzed,

DNA effector pmol

None
+X174
G4
Hoe I11 fragments of t$X

21
22
23
24
25-11*

<I
132
3

114
8
13
9
8

32P-labeled single-stranded 6X DNA (200 pg) was incubated with
450 units of Hue 111 nuclease in 50 mM Tris-HC1 (pH 7.5), 5 mM
MgC12, and 0.5 mM dithiothreitol at 37OC for 20 hr. Reactions were
stopped by additions of EDTA, Sarkosyl, glycerol, and bromphenol
blue to final concentrations of 50 mM, 270, 10% and 0.01%. respec-
tively. Digestion products were electrophoresed in a 2.5% agarose gel
in Tris acetate and stained with ethidium bromide (1 pg/ml). DNA
bands were sliced and extracted. Purified DNA fragments at 0.8 nM
covered by SSB (one SSB per nine nucleotides) were assayed as ef-
fectors for n'-ATPase activity by using the standard assay condi-
tions.
o Analyzed separately or collectively, the values for these Hue 111

fragments were 8 or less.

Laboratories, and polyethyleneimine-cellulose was from
Brinkmann. 1-[(m-Nitrobenzyloxy)methyl] pyridinium chlo-
ride was a gift from G. M. Wahl of this department.
Assays for Protein n'. (i) Reconstitution of a single-stranded
ctrcvhr DNA (SS) -. RF 4.X DNA replication reaction. The
reconstituted enzymatic reaction was as described (17).
(ti) DNA-dependent ATPase. The assay measures the pro-
duction of labeled ADP from [3H]ATP or [CY-~PJATP and could
also be applied to the production of 32Pi from [y-32P]ATP.
Standard assays were carried out in 25-pl reaction mixtures
containing 10 mM KCI, 1 mM MgC12, 1 mM labeled ATP or
dATP, 120 pmol of 4X DNA (as nucleotide), 50 mM Tris-HC1
(pH 7.5), 6% (wt/vol) sucrose, and bovine serum albumin at 0.2
mg/ml. Samples to be assayed were diluted in 50 mM imid-
azole.HC1, pH 6.8/25!% glycero1/100 mM ammonium sul-
fate/bovine serum albumin at 0.2 mg per ml/l mM EDTA.
When SSB was used, 1 pg was added, unless otherwise noted.
Reactions were carried out at 30°C unless otherwise noted.
Aliquots of 2 pl were applied to polyethyleneimine-cellulose
strips (0.6 X 6 cm) together with unlabeled ATP, ADP, and
AMP markers. The strips were developed with 1 M formic
acid/0.5 M LiCl at room temperature, dried, and examined
with UV light to locate and cut out the ATP and ADP spots.
Radioactivity was determined in scintillation fluid without

Abbreviations: &X, 4x174; SS, single-stranded circular DNA; RF,
double-stranded DNA of circular replicative form; SSB. single-
stranded-DNA binding protein; DBM, diazobenzyloxyrnethyl.

800 Biochemistry: Shlomai and Kornberg

Proc. Natl. Acad. Sci. USA 77(1980)

INCUBATION TIME, min

FIG. 1. Exonuclease VI1 resistance of DNA effector activity. The
Hae 111 21 DNA fragment (300 pmol in nucleotides, Table 1) was
incubated with 0.3 unit of E. coli exonuclease VI1 in 13 pl of 67 mM
potassium phosphate, pH 7.9/8.3 mM EDTA/IO mM 2-mercap-
toethanol at 37OC or 45'C. At indicated times, reactions were pre-
pared for ATPase assays by the following additions: MgCIz to 21 mM,
KCI to 10 mM, sucrose to 6%, bovine serum albumin to 0.1 mg/ml,
SSB to 10 pg/ml. and ["HJATP (20,000 cpm/pmol) to 1 mM. ATP
hydrolysis at 30°C was measured 15,30, and 45 min after the addition
trf protein n' (fraction VIII) (unpublished results). Values represent
rates of ATP hydrolysis corrected for background ATPase activity
of the exonuclease VI1 preparation.

elution. One unit of n' ATPase activity represents the cleavage
of 1 pmol of ATP or dATP per min. The ATPase assay condi-
tions, designed to resemble those of the replication assay, were
not optimal with respect to pH (8540% of maximal activity)
or @X DNA concentration (about 65% of maximal activity).

OWA
FRAGMENT

ORIGIN -..

-3

FIG. 2. Separation of exonu-
clease VII-resistant sequences in
Hae 111 DNA fragment Z1 in a
polyacrylamide gel. 32P-labeled
Hoe I11 21 fragment (2.9 pg) was
incubated at 37'C for 60 min with
5 units of E. coli exonuclease VI1 as
in Fig. 1. Reaction products were
precipitated overnight at -20°C in
0.3 M sodium acetate (pH 5.5) with
2 vol of isopropanol. The precipi-
tate was collected by centrifuga-
tion in the SW56 rotor at 50,000
rpm and 0°C for 2 hr, dissolved in
80% deionized formamide, heated
for 3 min at 100OC and cooled in an
ice-water bath, adjusted to 0.01%
in bromphenol blue and xylene
cyano1 FF, and electrophoresed on
a preparative 12% polyacrylam-
ide/7 M urea gel (20) at a constant
voltage of 200 V for 8 hr at room
temperature. DNA bands were
detected by autoradiography of the
wet gel. Numbers in parentheses
indicate fragment size in resi-
dues

I I I I I

3.4 1

SB

15 30 45 60 90

TIME, min

FIG. 3.

     DNA effector activity for n'-ATPase in exonuclease
VII-resistant DNA sequences of Hae I11 Z1 fragment. The Hoe 111
Z1 fragment (Table 1) was digested with exonuclease VII; the products
were separated by electrophoresis in 12% polyacrylamide/'i M urea
gels as in Fig. 2. DNA fragments 1,2,3, and 4 were electroeluted from
the gel, concentrated by the hydroxyapatite procedure, and assayed
for effector activity in the n'-ATPase reaction. Assays covered a range
of DNA concentrations (0.2-1.6 nM) in the presence of SSB (one SSR
per nine DNA nucleotides). Values were obtained at 1.6 nM DNA.

Exonuclease VI1 Assay. E. coli exonuclease VI1 was assayed
as described by Chase and Richardson (18, 19). Reaction
mixtures of 25 pl were used at 37"C, unless otherwise noted.

Gel Electrophoresis. Electrophoresis of DNA was carried

250 I I I I I

I

200

/gomil

DNA, nM

FIG. 4.

     Influence of concentration of fragment 2 on n'-ATPase
reaction. Exonuclease-resistant fragment 2 (Fig. 3) was assayed for
its activity as a DNA effector in the n'-ATPrse reaction after 30,60,
and 90 min of incubation under standard assay conditions.

Biochemistry: Shlomai and Kornberg

Proc. Natl. Acad. Sci. USA 77 (1980)

801

out in 0.7-3.5% agarose gels at constant voltage (1 V/cm) at
room temperature in either 90 mM Tris borate, pH 8.3/2.5 mM
EDTA or in 40 mM Tris acetate, pH 7.8/5 mM sodium ace-
tate/l mM EDTA. Electrophoresis of DNA fragments in 12%
or 20% polyacrylamide/7 M urea gels was as described (20).
Other Procedures. Labeling of DNA 5' ends with 32P by T4
polynucleotide kinase and determination of DNA sequence
were as described by Maxam and Gilbert (21). Preparation of
diazobenzyloxymethyl (DBM) paper, transfer of DNA to DBM
paper, and hybridization procedures were as described by Al-
wine et al. (22) and by Wahl et al. (23). To isolate DNA frag-
ments from agarose gels, the gels were digested with 1.5 vol of
6 M NaClOd at 65°C for 10-20 min (or at 37°C for 2 hr). The
DNA was adsorbed on hydroxyapatite columns in 10 mM so-
dium phosphate buffer (pH 7) and maintained at 65°C. Loaded
columns were washed with several bed volumes of this buffer
and eluted with a gradient of 0.3-0.5 M sodium phosphate
buffer (pH 7). Alternatively, the same procedure was used
batchwise at room temperature (G. Weinstock, personal com-
munication); DNA was extracted from gels by electroelu-
tion.

RESULTS

A Unique Region in 4X Chromosome is Recognition Site
for Protein n'. ATP or dATP hydrolysis catalyzed by the E. coli
prepriming enzyme n' was DNA-dependent, used 4X DNA
preferentially as an effector, but was inactive with other DNAs
in the presence of SSB (Table 1). Among the separated single-

A

stranded Hae 111 fragments of 4X DNA assayed as effectors for
n'-ATPase activity, only one (Z1, which includes about one-
fourth of the @X chromosome as Seen in Fig. 6) could serve and
was as effective on a molar basis as intact @X DNA.
Recognition Site for Protein n' in 4X SS Has Secondary
Structure. Activity of an SSB-coated 4X DNA fragment as an
effector in the n'-ATPase reaction, when other coated single-
stranded DNAs are completely inactive (unpublished obser-
vations), suggests a recognition site with persistent duplex
structure. For this reason, the isolated Hae I11 Z1 fragment was
digested by exonuclease VII, which degrades single-stranded
DNA from both the 3' and 5' ends but does not digest double-
stranded DNA (19). The digest retained effector activity
(>45%) even after prolonged incubation at 37"C, but at 45°C
the activity of Z1 was destroyed (>95%) (Fig. 1). Resistance of
the activity of the Z1 fragment at 37°C and susceptibility at
45°C suggested that the effector activity of this single-stranded
DNA fragment is in a duplex denatured at 45°C.
Isolation of Duplex DNA Region that Carries Protein n'-
Recognition Site. The limit-digestion products of exonuclease
VI1 hydrolysis of fragment Z1 separated by electrophoresis were
mainly oligomers 8-12 nucleotides long (Fig. 2). However, 7.5%
of the digested DNA was found in longer chains: fragment 1,
65-70 residues (2.5%); fragment 2,55-60 residues (3.6%); and
fragment 3, 18-22 residues (1.4%). Only fragment 2, repre-
senting about 1% of the original $X chromosome, was active
as an effector for n'-ATPase (Fig. 3); the longest fragment
(fragment 1) was inactive. ATP hydrolysis rate with fragment
2 (Fig. 3) was 52% of that with intact 4X DNA. ATPase activity

8

ORIGIN

-53U6-

-1353-

-812-

-413-


- 250-

- -

.

n

FIG. 5L

    Sequence homology of exonuclease VII-resistant fragment 2 as judged by hybridization to various regions of gX DNA. gX RFI DNA
(0.8 pg) was incubated at 37OC for 1 hr with restriction endonucleases as follows: Hae I11 (3 units) in 50 mM Tris.HC1, pH 7.5/5 mM MgC12/0.5
mM dithiothreitok HinfI (3 units) or Hoe I11 and HinfI simultaneously (each 3 units) in 6 mM Tris-HCI. pH 7.5/6 mM MgCln/6 mM 2-mercap-
toethanoU100 mM NaCI; or Mbo I1 (3 units) or HinfI and Mbo I1 simultaneously (each 3 units) in 10 mM Tris.HC1, pH 7.9/6 mM KCI/10 mM
MgCIJ1 mM dithiothreitol. Reactions were stopped by addition of EDTA, Sarkosyl. glycerol, and bromphenol blue to final concentrations
of 50 mM, 296, 10% and 0.0196, respectively. (A) DNA fragments were electrophoresed in 2% agarose gels and stained with ethidium bromide
(1 pg/ml). DNA was denatured in the gel and transferred to DBM paper (22). (R) 5'-:v2P-labeled exonuclease VII-resistant DNA fragment 2
(Fig. 2) was hybridized (23) in the presence of 10% dextran sulfate-500 at 42'C for 40 hr to the DNA fragments bound to DBM paper; the paper
was washed (23) and subjected to autoradiography at -80°C by using an intensifying screen (Cronex, Du Pont). In the far right lane, :"P-labeled
@X DNA was used as a size and transfer marker.

802 Biochemistry: Shlomai and Kornberg

PRDTflN n

Proc. Nut[. Acad. Sci. USA 77 (1980)

2394 2465

RECOGNITION
LOCIJS \, ---~

22x5,

,3731

T-T 2330

/\
TA
\/

T*A
II
A*T
II

2325 A-T
   II
   A'T 2335
   11
   A-T
   II
   A*T
   I1

2320 G :P *C

II

j* 4949 `4877

5ia

     Location of protein n' recognition locus on @X chromo-
some. Heavy lines represent restriction fragments of @X RFI DNA,
to which exonuclease VII-resistant DNA fragment 2 was hybridized
(Fig. 5B). Broken lines indicate products of a partial digestion of an
M60 I1 fragment (1653-2465) by HinfI, to which the radioactive DNA
probe was hybridized (Fig. 58). The magnified region (2264-2465)
closest to the center of the diagram is the hybridized sequence com-
mon to all three restriction digests. The n' recognition site is located
within this 201-nucleotide region. It contains an untranslated se-
quence of 110 nucleotides (22s2394) between structural genes F and
G; within this intergenic region is the hairpin sequence of 44 nucleo-
tides (see Fig. 7) in the exonuclease-resistant DNA fragment 2

FIG. 6.

(230a235i).

is a function of the concentration of the DNA effector, but the
dependence is more nonlinear at low concentrations (Fig. 4).
Location of Isolated Protein n` Recognition Sequence in
4X Chromosome. Molecular hybridization and sequence
analysis were used to map the location of the exonuclease VII-
resistant fragment 2 (above). Products of digestion of @X RFI
DNA by the restriction endonucleases Hoe HI, HinfI, and Mbo
I1 were separated on 2% agarose gels (Fig. 5A), denatured, and
transferred to DBM paper (22). Fragment 2, labeled with 32P
at its 5' end by polynucleotide kinase, was hybridized to the
denatured, paper-bound @X DNA fragments (Fig. 5B) (23).
with the Hoe 111 digest separated into nine bands (Fig. 5A),
the fragment 2 probe hybridized, as expected, only to fragment

Zl (24). 1353 residues at position 1777-3129 (Fig. 6).

Of the ten bands produced by HinfI digestion (Fig. 5A), the
probe hybridized mainly to fragment F5b (24), 413 nucleotides
long (Fig. 5B) at position 2265-2677 (Fig. 6). F5b was part of
a triplet band with fragments F5a and F5c (24), 417 and 427
nucleotides long, respectively; but these two fragments (at
positions 3732-4148 and 4702-5128) are well outside the Hue
111 Zl sequence. Fragment F5a is also excluded by the results
of the double digestion by Hue I11 and Hinfl. Although the F5a
sequence contains three Hue 111 sites (at positions 4759,4877,
and 4949) (Fig. 6), hybridization of the probe to a fragment
about 413 residues long was not affected. Slight hybridization
to a HfnfI (or HinfI plus Hoe 111) digestion product about 250
nucleotides long (Fig. 5B) will be considered below.
With the Mbo I1 digest, which yielded six bands (Fig. 5A),
the fragment 2 probe hybridized to fragment M4 (24), 812

A*+ 2350 2354

2301 2305 11

5'-A-G-G -T- T-A -T- A o T-G -A-C - C -3

FIG. 7.

     Possible secondary structure in recognition locus of
protein n'. The proposed structure of base-paired regions separated
by two internal loops and a hairpin loop is based on a calculation of
free-energy contributions of base-paired regions and loops of the se-
quence at position 230a2351 (Fig. 6) within the 55-nucleotide frag-
ment possessing the protein n' recognition site.

nucleotides long (Fig. 5B) at position 1654-2465 (Fig. 6). M4
migrated in the agarose gel as part of a doublet band with M3,
837 nucleotides long (at position 817-1653) (Fig. 6). Not only
is M3 outside the Hoe 111 Z1 sequence, but it is also eliminated
by the results of a double digestion (completely with Mbo I1 and
partially with HinfI). Fragment 2 hybridized to another set of
fragments generated by the incomplete digestion of the Mbo
I1 fragment M4 by HinfI (Figs. 5B and 6). Hybridization was
observed with the 812-nucleotide fragment when no cuts were
introduced in this region by HinfI, and with fragments 201,
450, and 568 nucleotides long, produced by HinfI cuts within
M4 at positions 2264,2015, and 1897.
In summary, the hybridization experiments limit the location
of the exonuclease VII-resistant fragment 2 to a 201-residue
region at position 2265-2465. It remained to be determined
whether the sequence of fragment 2 contains the potential for
secondary structure.
A Hairpin Structure in Recognition Site. Exonuclease
VII-resistant fragment 2 upon electrophoresis in 20% polyac-
rylamidej7 M urea contained a major component 55 nucleot-
ides long and a minor one, one or two nucleotides shorter. To
map this fragment precisely, the DNA sequence was deter-
mined. Analysis of a 5%nucleotide region in this fragment (data
not shown) and comparison of the sequence with the Sanger
map (24) locates it at position 2304-2355. Within this fragment
is a 44-nucleotide-long sequence at position 2308-2351 with
a strong potential for secondary structure (Fig. 7). Calculations
of the free energy contributions of the base-paired regions and
loops, based on studies with polyribonucleotides (E), yield a
value of -14 kcal/mol at 25°C and 1 M NaCI. This value is
consistent with a duplex form for this sequence (Fig. 7) under
the experimental conditions in which it resists destabilization
by SSB and digestion by exonuclease VI1 (Fig. 2).
The HinfI (or HinfI plus Hue 111) fragment about 2-50
nucleotides long that showed some homology to fragment 2 (see

Biochemistry: Shlomai and Kornberg

Proc. Natl. Acad. Sci. USA 77 (1980)

803

above) is the 249 nucleotide-long HinfI fragment F7 (24) at
position 2016-2264, as judged by its size and its location within
a sequence that overlaps both the Hae 111 Z1 and the Mbo II M4
DNA fragments that were shown to be the only Hae 111 and
Mbo I1 4X DNA fragments with homology to the DNA probe
(Fig. 5B). The F7 DNA fragment contains (in its viral strand)
a sequence of eight nucleotides, GCT-GA-GGG (at position
2039-2046), identical to one on the exonuclease VII-resistant
DNA fragment (position 2341-2348); this may explain its hy-
bridization to the DNA probe.

DISCUSSION

A prepriming system reconstituted from six E. colt proteins is
needed to prepare the single-stranded, binding-protein-covered
$X DNA circle for primer synthesis by primase to initiate DNA
replication (26, 27). This system is specific for @X DNA and
does not operate on the SSs of phages G4 and M13 (26, 27).
Unlike phages G4 and M13, which posses unique origins for
complementary strand starts, both in utvo and in uttro studies
indicate multiple starts on $X DNA (7,9, 11, 14). However, the
strict specificity of the prepriming system for 4X DNA suggests
that a particular locus is recognized by one or more of the
proteins of the prepriming system. Protein n` is a candidate for
this role.
Protein n', now available in homogeneous form (unpublished
observations), is an ATPase dependent on the presence of DNA.
Unlike most other such ATPases, protein n' is strikingly specific
in its DNA dependence. Among a large number of binding-
protein-coated single strands and duplexes, only 4X DNA is
active (unpublished observations). Because recognition of
coated G4 DNA by primase and M13 DNA by RNA polymer-
ase depends in each instance on a unique hairpinlike structure,
it seemed that protein n' might also depend on such a helical
structure that resists destabilization by SSB. Exonuclease VII,
an enzyme that acts on single-stranded DNA from both 3' and
5' termini (18, 19). leaves a resistant .%-nucleotide stretch that
retains the full effector capacity of the #X circle to support
protein n' ATPase activity (Figs. 1-4).
Within the exonuclease VII-resistant, 55-nucleotide fragment
,there is a 44-nucleotide sequence with a potential for forming
a relatively stable hairpin structure (Fig. 7). Because its size
distinguishes this sequence neither from other duplex regions
in $X that also resist exonuclease VI1 action (Fig. 2) nor from
the duplex origins in G4 and M13 DNAs, the basis for recog-
nition by protein n' must depend on other features, such as a
particular sequence within the hairpin, mismatched regions,
the terminal loop, or neighboring regions of the hairpin.
Available methods for chemical synthesis of polydeoxynu-
cleotides (28) offer an approach for settling this question.
The 55-nucleotide recognition sequence for protein n` is
contained within a 107-nucleotide, untranslated region of the
$X chromosome between genes F and G, a location analogous
to that of the complementary strand origin of phage G4 (12).
This is remarkable in view of the low degree of overall sequence
homology between these phages and the lack of resemblance
between the hairpins recognized by protein n' in $X and by
primase in G4. It seems likely that the series of reactions that
culminates in the priming of 4X complementary strand syn-
thesis is initiated by protein n' at a specific locus, and that
subsequent events, such as movement of dnaB protein around
the $X circle, may obscure this unique start.

We thank Profesor 1. Tinoco for a discussion of stability of secondary
structures in DNA. This work was supported by grants from the Na-
tional Science Foundation and National Institutes of Health. J.S. was
a Fellow of the Fogarty International Center of the National Institutes
of Health.

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