(Received for publication, April 7, 1995; and in revised form, July 12, 1995)
From the
The coliphage N4-coded single-stranded DNA-binding protein
(N4SSB) is essential for phage replication and for expression of the
phage late genes, which are transcribed by the Escherichia coli RNA polymerase. As a first step in
investigating the role of N4SSB in replication and transcriptional
activation, we have identified and sequenced the N4SSB gene. The gene
encodes a 265-amino acid protein with no apparent sequence homology to
other single-stranded DNA-binding proteins. We present data indicating
that N4SSB is also essential for phage recombination. Mutational
analysis of the carboxyl terminus of the protein indicates that this
region is required for protein-protein interactions with the N4
replication, N4 recombination, and E. coli transcriptional
machineries, while the rest of the protein contains the determinants
for single-stranded DNA binding.
Proteins that bind nonspecifically to single-stranded DNA with high affinity have been purified and characterized from several sources (1, 2) . Single-stranded DNA-binding proteins are present in high concentration in vivo and are essential components in a variety of DNA metabolism processes. They are required for DNA replication and are also involved in repair and recombination (1) . They bind to single-stranded DNA stoichiometrically and, in most cases, with positive cooperativity(3) .
The N4-coded
single-stranded DNA-binding protein (N4SSB) ()was
originally detected as an activity capable of complementing the defect
of mutant phage N4am7 in replication(4) . The purified
protein has a monomer M
of 32,000 and binds
single-stranded DNA more tightly than RNA, with a binding site size of
11 nucleotides, an intrinsic binding constant of 3.8
10
M
, and a cooperativity of 300
(
) in 0.22 M NaCl at 37 °C(5) . Although
N4SSB is able to lower the melting transition of poly(dA)
poly(dT)
by at least 60 °C, it cannot lower the melting transition or assist
in the renaturation of natural DNAs. In vitro, N4SSB
specifically stimulates the N4 DNA polymerase by increasing its
processivity 300-fold and by melting out hairpin structures that block
polymerization(5) . Surprisingly, N4SSB is also required for
the synthesis of N4 late RNAs, which is catalyzed by the Escherichia coli
RNA
polymerase(6) . In vitro, N4SSB allows E. coli
RNA polymerase to utilize efficiently N4 late
promoters(6) . Therefore, in addition to playing an essential
role in N4 DNA replication, N4SSB is a transcriptional activator,
suggesting that it is a multifunctional protein. To dissect the domains
of N4SSB responsible for activation of N4 DNA polymerase on a primed
template and of E. coli
RNA polymerase at
N4 late promoters, we cloned and sequenced the gene encoding N4SSB and
constructed an N4SSB expression vector. Expression of the cloned
protein complemented N4am7 phage, carrying a mutation in the
N4SSB gene, for N4 DNA replication and late transcription.
Additionally, we have found that N4SSB is essential for N4 DNA
recombination. Analysis of the different phenotypes of
carboxyl-terminal deletion mutants indicates that determinants of
protein-protein interactions reside in a short, basic,
carboxyl-terminal domain, while the rest of the protein contains the
determinants of single-stranded DNA binding. Moreover, the phenotype of
certain mutations indicates that the different functions of N4SSB are
carried out by separate determinants.
PCR amplification was performed on template DNAs (20 ng of plasmid
DNA and 200 ng of wild-type N4 and am7 genomic DNAs) suspended
in a mixture containing 50 mM KCl, 10 mM Tris-HCl (pH
8.3), 1.5 mM MgCl, 0.01% gelatin, 0.2 mM dNTPs, 1 mM primers, and 5 units of Taq polymerase (Perkin-Elmer) in a total volume of 100 µl, in an
amplifying thermocycler (Perkin-Elmer). The DNA sequence of all PCR
products was confirmed by the dideoxy chain termination method of
Sanger et al.(12) after cloning of the fragment into
M13.
The DNA fragment containing the N4SSBam7 open reading frame (ORF) and the distal 100 noncoding bases was amplified by PCR using N4am7 DNA as a template and the following oligonucleotides as primers.
N4SSB-N generates an additional valine residue as the second amino acid (underlined). After confirmation of the DNA sequence, the desired DNA fragment was inserted into pT5 (provided by Steve Eisenberg, Synergen) to yield pMC3, which carries the N4SSBam7 ORF under T7 RNA polymerase promoter control. Clones expressing the wild-type ORF were not recovered by this procedure (see ``Results'').
To clone the wild-type N4SSB ORF, a DNA fragment carrying the Shine-Dalgarno (SD) sequence present in pT5 and the amino-terminal region of the N4SSB gene (120 amino acids) was generated by PCR, using N4 DNA and the following oligonucleotides as primers.
The PCR fragment was restricted with XbaI and HindIII and inserted into the same sites of mp19 to verify the
DNA sequence. The XbaI/BstBI fragment
(N4SSB) was used to replace the corresponding fragment
(carrying the am7 mutation) in pMC3, yielding pMC5. Finally,
pMC5 was treated with BglII and XbaI to release the
fragment containing the T7 RNA polymerase promoter. This fragment was
replaced with a 66-base-long DNA fragment containing the T7 minimal
promoter and lac operator, isolated from pET-11a (Novagen) by
restriction with the same enzymes, to yield pMC6. N4SSB was purified as
described (5) with some modifications. (
)Deletion
mutants were generated by oligonucleotide-directed, site-specific
mutagenesis of pMC6.
To measure N4 DNA synthesis, at the indicated times after infection, 100-µl samples of cells were removed and incubated for 2 min with 10 µCi of thymidine (Amersham Corp.). Samples were processed as described previously(16) . Late RNA synthesis was measured by primer extension as described(6) .
Positive clones were tested for rescue of N4am7, which carries a mutation in N4SSB, by recombination between the N4am7 genomic DNA and the cloned fragment(14, 16) . One clone, pMC1, containing a 2.5-kb fragment was able to rescue N4am7. The frequency of wild-type plaque-forming units/total plaque-forming units of a lysate from pMC1-containing W3350 cells was 1000-fold higher than the frequency in hosts carrying pKK232-8, indicating that at least part of the N4SSB gene was present in pMC1 (data not shown). Although pMC1 contains only 2.5 kb of the N4 HpaID fragment, the cloned fragment is not the result of a rearrangement since the same PCR products were generated using pMC1, wild-type N4, or N4am7 genomic DNA as templates for amplification with two oligonucleotides that hybridize to each end of the 2.5-kb fragment (data not shown). The presence of the N4SSB gene in pMC1 was confirmed using an oligonucleotide probe (oligonucleotidea in Fig. 1) derived from the sequence of the amino-terminal 35 amino acids of the N4SSB protein purified from N4-infected cells. This probe hybridized to both pMC1 and the N4 HpaID-a fragment on genomic blots (data not shown).
Figure 1: DNA sequence of the N4SSB-coding region. The sequence of the N4SSB-coding region reveals a 265-amino acid open reading frame. The oligonucleotide used for hybridization and as a primer for DNA sequencing is shown (boldface). The N4am7 mutation occurring at amino acid 54, a CAG (Gln) to TAG (amber) transition, is shown (boldface). The amino acid sequences that coincided with the amino-terminal 34 amino acids of the purified N4SSB protein are underlined.
The same oligonucleotide probe was used as a
primer for double-stranded DNA sequencing of pMC1, wild-type N4, and
N4am7 genomic DNAs. The sequence revealed a 265-amino acid ORF (Fig. 1). The N4am7 mutation mapped at amino acid 54, a
CAG (glutamine) to TAG (amber) transition, verifying that this ORF
codes for N4SSB. The DNA sequence is in agreement with the determined
amino acid sequence of the amino-terminal 34 amino acids of the
purified N4SSB protein, underlined in Fig. 1. ()The
predicted sequence agrees well with the determined amino acid
composition of the purified protein(5) . Analysis of the
derived protein sequence does not reveal sequence homologies to T4 gp32
or any other single-stranded DNA-binding proteins.
Cloning of the wild-type N4SSB ORF (pMC6) was successfully achieved using a tightly regulated system in which the N4SSB gene is under the control of a T7 RNA polymerase promoter and the lac operator. In addition, the recombinant plasmid was introduced into a host strain carrying the pcnB mutation, which reduces the copy number of pBR322 derivatives(8) , and pLysE, which synthesizes the T7 lysozyme and is an inhibitor of T7 RNA polymerase. Fig. 2shows the expression of N4SSB in E. coli W3350pcnB(DE3)/pLysE, which was used as a host for N4 phage infection to test the effect of cloned N4SSB on N4 DNA recombination, N4 DNA replication, and N4 late transcription in vivo, and in BL21(DE3)/pLysE, which was used for the overexpression and purification of cloned N4SSB. The size of the N4SSB protein produced by the T7 RNA polymerase-directed, expressing clone was the same as that from N4-infected cells. The amount of N4SSB is higher in E. coli BL21(DE3)/pLysE than in E. coli W3350pcnB(DE3)/pLysE due to a higher copy number of pMC6 in the former strain.
Figure 2: Expression of N4SSB in E. coli W3350pcnB(DE3)/pLysE carrying pMC6 and in E. coli BL21(DE3)/pLysE carrying pMC6 in the absence or presence of 1 mM IPTG. For experimental details, see ``Materials and Methods.'' The arrowhead indicates the N4SSB polypeptide.
The ability of the cloned and
expressed N4SSB protein to complement N4am7 for N4 DNA
replication was examined. Fig. 3(leftpanel)
shows the rate of [H]thymidine incorporation into
DNA after N4am7 infection of the following cells: suppressor
(W3350supF), non-suppressor (W3350),
W3350pcnB(DE3)/pLysE carrying pMC6, or pMC6
without or with preincubation with 0.4 mM IPTG for 30
min. Even though the rate of [
H]thymidine
incorporation in cells expressing cloned N4SSB was lower than in
suppressor-containing cells, it was
4-fold higher than in
noninduced or N4SSB
-expressing cells. The increased rate
of thymidine incorporation occurred in N4SSB-expressing cells when
cells had been preincubated with 0.2-0.5 mM IPTG for
20-45 min. The amount of N4SSB, synthesized from the T7-directed
clone induced with 0.1-1 mM IPTG for 15-90 min,
was found to be comparable to that produced in N4am7-infected
suppressor-containing cells (data not shown).
Figure 3:
Leftpanel, ability of
cloned N4SSB to complement N4am7 for DNA replication. Shown is
the rate of [H]thymidine incorporation into DNA
after N4am7 infection of W3350supF (
), W3350
(
), E. coli W3350pcnB(DE3)/pLysE carrying pMC6
without (
) or with (
) 30 min of induction with 0.4 mM IPTG, and E. coli W3350pcnB(DE3)/pLysE carrying
pMC6
without (
) or with (⧫) 30 min of induction
with 0.4 mM IPTG. Rightpanel, Southern
hybridization of N4am7-infected intracellular DNA and XbaI map of N4 genomic DNA. Top, the amount of N4
genomic DNA in infected cells was determined by hybridization of
intracellular DNAs prepared from E. coli W3350pcnB(DE3)/pLysE carrying pMC6 that had been
preincubated without or with 0.4 mM IPTG for 30 min at 8, 18,
and 35 min after N4am7 infection. The DNA was digested with XbaI and SalI, blotted onto nitrocellulose, and
probed with an excess amount of
P-labeled N4 DNA (5
10
cpm/lane). *, N4SSB ORF-bearing DNA fragment
generated by digestion of pMC6 with XbaI/SalI; JF, fragment generated during N4 DNA replication and
containing unique sequences of fragments E and F and one copy of the
terminal redundancy(18) . LaneM, XbaI-digested N4 DNA and XbaI/SalI-digested
pMC6. There are no SalI restriction sites on N4 DNA. Bottom, XbaI map of N4 genomic
DNA.
Southern blot analysis
was performed to test whether the increased rate of thymidine
incorporation in N4SSB-expressing cells was due to active N4 DNA
replication, recombination, or repair. Total intracellular DNAs (host
chromosomal, N4 genomic, and plasmid DNAs) were prepared from wild-type
N4SSB-expressing cells that had been preincubated in the absence or
presence of 0.4 mM IPTG for 30 min at three different times
(8, 18, and 35 min) after N4am7 infection. DNAs were
restricted with XbaI and SalI, and fragments were
separated on an agarose gel, blotted, and hybridized to an excess
amount of P-labeled N4 genomic DNA (see ``Materials
and Methods''). Fig. 3(rightpanel)
shows that the amount of N4 DNA increased (as indicated by the
increased amount of N4 XbaI fragments and the joint fragment,
a product of N4 DNA replication(18) ) only when IPTG was
present, i.e. when N4SSB was induced. The decrease or absence
of N4 DNA XbaI fragments in noninduced cultures at times late
after infection is indicative of DNA degradation. These results
demonstrate that cloned and expressed wild-type N4SSB protein can
complement N4am7 for N4 DNA replication. In contrast,
N4SSB
is inactive.
N4SSB enhances the rate of DNA
synthesis catalyzed by N4 DNA polymerase by increasing the processivity
of N4 DNA polymerase and by melting out hairpin structures that block
polymerization(5) . The ability of N4SSB purified from
overproducing cells to activate N4 DNA polymerase on a primed template
was examined. Fig. 4shows that N4SSB purified from
overproducing cells activates N4 DNA polymerase on a primed template as
efficiently as the protein purified from N4 phage-infected cells, while
N4SSB is inactive.
Figure 4:
Cloned
N4SSB stimulates N4 DNA polymerase as efficiently as the protein
purified from N4-infected cells. Reactions contained X174 viral
single-stranded DNA primed with replicative factor HaeIII
fragment 7, N4 DNA polymerase, and increasing concentrations of N4SSB
purified from T7-directed overexpressing clones (
), N4SSB
purified from N4-infected cells (
), or N4SSB
(
).
To define the role of the carboxyl
terminus, we generated two types of deletions by site-specific
mutagenesis: carboxyl-terminal truncations and internal, in-frame
deletions. The sequence of the mutant N4SSB ORFs in each plasmid was
confirmed by double-stranded DNA sequencing. Mutant proteins were
cloned for overexpression in W3350pcnB(DE3)/pLysE. The size
and amount of expressed mutant protein were determined, following IPTG
induction and [S]methionine labeling, by
SDS-polyacrylamide gel electrophoresis and autoradiography. All mutants
were of the expected size, were expressed to the same degree, and were
as stable as the wild-type protein (Fig. 5).
Figure 5: Expression of cloned mutant N4SSB proteins in E. coli W3350pcnB(DE3)/pLysE carrying wild-type N4SSB (wt) or the indicated N4SSB mutants. Expression plasmids were incubated in the absence or presence of 1 mM IPTG, and the expressed proteins were labeled and analyzed as described under ``Materials and Methods.''
The ability of N4SSB mutants to complement N4am7 for N4 late transcription, to support N4 DNA recombination, and to activate N4am7 DNA replication was measured. The results of these experiments are presented in Table 2.
Deletion of the three carboxyl-terminal
amino acids generated a protein (N4SSB)
active in supporting replication, but inactive in recombination or late
transcription. Deletion of an additional residue
(N4SSB
) abolished all three activities.
These results suggest that the carboxyl-terminal region of N4SSB is
required for interactions with the N4 DNA replication, recombination,
and transcriptional machineries. Furthermore, the properties of
N4SSB
indicated that it is possible to
isolate N4SSB mutants in which the determinants for catalyzing all
three activities can be separated. This hypothesis was confirmed when
we characterized two internal, in-frame deletions:
N4SSB
and
N4SSB
. N4SSB
shows reduced ability to support late transcription, whereas
N4SSB
is fully proficient in supporting
recombination, while it cannot complement N4am7 in replication
and late transcriptional activation to wild-type levels.
The defect
in all mutants discussed above can be explained by impaired
single-stranded DNA binding activity and/or a differential involvement
of the single-stranded DNA binding activity of the protein in
recombination, replication, and activation of late transcription. To
rule out this possibility, the N4SSB protein, which is deficient in supporting replication,
recombination, and late transcription, was purified, and its ability to
bind to single-stranded DNA was estimated from gel shift experiments.
N4SSB
eluted at the same salt
concentration (1.5 M NaCl) from single-stranded DNA-agarose as
the wild-type protein. Since the binding site size of N4SSB is 11
± 2 nucleotides, a single-stranded DNA oligomer containing one
binding site size (12-mer) was used as a template in gel shift
experiments. To stabilize the interaction of the proteins and 12-mer
DNA, complexes were covalently cross-linked by irradiation at 300
ergs/mm
. Single-stranded DNA-bound N4SSB complexes were
analyzed on a native polyacrylamide gel (Fig. 6). N4SSB binding
to a 12-mer produces a major retarded species and a minor species due
to protein-protein interactions. N4SSB
binds more efficiently than the wild type to the single-stranded
12-mer, but fails to form the second complex. These and other results
(data not shown) indicate that N4SSB
is
deficient in N4SSB-N4SSB interactions.
Figure 6:
Binding of N4SSB and
N4SSB to single-stranded DNA. Reaction
mixtures containing an excess of labeled 12-mer oligonucleotide and
increasing concentrations of wild-type (wt) N4SSB or
N4SSB
were treated and applied to a 8%
native polyacrylamide gel as described under ``Materials and
Methods.'' Labeled free probe is not
shown.
We have succeeded in sequencing and cloning the gene for N4SSB, a protein required for viral DNA replication, activation of late transcription, and, as we demonstrate in this paper, phage recombination. N4SSB is 265 amino acids in length, and no sequence similarity to other single-stranded DNA-binding proteins is evident. Specifically, the acidic carboxyl-terminal region present in several single-stranded DNA-binding proteins is absent in the predicted N4SSB sequence(19) . However, both N4SSB and T4 gp32 contain a series of similarly spaced aromatic and charged residues in the first 130 amino acids(20) .
Our inability to clone the N4SSB gene
suggests that it is highly toxic to E. coli. While it is not
yet clear what the determinants of lethality are, two SSB functions
might be involved: its single-stranded DNA binding activity and its
ability to activate RNA polymerase at the N4 late promoters. Successful
cloning of N4SSB required a tightly regulated expression system
encompassing (a) deletion of weak E promoters present upstream of the N4SSB translational start site, (b) use of the chromosomal pcnB mutation to reduce
the plasmid copy number, (c) introduction of the T7 lysozyme
carried on plasmid pLysE to inhibit T7 RNA polymerase, and (d)
introduction of the lac operator sequence immediately
downstream of the T7 promoter to prevent T7 RNA polymerase from binding
to its promoter until IPTG is added.
The cloned and expressed
protein was able to complement N4am7 in vivo for N4
DNA replication and N4 late transcription. In addition, expression of
wild-type N4SSB from T7-directed expressing clones increased N4 DNA
recombination 10-10
-fold, indicating that
N4SSB is required for N4 DNA recombination.
Even though cloned N4SSB
was able to complement N4am7 for N4 DNA replication and N4
late transcription, the level of activation did not reach those
observed after N4am7 infection of suppressor-carrying cells.
We have considered several alternative explanations that can account
for the lower level (20%) of replication and late transcription
activation in cloned N4SSB-expressing cells. N4am7 is not a
dominant negative mutation. The N4am7 mutation could exert
polarity, affecting the expression of downstream gene products that
might be required for N4 DNA replication and late transcription. The
downstream region (2 kb) of the N4SSB-coding region was sequenced. Two
ORFs (185 and 147 amino acids in length) are present. An expression
plasmid (pMC8) carrying the wild-type N4SSB or am7 allele and
the two downstream ORFs was constructed. The N4SSB protein and the
expected products from the two downstream ORFs were expressed in E.
coli W3350pcnB(DE3)/pLysE. These proteins were also
expressed from pMC8am7 in E. coli W3350pcnB(DE3)/pLysE. ()These results suggest
that the am7 mutation does not affect the expression of the
two downstream genes. The ability of cloned N4SSB and the two
downstream gene products to complement N4am7 for N4 DNA
replication and N4 late transcription was examined (data not shown).
The levels of both activities were similar to those observed when only
N4SSB was expressed. We suspect that the inability of cloned N4SSB to
fully activate DNA replication and late transcription is due to the
timing of N4SSB expression during N4 development under these
conditions. The lower level of late transcription might also be the
result of lower levels of template.
The N4SSB expression vector pMC6
was able to rescue N4am7, while pMC6 was not.
Increased expression of wild-type N4SSB from expressing clones
increased N4 DNA recombination
10
-10
-fold. This observation indicates
that N4SSB plays an essential role in N4 DNA recombination. Two other
lines of evidence indicate that N4SSB is essential for N4 recombination
independently of its requirement for N4 DNA replication. First,
N4SSB
supports DNA replication, while it
is defective in activating N4 recombination (Table 2). Second,
two additional N4SSB mutants (N4SSB
and N4SSB
), although deficient in
supporting replication, can activate recombination to wild-type or
nearly wild-type levels. N4 DNA recombination is independent of host
recombination genes, suggesting that N4 encodes its own recombination
functions. (
)T4 gp32 is also required for T4 DNA
recombination. (
)In contrast, E. coli SSB activates
host DNA recombination; its absence in ssb mutant strains
reduces recombination only 7-fold(21) .
N4SSB and E.
coli SSB differ from other well characterized single-stranded
DNA-binding proteins in that, in addition totheir involvement in
replication and recombination, they are transcriptional
activators(6, 22) . N4SSB and E. coli SSB are
unique among transcriptional activators in that they do not bind to a
specific double-stranded DNA site for activation to occur. How do they
accomplish their transcriptional activation tasks in the absence of a
cognate DNA-binding site? We have shown that E. coli SSB is an
activator of the N4 virion-encapsulated DNA-dependent RNA polymerase by
providing the correct DNA structure, a DNA hairpin on the template
strand, for N4 virion RNA polymerase
recognition(22, 23) . ()DNA-binding sites
for transcriptional activators serve at least two functions: 1) to
increase the local concentration of the activator at its target and/or
2) to position the activator so as to make proper contacts with the
transcriptional machinery. A DNA-binding site for N4SSB as a
transcriptional activator might not be required since it is expressed
at high levels during infection:
11,000 molecules of N4SSB/cell
(9-18 µM)(5) . Indeed, we have recently
proposed that an N4SSB-E. coli RNA polymerase complex forms,
which then binds to N4 late promoters(24) . Preliminary results
indicate that the single-stranded DNA binding activity of N4SSB is not
required for transcriptional activation. (
)
The isolation of N4SSB mutants differentially affected in DNA replication, recombination, and activation of late transcription (Table 2) suggests that different determinants of N4SSB are important for these different activities. The isolation and characterization of additional N4SSB mutants specifically affected in DNA replication, recombination, activation of late transcription, single-stranded DNA binding, and cooperativity are required to understand the role of N4SSB in these processes.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) N4 U29728[GenBank].