volume 9 Number 31981
Nucleic A c i d s Research
The nuclease specificity of the bacteriophage 0X174 A* protein
S.A.Langeveld1'2, A.D.M.van Mansfeld1'3, A.van der Ende 1 , J.H.van de Pol 4 , G.A.van Arkel2 and
P.J.Weisbeek2
' Institute of Molecular Biology, 2Department of Molecular Cell Biology, •*Laboratory for
Physiological Chemistry, and Academic Computer Centre Utrecht, State University of Utrecht, The
Netherlands
Received 24 November 1980
ABSTRACT
The A protein of bacteriophage 0X174 is a single-stranded DNA specific nuclease. It can cleave 0X viral ss DNA in many different places. The position
of these sites have been determined within the known 0X174 nucleotide sequence (1). From the sequences at these sites it is clear that the A protein recognizes and cleaves at sites that show only partial homology with
the origin of RF DNA replication in the 0X DNA. Different parts of the origin sequence can be deduced that function as a signal for recognition and
cleavage by the A protein. We conclude that different parts within the DNA
recognition domain of the A protein are functional in the recognition of
the origin sequence in single-stranded DNA. The existence of different DNA
recognition domains in the A protein, and therefore also in the A protein,
leads to a model that can explain how the A protein performs its multiple
function in the 0X174 DNA replication process (2).
INTRODUCTION
Gene A of bacteriophage 0X174 codes for the only phage protein that is involved in the DNA replication process of the virus. Other proteins required for
this process are provided by the host cell (3).
The main product of gene A is a protein of 55 kD and this A protein has
been investigated in great detail, both in vivo and in vitro (4-7). It has
been shown that this protein fulfills several functions during the phage replication process. The A protein initiates the RF DNA replication by introducing a nick in the viral plus strand of the supertwisted RFI DNA molecule. At
this site which is the origin of replication,a free 3' OH terminus is created
which functions as a primer for DNA synthesis. The A protein becomes covalently attached to the 5' end of the nicked strand and is thought to interact
with other proteins necessary for the initiation of the DNA synthesis. After
initiation the DNA replication proceeds in a rolling circle mode (2). The A
protein is also capable to linearize and circularize the displaced plus strand
DNA after each round of replication. These steps require a re-cleavage at the
origin site and ligation of the linear single-stranded DNA by the A protein.
© IRL Press Limited. 1 Falconberg Court. London W 1 V 5FG. U.K.
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The genetic organisation of the region in 0X DNA that contains gene A is
rather complex. Three different proteins, the A protein (55 k D ) , the A
pro-
tein (37 kD) and the B protein (13.8 k D ) , are coded for by this part of the
DNA molecule.The A and A
proteins use the same reading frame of the DNA. The
B protein is made in a different reading frame and is not involved in the
phage DNA replication (8,9). Whereas the A protein is coded for by the entire
gene A the A
protein is synthesized from a translational start signal within
gene A. The translation of the A and A
proteins terminates at the same stop-
codon (1, 10).
The region also contains the origin of replication. The sequence at the
origin of replication in 0X174 DNA is characterised by an AT-rich sequence of
30 nucleotides. This sequence is highly conserved in the bacteriophages 0X174,
G4 and Stl and is surrounded by GC-rich regions (1, 11-14). These features allow the AT-rich sequence at the origin to adopt a partially denatured structure when the RF DNA molecule is supertwisted. Thus recognition and cleavage
of the origin sequence in RFI DNA may resemble to some extent the recognition
and cleavage of the origin sequence in single-stranded DNA. WLthin the AT-rich
sequence of 30 nucleotides three different symmetrical sequences are found,
-CAACTTG-, -TATTAATA- and -CTATAG-. These sequences may be. important for a
specific recognition process between the RFI DNA or single-stranded DNA and
the A protein, as many enzymes interacting with DNA require such symmetrical
sequences.
In contrast to the A protein which has well characterized functions in the
viral DNA replication, the function of the A
stood. The A
protein is not clearly under-
protein has been characterized as a single-stranded DNA specific
endonuclease (lo), it cleaves 0X viral plus strand DNA at the origin of replication and at many other sites. It does not cleave supertwisted 0X RFI DNA
under conditions where the A protein is active on RFI DNA (lS,lb,20 ) . This
makes it less likely that the A
protein is also involved in the initiation
of the RF DNA replication. The in vitro properties of the A and A
proteins
are compared in Table 1 .
Since the aminoacid sequence of the A
protein is also present in the A pro-
tein the nucleolytic activity of both proteins should be strongly related.
This is confirmed by the similarities of the enzymatic activities of the two
proteins as shown in Table I. Both
proteins recognise the origin sequence,
nick at this site and have ligation activity.
We studied the properties of the A
protein to elucidate some of the basic
aspects of the nucleolytic activities that are found in the A and A
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proteins.
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Table I .
PROPERTIES OF THE A AND A* PROTEINS IN VITRO
A protein
cleaves RFI DNA (Mo**)
•
cleaves RFI DNA (Mn**)
+
A* protein
ref.
(2,5.6.11)
+
forms RFIII DNA (Mn**)
+
forms RFIV DNA (Mo**)
(15)
(15) (a)
(15)
forms RFIV DNA (Mn**)
*
cleaves ss DNA at origin
•
(15)
+
(16.17)
cleaves ss DNA at other sites
+
(16)
attaches covalently to 5' end
of the nick
•
(16)
+
(b)
binds to ds DNA
binds to ss JNA
+
(b)
(a) Highly purified A protein which was obtained after purification on a
heparine-sepharose column in addition to the procedure described in
(15), did not fora RFIII DNA upon incubation of it RFI DNA in the
presence of Mn .
(b) Results of DNA Dinding experiments are personal conmunications of
H. v.d. Avoort.
The extensive nuclease activity of the A
protein on viral ss DNA provided a
way to trace the specificity of this enzyme in the process of DNA recognition
and cleavage.
In this paper we present an analysis of the nucleotide sequences found at the
different cleavage sites for the A
protein in the 0X viral ss DNA.
The results presented here show that a specific sequence at the origin of
replication is essential for the recognition and cleavage by the A
protein.
This sequence can be divided into different parts each of which can be recognized separately by the A
protein.
The properties of the nuclease activity of the A
protein are extrapolated to
the A protein and a model based on these properties is presented for the multiple enzymatic activities of the A protein in the RF DNA replication process.
MATERIALS AND METHODS
Chemicals. |y-32P|ATP (specific activity 2000-3000 ci/nnnole) was purchased
from the Radiochemical Centre (Amersham, U.K.); forraamide was from Merck and
purified prior to use by stirring quantities of 100 ml with 5 g of the mixed
bedresin AG 501-X8 (D) from Bio-Rad Laboratories. Acrylamide was from Serva
and bisacrylamide (ultra pure) was from Eastman-Kodak Co; agarose was from
Serva.
Enzymes. Proteinase K was purchased from Merck; bacterial alkaline phospha-
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tase (BAPF) from Worthington and T4 polynucleotide kinase from Boehringer
(Mannheim). Haelll restriction enzyme was kindly provided by Dr. P. Baas. The
A and A
proteins were isolated as described in (15).
Preparation of single-stranded DNA restriction fragments. Viral singlestranded DNA was isolated from virus particles by phenol extraction. 30 ng of
viral ss DNA was digested with Haelll restriction enzyme in a 0.5 ml reaction
mixture containing 50 mM Tris-HCl at pH 7.5, 10 mM MgCl.,, 5 mM dithiothreitol
and 1 mM EDTA. Incubation proceeded for 16 hrs at 37 C. The reaction was terminated by the addition of EDTA to 50 mM and 500 yg proteinase K. Incubation
was continued for 30 min at 37°C. The mixture was treated with phenol followed
by precipitation of the DNA fragments with ethanol. Finally the DNA fragments
were dissolved in 100 pi 50 mM Tris-HCl at pH 7.5, treated with alkaline phosphatase and labelled at their 5' termini with | Y - 3 2 P | A T P by T4 polynucleotide
kinase as described by Maxam and Gilbert (18).
The overall recovery of single-stranded DNA fragments after labelling was
32
estimated to be approx. 50%.
p-labelled fragments were run on a neutral pre-
parative 5% polyacrylamide slab gel in 40 mM Tris-acetate, 20 mM Na-acetate
and 2 mM EDTA at pH 7.7. The gel was autoradiographed to visualize DNA fragments, which were eluted from the gel as in (18).
Assay for nuclease activity on ss DNA fragments. 10 pi reaction mixtures
contained 0.15 - 0.2 pinoles ss DNA fragments, 1 pinole A
protein, 50 mM Tris-
HCl at pH 7.5, 150 mM NaCl, 10 mM MgCl , 5 mM dithiothreitol and 1 mM EDTA unless otherwise stated and were incubated during 90 min at 37°C. Reactions were
terminated by addition of EDTA to 50 mM and 10 yg of proteinase K. The incubation was continued for 30 min at 37 C. Finally 20 ul formamide containing 20%
sucrose, 0.25% xylene cyanol, 0.25% bromophenol blue and 0.2% sarkosyl was
added. The mixtures were then heated for 3 min at 100 C and layered on a denaturing slab gel.
Agarose gel electrophoresis. Horizontal slab gels (1.4% agarose) were run
in 40 mM Tris-acetate, 20 mM Na-acetate and 2 mM EDTA at pH 7.7 in the presence of ethidium bromide (5 ug/ml) and photographed under ultraviolet light (366
nm) .
Denaturing gel electrophoresis. Denaturing gels (33 x 250 x 1.5 mm) were
all 6% polyacrylamide gels made up in 98% formamide as described by Maniatis
et al. (19).
Sequence analysis. Sequence analysis was according to the chemical degradation method of Maxam and Gilbert (18) . Specific cleavage at guanine residues
was achieved with dimethyl sulfate, at adenine residues by ringopening with
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alkali, at cytosine and thymine residues by hydrazinolysis and at cytosine
residues alone by hydrazinolysis in 2 M NaCl. Strand-scission was achieved by
incubation with piperidine in all cases. For resolution of the cleavage products, 20% acrylamide gels were run in 7 M urea, 50 mM Tris-borate and 1 mM
EDTA at pH 8.3.
RESULTS
A
protein nuclease activity on 0X viral single-stranded DNA
The purity of the A
protein preparation used in the experiments was tes-:
ted in two different ways. Firstly by electrophoresis on SDS polyacrylamide
gels. In this way no additional protein bands were detected. Secondly by incubation of 0X RFI DNA with A* protein in the presence of Mg + + (see Table I ) .
The A
protein does not nick RFI DNA under these conditions (15, 20), so that
contamination with A protein would be detected by conversion of RFI DNA into
RFII DNA. The A
protein itself is not inhibitory to the action of the A pro-
tein on RFI DNA (data not shown). In this way no traces of A protein were
found in our A
protein preparation, not even at a protein/DNA molar ratio of
50.
Incubation of circular single-stranded 0X DNA with increasing amounts of the
pure A
protein preparation and analysis of the reaction products on an aga-
rose gel gives the result shown in Figure 1. At first two distinct subfragments of about 2100 and 3300 nucleotides appear. With higher protein concentrations the DNA is found as a broad band with higher mobility. This indicates that substantial degradation of the DNA has occurred. Circular and linear
single-stranded DNA have the same mobility on the agarose gel used. Therefore
the first nick in the circular DNA remains unnoticed. The conclusion is that
two relatively strong cleavage sites for the A
protein must be present in
the single-stranded DNA, next to a large number of weaker sites. When the cir-
Figure 1.
Agarose gel electrophoresis of 0X circular single-stranded DNA incubated with
increasing amounts of A protein. 1.5
pmoles ss DNA were incubated with (b) 6
pmoles, (c) 30 pmoles, (d) 60 pmoles and
(e) 120 pmoles A protein, (a) is a control without A protein. Reaction conditions were as described in Materials and
Methods. Arrows indicate the discrete
subfragments.
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cular single-stranded DNA is treated with A protein under similar experimental conditions only one nick at the origin of replication is introduced (16).
Determination of the positions of the cleavage sites
The position of the cleavage sites was determined by digesting specific ss
DNA fragments with A
protein. Single-stranded 0X DNA isolated from phage par-
ticles was digested with the restriction enzyme Haelll which cleaves singlestranded DNA with the same sequence specificity as it does double-stranded
DNA (16, 2 1 ) . The single-stranded DNA restriction fragments were labelled at
32
their 5' termini with
P and purified as described in Materials and Methods.
All restriction fragments were separately incubated with A
protein, depro-
teinzed with proteinase K and applied to formamide-polyacrylamide gels. On
each gel a set of Haelll restriction fragments with known sizes was coelectrophoresed to serve as length markers. When the Haelll fragments are plotted
semilogarithmically versus their migration distances a good linear relationship is found for the fragments Z4 (603 nucleotides) through Z10 (72 nucleotides) on a 6% polyacrylamide gel containing formamide. Therefore the sizes
of the A
protein produced subfragments can be deduced with reasonable accu-
racy from their relative positions on the gel. Since only the 5' termini were
labelled the size of each subfragment determines the position of a cleavage
site in the 0X DNA sequence (1). An example of such an A
protein digestion
is given in Figure 2 for the fragments Z3, Z4 and the partial digestion product Z5-Z8. The cleavage sites determined in this way are listed in Table II.
Efficiency of cleavage
It is evident from Figure 1 that there is a large difference in cleavage
efficiency for the various sites. Furthermore, upon incubation of individual
restriction fragments with A* protein alsmost every fragment gives a certain
number of subfragments (see Table II), but in varying fields. We determined
the sites which are most efficiently cleaved by incubating mixtures of DNA
fragments with limited amounts of A
protein. An example of such an analysis
is shown in Figure 3. The results show that restriction fragments Z5 through
Z10 give two distinctive subfragments upon incubation with A
protein, one of
approx. 98 nucleotides and one smaller than 30 nucleotides. The 98 nucleotide
long subfragment results from a cleavage at the origin site in Z6B (16). The
small subfragment obviously results from a cleavage in the Z8 restriction
fragment, as the Z8 band shows a significant decrease of intensity on the
autoradiograph. Under similar conditions of incubation with a limited amount
of A
550
protein, a mixture of the restriction fragments Zl through Z6B only
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Figure 2.
Formamide polyacrylamide gel containing the
individual restriction fragments Z3, Z4 and
the partial digestion product Z5-Z8 incubated with A protein, (a): 0.3 pmoles of Z3,
0.3 pmoles of Z4 and 0.1 pmoles of the partial Z5-Z8 were incubated with 0.1 pmoles A
protein, (b): control without A protein.
Reaction conditions were as described in
Materials and Methods. Marker positions are
indicated.
Z
6A
gives the 98 nucleotide subfragment.
We conclude from these data that there are two extremely efficient cleavage
sites for the A
protein in 0X viral single-stranded DNA, one at the origin
of replication and another one in the Z8 restriction fragment. The positions
of these sites are in good agreement with the sizes of the two discrete subfragments of 2100 and 3300 nucleotides found when intact single-stranded DNA
is incubated with A
protein (Fig. 1).
The position of the site in Z8 was also determined by cleavage of a DNA fragment that contains Z5 and Z8. This partial digestion product is due to the
stable hairpin that can be formed in the GC-rich region in which the Haelll
cleavage site is located (22). Under our conditions approximately 50% of the
single-stranded DNA remains uncleaved at this site. It was found that the
cleavage site of the A
protein in Z8 is about 10 nucleotides away from the
Z5-Z8 junction. This position corresponds with the results obtained upon digestion of Z8 alone (data not shown).
A comparison of the sequence at the cleavage site in Z8 and the sequence at
the origin of replication in Z6B is given in Table III. Remarkable similari-
551
z
CD
o'
>
TABLE II. CLEAVAGE SITES OF THE A* PROTEIN IN 0X VIRAL SINGLE-STRANDED DNA
Fragment
Length of
subfragment
Position of
cleavage site
21
235 - 245
190 - 200
2016 + 5
Z2
Fragment
Z3
Length of
subfragment
740 - 760
Position of
cleavage site
313 +
153 +
Fragment
Lenght of
subfragment
10
Z5
180 - 205
10
Z6A
165 - 170
lt>0 - 170
1971 + 5
1941 ± 5
500 - 510
68 + 5
100 - 120
1886 + 10
380 - 400
5339 t 10
90 - 95
1869 + 3
285 - 295
5239 + 5
84 - 86
1861 + 1
195 - 200
5147 + 3
670 - 690
3809 + 10
170 - 175
5122 + 3
150 - 160
3284 + 5
145 - 155
5099
95 - 105
3229 + 5
85 - 95
3219 + 5
50 - 60
3184 i 5
< 30
3159 - ?
580 - 600
90 - 100
7*
Z4
130 - 135
i5
Z6B
Z5-Z8
Z9
5044 + 5
Position of
cleavage site
362 +
13
4656 *_ 3
130 - 135
4621 + 3
30 - 35
4521 + 3
98*
320 - 325
4305
992 + 3
29*
4826
4788
60*
4937
67*
4956
Z10
1306 + 3
±3
80 - 85
1256
60 - 65
1236 + 3
40 - 45
1226 + 3
Restriction fragments were incubated with A* protein as described in Materials and Methods. The restriction
fragment Z7 was not cleaved under these conditions. The lengths of the subfragments derived from the
formamide polyacrylamide gels are expressed in nucleotides. The values determined in different experiments
fall in the range that is indicated for each subfragment. Asterisks mark the subfragments of which the
length has been established by sequence analysing techniques.
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Figure 3.
A mixture of restriction fragments Z5 through
Z10 was incubated with A protein, (a): 0.8 1.0 pmoles ss DNA fragments were incubated
without A protein and (b): with 0.3 pmoles
A protein.
Arrows indicate the subfragments.
ties exist between the two sequences: i) 13 nucleotides in a stretch of 18 nucleotides are identical, i i ) a pyrimidine-rich sequence is present at the left
side of the cleavage s i t e , i i i ) an AT-rich region is present at the right side.
The sequence -ACTTGA- that contains the point of cleavage at the origin site
is also found in the Z8 cleavage s i t e . These are the only two sites in the 0X
viral ss DNA where this sequence is present. I t suggests that the hexanucleotide -ACTTGA- is important for the A protein - ss DNA interaction.
TABLE I I I .
MUCLEOTIDE SEQUENCES AT THE CLEAVAGE SITES
Fragment
Sequence
Z5B
4293 - c T C C C CC A A C T T G'A T A T
Z5-Z8
978 - G G,
C C C CT T A C T T G A
IN Z6B AND Z5-Z8
T A A T A A C A - 4317
G G A T A A A T T A T - 1002
Haelll
Nucleotides ideadeal in both sequences are represented in boxed
areas. The AT-rich regions scare at positions ^306 and 995, GT-rich
regions scare at positions 4299 and 983 and proceed leftward.
The arrow indicates the poinc of cleavage ac che origin of
replication.
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The best cleaved sites in the indivivual restriction fragments Z2 and Z3 were
also determined. The fragments were incubated with an amount of A
protein
tenfold less than normal (see Materials and Methods). Under these conditions
both fragments give only one subfragment with a low yield per subfragment.
The sequences at these cleavage sites together with the cleavage sites in
Z6b and Z8, are listed in Table IV. The other weaker cleavage sites in Z2
and Z3 are listed in Table VI. From these tables it is clear that the best
cleaved sites have the best homology with the origin sequence. These sites
also have the sequence -TTGA- in common. The tetranucleotide -TTGA- is present at 40 different places in the 0X viral single-stranded DNA. Only seven
cleavage sites, including the best cleaved sites listed in Table IV, have this
sequence in it (see Table VI). This means that the tetranucleotide -TTGA- is
not necessary nor sufficient for cleavage by the A
protein. It can therefore
be concluded that there is no sequence common to all cleavage sites. However
almost all sites show homology with the sequence around the origin to some
extent. The best cleaved sites have contiguous sequences that are homologous
to the sequence -ACTTGATATT- at the origin. This sequence therefore is expected to have an important role in the recognition and cleavage process of
the single-stranded DNA by the A* protein.
Determination of exact cleavage sites
The lengths of the subfragments as derived from the migration distances in
the formamide gels give the approximate positions of the cleavage sites. For
a limited number of sites the exact position of cleavage was determined by
more accurate methods. Subfragments obtained from the individual restriction
fragments Z3, Z6B, Z9 and Z10 after incubation with A
protein were isolated
and subjected to sequence analysis, following the chemical degradation method
TABLE [V. MUCLEOTIOE SEQUENCES AT THE BEST CLEAVAGE SITES
Fragment
Sequence
Z63
42y3
- C T C C C C C A A C T T G A
Z8
978 -
.
. C C C C .
. A C T T G A
Z2
3804 -
T T G A
Z3
69 -
A . C T T G A
T A T T A A T A
.
.
. T A A .
C A - 4317
.
.
. -
T A T T . . T . A . . T A
A -
1002
3823
83
The sequences at the besc cleaved sices are compared with the
origin sequence. Docs mark che positions oc the non-homologous
nucleocides. The coasaon sequence is represented in a boxed area.
The poinc of cleavage ac che origin in che restriction Srasnnenc
Z6B is indicated by che arrow.
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of Maxam and Gilbert (18). Subfragments were selected on appropriate length
for sequence analysis. The subfragments were degraded directly without relabelling of their 5' ends and were analysed on sequencing gels. An example of
such an analysis is shown for the 29 nucleotide subfragment of Z9 (Fig. 4 ) .
The nucleotide sequence up to the nucleotide at the 3' OH end of the subfragments could be determined. The exact cleavage sites determined in this way
are present in Table V. The sequences are arranged in such a way that optimal
homology to the origin sequence in Z6B is achieved. It is clear that the A
protein does not cleave in the homologous sequences at a fixed position. In
Z3 and Z9 the sites with the best homology to the origin sequence are cleaved
at positions other than those expected from the similarity with the origin
sequence.
Remarkably, the sequence at the cleavage sites in Z9 (4826) and Z10 (4937)
differ considerably from the origin sequence, but they resemble each other
strongly. The heptanucleotide -TTCTGGT- in Z10 was also found at other cleavage sites of the A
protein that have no or very little resemblence to the
sequence at the origin. From the exact cleavage sites given in Table V it becomes evident that the A
protein does not cleave between two specific nucleo-
C
T
A
G
Flgure 4-
v*
•
"
\a
Autoradiograph of a sequencing gel containing the
chemical degradation products of the smallest subfragment (29 nucleotides) of Z9 obtained after incubation with A protein.
SSS:
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TABLE V.
CLEAVAGE SITES DETERMINED BY SEQUENCE ANALYSIS
Fraqment
Sequence
Z6B
4299 - C A
Z3
4950 - C c t 9
Z9
4781 - g c
Z9
g - 4835
Z10
g - 4946
A C T T G•A T A T T A A
T - 4313
T T G A•T 9 c T A A
a - 4964
c - 4795
4821 - a
A C T T t A*T 9 c g 9 A
A t t T TJG g T c g T c 9
4932 - C
9 t t c T^G g T t g g t t
Points of cleavage are indicated by arrows.
Nucleotides which are homologous with the
origin sequence are represented by uppercase characters.
tides. Furthermore the sequences in Table V in addition confirm the previous
observation that there is no common sequence required for the cleavage activity of the A
protein on ss DNA.
Sequences at the cleavage sites
The cleavage sites presented in Table II have sequences that can be divided
into two classes, one class of sequences with homology to the origin sequence
and another class of sequences resembling the sequence -TCTGGT- as found in
Z10.
The origin—like sequences are listed in Table VI and are aligned in such a way
that the homology with the origin is maximal. The pattern of homologous sequences shows that the sequence -CAACTTGATATT- comprises almost all homologous
sequences, but that there is a broad variation in homology between the two
terminal sequences -CC.CAACTT- and- -GATATT-. Apparently only nucleotides in
this part of the origin region are crucial for recognition and cleavage by the
A
protein. However, no unique sequence that is part of this sequence can be
deduced from the cleavage sites. It appears that only a small degree of homology with the origin sequence suffices as a signal for cleavage and, as was
found for Z2 and Z3, that the efficiency of cleavage depends on the degree of
homology.
The second class of sequences resemble the sequence -TTCTGGT-, a sequence also
found at two other cleavage sites in the 0X viral ss DNA. This sequence does
not correspond with any sequence at the origin of replication. However, when
nucleotides in this sequence are represented as pyrimidines or purines a part
of it can be written as -PyPyTGPuT- which is identical with the sequence
-CTTGAT- at the origin. All sequences of cleavage sites resembling the sequen-
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TABLE VI.
HOMOLOGY OF ORIGIN-LIKE CLEAVAGE SITES
Fragment
Sequence
Z6B
4293
Z3
5138 - .
Z3
5090 -
Zl
1960 - . T . . . . C A A C T T G
Z3
- C T C C C C C A A C T T G A T A T T A A T A A C A - 4317
. . C C . C A ACTT
T . A . A A .
. - 5162
C CAA . . T G . . . . T A . T . . C . -
5114
A - 1984
144 - C . . C C . . . .C T T G
A . A -
168
Zl
1850 - . T
C T T G . . . T T . . T . . . . -
1874
Zl
1856 - . . . C
C T T G . T . . T . . T . . . . -
1880
Z2
3171 -
Z4
1246 -
Z3
5037 -
Z8
C . .C T T G A . . . T A
- 3195
C . T G A
- 1270
A. C T T G . T . . T A A
- 5061
978 - . . C C C C . . A C T T G A . . . T A A
- 1002
Z4
1214 - . T C C C . C A . C . T . A . . T T . A T . . . . -
Z3
5228 - . . C . . . C A A C . . G A T A T T . A
Z9
4775 - . . C
Z2
3273 -
C . T G A T . . T . . T . . . . -
3297
Z2
3219 -
C T T G A T . T . . . T . . . . -
3243
Z3
59 -
Z2
3804 -
Z3
A CTT
. AT . .
. . A . A . .
A. C T T G A T A
1238
- 5252
. - 4799
A -
83
. T T G A T A T T . . T . A . . -
3828
310 - . . . . C . C . . . T T G A . A T T . . . A A . . -
334
Z6A
4506 - C
. T T G . T A T T . A
- 4530
Z3
4944 - . T . . . . C . . . T T G A T . . T A A
- 4968
Z6A
4644 - . . C
Z6A
4611 - . . . . C . . . . . . T G A T A . T . . . A A . . -
C . T G A T A T T . . T . . . A - 4668
4635
Zl
1874 - . T . C C . C . .. , T G . . A . T A A .
. A . A - 1898
Z2
3207 - . . . . C C . . . . . T G A T A
Zl
1925 - . T . . C . . A . . . . G A T A T T . . T . . C . - 1949
- 3231
Sequences at the cleavage sites listed in Table II which are
homologous with the sequence around the origin in Z6B, were
aligned with the origin sequence. Nucleotides which are homologous to the origin sequence are represented by upper-case
characters, dots mark the positions of aberrant nucleotides.
ce -TTCTGGT- are listed in Table VII. Cleavage at the sequences resembling the
sequence -TTCTGGT- is at identical positions in the restriction fragments Z9
and Z10 as is shown by the alignment of sequences in Tables V and VII.
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TABLE VII.
CLEAVAGE SITES WITH SEQUENCES HOMOLOGOUS t'lTH -TTCTGGT-
Fragment
Sequence
Z10
4929 - T C A C GT T C T*G G T T G G T T G T G -
4948
Zl
2003 - T t c C GT T C T G G T g a t T c G T c -
2022
Z2
3141 - T t t g t T T C T G G T g c t a T G g c - 3160
Z5
863 - a a A C GT T C T G G c g c t c g c c c -
882
Z4
1226 - c a c g t T T a T G G T g a a c a G T G - 1245
Z9
4818 - c C t a a 7 T t T*G G T c G t c g G g t -
4837
Z4
1295 - a a c a c T a C T G G T T a t a T t g a -
1314
Z3
5327 - g a c a a a T C T G c T c a a a T t T a -
5346
Z3
5114 - c a A g a a g C T G t T c a G a a t c a - 5133
Sequences of cleavage sites listed in Table II which are
homologous to the sequence -TTCTGGT-, are compared with
the sequence around the cleavage site in the restriction
fragment Z10. Homologous nucleotides are represented by
upper-case characters, non-homologous nucleotides are
represented by lower-case characters. Arrows indicate
the cleavage points in Z9 and Z10 which were determined by
sequence analysis (see also Table V ) . The boxed area
includes the -TTCTGGT- like sequences.
DISCUSSION
The a n a l y s i s of the nuclease a c t i v i t y of the A p r o t e i n on 0X v i r a l
stranded DNA has revealed 35 d i f f e r e n t
single-
cleavage s i t e s . I t i s c l e a r t h a t not
a l l p o t e n t i a l cleavage s i t e s of the A p r o t e i n on v i r a l ss DNA could be d e t e c ted with our method. E f f i c i e n t l y
cleaved s i t e s near the 5 ' ends of the r e s -
t r i c t i o n fragments can easily mask l e s s e f f i c i e n t l y
cleaved s i t e s further away
from the l a b e l l e d 5 ' terminus. The sequences of the majority of these c l e a vage s i t e s show s i g n i f i c a n t homology with the sequence at the r e p l i c a t i o n o r i gin of the phage DNA. Eight of these s i t e s however, have b e t t e r homology with
the sequence -TTCTGGT-. I t was found t h a t the s i t e s which a r e most
efficient-
ly cleaved show good homology with the sequence a t the o r i g i n . This was demonstrated for the cleavage s i t e in Z8 and the b e s t cleavage s i t e s in Z2
and Z3.
The sequences found a t the many cleavage s i t e s of the A p r o t e i n show
c l e a r l y that the sequence of 30 nucleotides at the o r i g i n i s not required
for s p e c i f i c recognition by t h e A p r o t e i n . All cleavage s i t e s ,
including
the best ones, have sequences t h a t are homologous to p a r t s of the sequence
-CAACTTGATATT- a t the origin of r e p l i c a t i o n . No common or unique sequence
emerged from the sequences a t the cleavage s i t e s . This was already exprected
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from the large difference in efficiency of cleavage at the various sites.
Less efficient cleavage activity of the A
protein at a certain site is re-
flected in a lower degree of homology with the origin sequence. The symmetrical sequences around the origin are not required for recognition and cleavage
of single-stranded DNA by the A
protein. This is in agreement with the fin-
dings of Van Mansfeld et al. (23), who recently demonstrated that the synthetic oligonucleotide -ACTTGATA- can be cleaved specifically by the A
protein
of 0X174. The actual cleavage at the cleavage sites occurs however at positions that differ from what is expected from the homology with the originsequence. Therefore it can be concluded that the A
protein has a more relaxed
sequence specificity than the larger A protein which nicks only in the originsequence and nowhere else in the 0X viral single—stranded DNA (16).
We observed that entirely different parts of the sequence -CAACTTGATATT- can
function as a signal for recognition and cleavage by the A
protein. It is re-
markable that the sequences -CAACTTG- and -GATATT- which represent the parts
left-ward and right-ward from the point of cleavage at the origin of replication can be recognized and cleaved by the A
sequence the A
protein individually. As a con-
protein must have DNA recognition domains which are able to
recognise these parts of the origin sequence separately. This suggestion is
supported by the ligation activity of the A
protein. It has been demonstra-
ted that relaxed RFIV DNA is formed upon incubation of 0X RFI DNA with A
protein in the presence of Mn
(15). In this reaction the A
protein cleaves
the RFI DNA at the origin of replication. While it is covalently bound to the
5' end of the DNA, it recognizes the 3' OH end of the nick after the release
of supertwists from the RF DNA and it ligates both termini.
Since the A
and the A protein have very identical fundamental properties;
cleavage of viral single-stranded DNA between two specific nucleotides at the
origin of replication, covalent attachment to the 5' end of the cleaved DNA
molecule and ligase activity, we expect that recognition of the specific sequence around the origin is basically the same for the A
and the A protein.
The independent recognition of the extreme parts of the sequence CAACTTGATATTat the origin by the A
protein therefore provides an explanation for the
multifunctional behaviour of the A protein in the phage DNA replication process as described by Eisenberg et al.(2). A model for the role of the different DNA recognition domains in the A protein
which can recognise the different parts of the origin sequence
during the bacteriophage RF DNA replication and viral strand DNA synthesis is
presented in Figure 5.
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Figure 5.
See text for explanation
The first function of the gene A protein in the phage DNA replication is the
recognition and cleavage of the sequence at the origin in RFI DNA (Figure 5,
A ) . In RFI DNA the duplex DNA structure of the AT-rich origin is thought to
be destabilised (24). Recognition and cleavage of the origin sequence in RFI
DNA and viral single-stranded DNA are therefore very similar. When the DNA is
cleaved the protein becomes covalently bound to the 5' end of the cleavage
site and a 3' OH terminus becomes available for the priming of the DNA synthesis (Figure 5, B) .
After one round of replication the displaced plus strand DNA is split off into a genome length circular molecule by the A protein. In this reaction the
origin sequence must specifically be recognized by the A protein that is covalently bound to the right part of the origin sequence (Figure 5, C ) . Therefore a free DNA recognition domain of the A protein is supposed to interact
with the left part of the sequence at the origin. The displaced plus strand
DNA is subsequently cleaved and the A protein switches from the 5' end of the
old DNA strand to the 5' end of the new strand under simultaneous ligation
of the 51 and 3 1 ends of the displaced strands. In this process a new circular DNA molecule is formed which is used as a template for the complementary
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strand, or is encapsidated during phage maturation (2). The sequence that is
recognized by the free part of the A protein actually functions as a signal
for the termination of the plus strand DNA synthesis. If this model is also
true for the filamentous phages it might offer an explanation for recent results obtained by Horiuchi (25). Ke found that when a second origin is cloned
into f] the area between these origins is always deleted in progeny phage,
suggesting that the origin sequence functions as a termination signal. From
the model described above it is clear that the first origin sequence that is
reached by the replication fork will act as a signal for the A protein or in
the case of fl the gene II protein to split off and circularize the displaced
strand.
Consequently, the presence of additional sequences in the plus strand DNA
that can function as a termination signal will abort the synthesis of intact
plus strand DNA. This means that the sequences with the best homology to the
origin sequence, listed in Tables IV and VI are not able to function as a termination signal during the in vivo DNA replication. Therefore the sequence
-CAACTTG- alone is not enough for termination although it is recognized by
the A
protein. The minimal sequence to function as a termination signal
therefore has to be larger that -CAACTTG-. The model presented in Figure 5
accounts for a processive action of a monomeric A protein molecule in the RF
DNA replication and viral DNA synthesis (2, 17). Experiments are in progress
to analyse in more detail DNA recognition domains in the A and A
The sequences at the cleavage sites of the A
proteins.
protein have given information
about sequences at the origin site that are essential for recognition and
cleavage by the A
and A proteins. Other sequences at the origin, however,
may be required for a specific interaction with host DNA replication enzymes
like the rep unwinding protein and DNA polymerase III.
This might explain why a much longer sequence of 30 nucleotides is conserved
in the phages 0X174, G4 and St-1. The RFI DNA of the phages G4 and St-1 is
also cleaved by the 0X gene A protein (13).
The activities of the A
protein determined in vitro do not provide a clear
in vivo function, but do point to an involvement of the A
protein in the
single-stranded DNA synthesis and/or maturation of the virus. It was suggested
by Martin and Godson (26) that the A
protein is also involved in the arrest
of host cell DNA synthesis.
The experiments presented here give a possible mechanism for such an interaction. If the A
protein nicks in chromosomal single-stranded DNA e.g. in the
replication fork, then the host DNA synthesis will be affected.
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We conclude that the nuclease activity of the A protein is analogous to the
A
protein nuclease activity and that the higher specificity of the A protein
comes from the extra NH_-terminal part of the A protein.
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