Previous Article | Next Article 
Journal of Bacteriology, May 1999, p. 3123-3128, Vol. 181, No. 10
0021-9193/99/$04.00+0
Bacteriophage T4 rnh (RNase H) Null Mutations: Effects
on Spontaneous Mutation and Epistatic Interaction with
rII Mutations
Anna
Bebenek,
Leslie A.
Smith, and
John W.
Drake*
Laboratory of Molecular Genetics, National
Institute of Environmental Health Sciences, Research Triangle Park,
North Carolina 27709
Received 22 January 1999/Accepted 15 March 1999
 |
ABSTRACT |
The bacteriophage T4 rnh gene encodes T4 RNase H, a
relative of a family of flap endonucleases. T4 rnh null
mutations reduce burst sizes, increase sensitivity to DNA damage, and
increase the frequency of acriflavin resistance (Acr)
mutations. Because mutations in the related Saccharomyces
cerevisiae RAD27 gene display a remarkable duplication mutator
phenotype, we further explored the impact of rnh mutations
upon the mutation process. We observed that most Acr
mutants in an rnh+ strain contain
ac mutations, whereas only roughly half of the Acr mutants detected in an rnh
strain bear
ac mutations. In contrast to the mutational specificity
displayed by most mutators, the DNA alterations of ac
mutations arising in rnh and
rnh+ backgrounds are indistinguishable. Thus,
the increase in Acr mutants in an rnh
background is probably not due to a mutator effect. This conclusion is
supported by the lack of increase in the frequency of rI
mutations in an rnh background. In a screen that detects
mutations at both the rI locus and the much larger rII locus, the r frequency was severalfold lower in an
rnh background. This decrease was due to the phenotype
of rnh rII double mutants, which display an r+
plaque morphology but retain the characteristic inability of rII mutants to grow on 
lysogens. Finally, we summarize
those aspects of T4 forward-mutation systems which are relevant to
optimal choices for investigating quantitative and qualitative aspects of the mutation process.
| |
INTRODUCTION |
The bacteriophage T4 rnh
gene encodes Rnh, an RNase H that removes the RNA primers of DNA
replication (11). This nuclease, whose structure has been
determined by X-ray crystallography (21), acts as a 5'-to-3'
exonuclease on RNA-DNA and DNA-DNA duplexes and also as a flap
endonuclease (2). An rnh null mutation
(rnh) reduces total DNA synthesis only slightly, causing
instead an accumulation of short, nascent DNA fragments and reducing
the burst size roughly twofold (10). rnh is
partly complemented by the host rnhA gene, whose impairment
reduces the rnh mutant burst size to about 10% of
normal, while rnh is more strongly complemented by the
5'-exonuclease function encoded by the host polA gene, whose
impairment almost abolishes the rnh mutant burst size
(10). In addition to its role in DNA replication, Rnh acts in recombination repair: rnh increases sensitivity to the
lethal effects of both UV radiation and the topoisomerase inhibitor
4'-(9-acridinylamino)methanesulfon-m-anisidide (m-AMSA), and this survival defect is epistatic to mutations
in two genes required for T4 recombination repair, uvsW and
uvsX (33).
T4 Rnh is related by sequence and/or structure to a family of flap
endonucleases that includes examples from the archaebacteria Methanococcus jannaschii (13) and
Pyrococcus furiosus (12), a eubacterium, a
mammal, and the Saccharomyces cerevisiae DNA repair protein
RAD27 (reviewed in references 26 and
30). These endonucleases remove the single-stranded
flaps that can arise either as a result of displacement synthesis past
the beginning of an Okazaki fragment or during genetic recombination
and/or repair. However, T4 Rnh flap endonuclease is strongly inhibited by the gene-32 single-stranded-DNA-binding protein whereas
its RNase H activity is not (2). In the T4 multiprotein in
vitro DNA replication system, Rnh preferentially uses its
5'-exonuclease activity rather than its flap endonuclease to remove
primers and adjacent DNA from the 5' end of lagging-strand fragments
(3). A rad27 mutation has a strong mutator
phenotype at microsatellite and minisatellite sequences (14,
16) and a remarkable duplication mutator activity
(31). These duplications comprised 5 to 108 bp, arose at
sequences flanked by repeats of 2 to 12 bp, and were proposed to occur
in a process triggered by the failure to excise DNA flaps bearing
short, separated sequence repeats.
Defects in genes of T4 DNA metabolism often result in a mutator
phenotype (8). Thus, T4 rnh mutations might
display mutator activity, and this activity might include a bias toward
duplications. An rnh null mutation was reported previously
to increase the frequency of T4 acriflavin-resistant (Acr)
mutants (10). While such an increase might result from
mutator activity, the relation between mutant frequency and mutation
rate is often complicated and can be strikingly distorted when a
mutational target (such as the ac gene whose knockout
generates acriflavin resistance) interacts with a gene (such as
rnh) whose product is involved in DNA metabolism. Acriflavin
itself is both a topoisomerase inhibitor (19, 24) and a
powerful mutagen (22). Because of the unknown impact of
these potential interactions and also in order to determine whether a
T4 rnh mutator activity had a duplication bias, we decided
that the increase in Acr mutant frequency in an
rnh
background should be examined in some
detail. The unexpectedly complex results of this investigation are
described here.
| |
MATERIALS AND METHODS |
Strains.
The translated portion of the rnh gene
comprises 915 bp. The rnh(10-777) mutation, obtained
from Ken Kreuzer, is an in-frame deletion of codon-encoding bases 10 through 777 which leaves intact a middle-mode promoter near the end of
rnh and is thus unlikely to affect downstream genes
(33). The rnh(352-847) mutation (renamed from
the original) (10) removes this middle-mode promoter; in
addition, because it disrupts the reading frame, it retains the first
117 rnh codons and then adds VAKFIHIL before encountering an
ochre stop codon. The rI, rII, and rV
mutants are from the Drake collection; rIIUV58 is
hyperrevertible by proflavin (5). The rnh mutants
are in a T4D background, and the r mutants are from the
Drake collection in a T4B background.
The Escherichia coli strains are from the Drake collection.
B cells are su, and tight mutations of all the
major T4 r loci produce the r plaque morphology (large,
sharp-edged plaques) on B cells. B40 suI+ cells
are B cells containing an amber-suppressing allele. BB cells are
su; rII mutants produce an
r+ plaque morphology on BB cells. KB cells are K-12()
and restrict the growth of T4 rII mutants.
Media and growth conditions.
Luria-Bertani (LB) medium and
Drake agars (4) were used throughout at 37°C. Stocks were
grown with BB cells.
Scoring rnh alleles.
Individual plaques were
resuspended in 40 µl of water, and the rnh gene in each
was amplified by PCR. A 1,034-bp sequence (including primers) was
amplified from 
99 through 935 where the AUG initiation codon begins
with bp 1. The upstream primer was 5'-TGAAAACACAATAGGAGCCCG-3'
(99 through 79), and the downstream primer was
5'-TTTAGCCATTATTCACCTC-3' (917 through 935). The PCR consisted of 25 cycles of 1 min at 94°C, 1 min at 62°C, 1 min at
72°C, and a final extension time of 10 min at 72°C with Display Taq polymerase (Display System Biotech). PCR products were
separated on a 1.2% agarose gel. The rnh(10-777)
mutation produces a product of 266 bp; the rnh(352-847)
mutation produces a product of 538 bp.
Screening and sequencing Acr mutants.
T4 stocks
were plated on BB cells with acriflavin neutral (Sigma) at 0.2 µg/ml
in the top agar and 0.5 µg/ml in the bottom agar to select
Acr mutants and on unsupplemented agars to score total
phages when an Acr mutant frequency was desired; the plates
were incubated overnight. To obtain Acr mutants of
independent origin, individual acriflavin-sensitive plaques were
isolated and a single Acr mutant was isolated from each;
the rnh+ background was T4B.
The sequence of the
ac gene (formerly open reading frame
52.2) (
32) comprises 156 bp encoding 51 amino acids plus an
ochre
termination codon and extending from T4 genomic coordinate 165493
through 165338 (
17). To determine their
ac
sequences, each mutant
plaque was resuspended in 40 µl of water and
its
ac gene was PCR
amplified with the upstream primer
5'-TCGAAGAAATGAACCGTATGT-3'
(complementary to 165618 through
165638) and the downstream primer
5'-CTACCAATAAAGCAGCAAGGG-3'
(complementary to 165294 through 165314).
The PCR consisted of 25 cycles of 1 min at 94°C, 1 min at 50°C,
1 min at 72°C, and a
final extension time of 10 min at 72°C with
Display
Taq
polymerase. PCR products were purified with the Qiagen
purification
kit. Sequencing was performed with an ABI Prizm 377
automatic sequencer
with the above upstream PCR primer and the
dRhodamine terminator cycle
sequencing kit (PE Applied
Biosystems).
Test for acriflavin mutagenicity.
Working under yellow light
to avoid photodynamic effects, we used rIIUV58 in an
rnh+ or an rnh(352-847)
background to infect concentrated log-phase BB cells at 5 × 108/ml at a multiplicity of infection of 5 in LB broth at
37°C on a rotary shaker. At t = 6 min after
infection, another multiplicity of infection of 5 was applied. At
t = 10 min, acriflavin was added to 0 or 1 µg/ml. At
t = 30 min, the mixture was diluted 80-fold in LB
broth, and incubation was continued. Phage development was terminated
at t = 90 min by adding chloroform.
rIIUV58+ revertant frequencies were determined
by plating phages on KB cells (on which only
rII+ phages grow) and on BB cells (on which all
phages grow).
Screening r mutants.
When T4 is plated on B cells, large,
sharp-edged plaque morphology mutants can be observed. These are called
r mutants for rapid growth, although the number of phage particles per
plaque is reduced. The r phenotype results from loss of lysis
inhibition, and r mutations can arise at any of several
loci. About 70% of spontaneous r mutants contain mutations at the
rII locus; about 25% contain mutations at the rI
locus; and the remainder contain rIII, rV,
unmapped, or leaky or rapidly reverting rII mutations. Although the rII locus is about 10-fold larger than the
rI locus, rII missense mutations are poorly
detected whereas rI missense mutations are better detected,
so that the observed ratio of rII to rI mutants
is only about threefold. A typical median r mutant frequency scored by
plating on B cells is roughly 6 × 104 in a
high-titer stock grown in BB cells but is twofold to threefold lower in
a resuspended plaque. We picked and restreaked all r and ambiguous
plaques to verify their phenotypes.
rII mutants produce the r
+ plaque morphology on
BB cells. Thus, when T4 is plated on BB cells, most of the r mutants
contain
rI mutations. A typical median r mutant frequency in
a resuspended
plaque plated on BB cells is roughly 2 × 10
5.
Details of plating procedures for detecting r mutants have been
described elsewhere (
4). Because of the clonal nature of
the
distribution of mutant frequencies among stocks, the best
measure of a
mutant frequency is the median rather than the mean;
the mean is
excessively sensitive to the small proportion of stocks
with high
mutant frequencies, particularly jackpots. Medians from
five stocks are
about 95% reproducible to within twofold (
25),
and those
from seven stocks are about 95% reproducible to within
about 1.5-fold.
When stocks contain similar numbers of T4 particles,
ratios of mutation
rates are the same as ratios of mutant frequencies.
For simplicity,
therefore, we present frequencies rather than
rates.
| |
RESULTS |
Mutation to Acr.
We wished first to redetermine
Acr mutant frequencies in the wild-type and
rnh backgrounds and then to characterize the mutations at
the level of DNA sequence. The Acr mutant frequency was
previously estimated to increase 10-fold (from about 4 × 106) in an rnh(352-847) background
compared to an rnh+ background (10).
Using the treatment protocol described in reference
10 but substituting BB cells for B cells, we
observed a wide range (2 × 106 to 20 × 106) of median Acr frequencies in several
experiments in an rnh+ background; in
particular, we were plagued by a variable background of tiny plaques on
the selection plates. Our Acr frequencies were 5 × 106 to 37 × 106 in
rnh(10-777) and 1 × 106 to 50 × 106 in rnh(352-847). Ratios of
Acr mutants in rnh versus
rnh+ were 0.5 to 20 for
rnh(10-777) and 0.5 to 10 for
rnh(352-847).
In order to identify possible changes in the rates of specific kinds of
Ac
r mutations, we turned to DNA sequencing. We plated
wild-type and
(both)
rnh strains in the absence of
acriflavin, suspended well-separated
plaques (of large to small rather
than pinpoint size), replated
these low-titer stocks in the presence of
acriflavin, picked a
single Ac
r mutant from each stock, PCR
enriched the
ac DNA, and sequenced.
[We analyzed similar
numbers of Ac
r mutants in
rnh(10-777) and
rnh(352-847) backgrounds and discerned
no differences
between the two backgrounds in all that follows.]
T4-infected cells
take up acridines at an increased rate compared
to uninfected cells,
and
ac mutations arise in a gene whose inactivation
prevents
this uptake (
27). The fractions of
ac mutants
among
Ac
r mutants were different in the
rnh+ and
rnh backgrounds (Table
1). In the wild-type background,
94% of
Ac
r mutants contained
ac mutations, a result
indistinguishable from
the 68 of 76 mutants (89%) reported previously
(
32). In the
rnh background, however, only
57% of Ac
r mutants contained
ac mutations. The
locations and Ac sensitivities
of the non-
ac mutations were
not investigated and remain unknown,
but our primers amplify 125 nucleotides before the start codon
and 23 nucleotides after the
termination codon, so that the observed
acriflavin resistance is
unlikely to result from a defect in
ac function; similar
mutations described previously arose in a small
region linked to
ac (
32). Our result suggests that roughly half
of
the previously described increase in Ac
r mutants in an
rnh background (
10) has nothing to do with
mutation
at the
ac locus.
The two
ac mutational spectra are shown in Fig.
1 (which does not display several large
mutations that are described in the
figure legend). None of the
mutations are complex (that is, mixtures
of two or more base pair
substitutions, insertions, and/or deletions).
These spectra do not
approach saturation, so that many of the
smaller differences tend to be
uninformative. However, the spectra
are notable for the several sites
and regions sporting multiple
mutations in both
rnh+ and
rnh backgrounds. In
addition, there is one apparent difference
between mutation frequencies
in the two
rnh backgrounds at position
108 (three additions
and four deletions in
rnh+ versus no mutations
in
rnh). A notable observation of multiple
differences
with little change in total mutation rate has been
described elsewhere
for differing gene-
43 backgrounds (
32).
A much
larger collection of
ac mutations would have to be
characterized
to determine whether any of the differences in the two
rnh backgrounds
are real.
View larger version (14K):
[in this window]
[in a new window]
|
FIG. 1.
Spectra of spontaneous ac mutations in
rnh+ (above sequence) and rnh
(below sequence) genetic backgrounds. Capital letters indicate base
pair substitutions, boldface indicates insertions, and lowercase
letters indicate deletions. The sequence is the ac gene from
ATG (positions 1 through 3) encoding the initiation codon through TAA
(positions 153 to 156) encoding the ochre termination codon; every 10th
base is underlined and numbered beneath when possible. When an
insertion or deletion could have arisen between two or more short
repeated sequences, the mutation is placed in the middle of the
wild-type repeats. Two or more adjacent added or deleted bases
represent a single mutation, and repeated mutations are stacked. There
are no complex mutations containing mixtures of base pair
substitutions, insertions, and/or deletions. Note that several
mutations are not shown: deletions of 32 bases between TTTT at 59 through 62 and at 91 through 94 that occurred three times in
rnh+ and twice in rnh and one
addition of 99 bases that occurred once in rnh (see
text).
|
|
The classes of mutations are summarized in Table
2. In contrast to Table
1, there is no
significant difference between the
frequencies of the various kinds of
mutations recovered from the
two genetic backgrounds; when comparisons
were made between the
11 categories of observed base pair substitutions
and additions
and deletions of base pairs, df = 10,
2 = 10.5, and
P = 0.40. Few mutator mutations produce mutations
identical to those arising in the nonmutator background. It therefore
seems likely that the previously reported increase in Ac
r
mutants in an
rnh background (
10) was due at
least in part
to the non-
ac mutants that are specifically
selected in that background
and perhaps to other less well
characterized factors.
Given the duplication propensity of the yeast
rad27 mutator
mentioned earlier, it is notable that the proportion of duplication
mutations in the two sets of
ac mutations is virtually
identical
(10 of 61 in the
rnh+ background and 5 of 30 in the
rnh background). These duplications
are
shorter than most of those observed in the yeast
rad27
mutator:
the T4 duplications included 12 of 1 bp, 6 of 2 bp, and 1 each
of 3, 4, 6, and 10 bp in the
rnh+ background and
3 of 1 bp, 2 of 2 bp, and 1 each of 4, 5, and
99 bp in the
rnh background. All of the addition mutations were
tandem
duplications. These can be of two general types: those
in which no
flanking sequence repeats existed in the wild-type
sequence and those
apparently arising from repeats. The latter
can be described as RiR
RiRiR where R is a repeated sequence
(which can be as short as a single
base pair) and i is an intervening
sequence between the repeats. Such
mutations can arise by slipped
mispairing (
29) and are
characteristic of the yeast
rad27 duplication
mutations
(
31). Of the 22 insertions in the
rnh+ background, 12 have slippage-like structure
and are additions
of only 1 or 2 bases. Of the eight insertions in the
rnh background,
three have slippage-like structure, three
are additions of only
1 or 2 bases, and one is an addition of 99 bases
where R is CATTT
at positions 32 through 36 and 131 through 135. Only
this single
mutation of the 30 additions resembles the duplications
arising
in the yeast
rad27 mutator.
Tests for acriflavin mutagenicity in the screen for Acr
mutants.
Acriflavin is a mixture of
3,6-diamino-10-methylacridinium chloride and 3,6-diaminoacridine
(proflavin), both of which are strong frameshift mutagens when present
during T4 replication (22). Thus, the acriflavin test might
be intrinsically mutagenic. Furthermore, because acridines
preferentially mutate and promote breakage at topoisomerase sites in T4
DNA (19, 24), and because an rnh mutation may
interfere with the repair of such breakage (33), the
acriflavin test may preferentially kill or mutate replicating genomes
carrying an rnh defect.
This possibility was tested by comparing acriflavin-induced reversion
of
rIIUV58 in
rnh and
rnh+ backgrounds (see Materials and Methods).
rIIUV58 was chosen because
its reversion is unusually
strongly promoted by proflavin mutagenesis
(
5). The results
of three experiments appear in Table
3.
The
variation is typical of experiments involving mutagenesis with
acridines but the trend is clear: acriflavin (at a concentration
twice
that used in our measurements of Ac
r frequencies, but for a
shorter time) is slightly more mutagenic
(2.1-fold on average) in an
rnh background than in an
rnh+
background. Although
rnh mutations typically reduce burst
sizes,
no reduction was seen in these particular experiments. Proflavin
treatments also typically reduce burst sizes, and the reduction
was slightly greater (85 versus 74%) in the
rnh
background. The
small difference in relative net mutagenesis
observed with this
very sensitive target suggests that the Ac test is
not significantly
more mutagenic to
rnh than to
rnh+ strains. This surmise is supported by the
lack of any increase
in the frequency of base pair additions or
deletions among
ac mutants in the
rnh
background compared to the
rnh+ background
(Table
2).
Absence of an rnh effect on mutation from
r+ to rI.
Most r mutants detected on
E. coli BB cells bear rI mutations. The
rI-encoded protein appears to sense superinfection and to
carry out the first step in establishing lysis inhibition
(23) but is not obviously involved in DNA metabolism. The
97-codon rI locus is large enough to fairly sample average
mutation rates. Therefore, in order to determine whether an
rnh mutation affects rates of spontaneous mutation in a
gene other than ac, we scored frequencies of r mutants on BB
cells. The results appear in Table 4. Two
entries per rnh genotype appear for mutant frequencies: values for sharp-edged plaques displaying the classical r
phenotype and values also including slightly fuzzy-edged
plaques (sr, for "semi-r") which we anticipated would be caused by
leaky missense mutants. To confirm this conjecture, we isolated eight
such sr plaques and sequenced them; four contained rI
missense mutations, one contained a frameshift mutation near the end of
the gene, and three contained no rI mutation. Therefore, the
values in the "r + sr" column were computed by multiplying the
sr contribution by five-eighths. Mutant frequencies such as those in
Table 4 based on seven stocks are usually reproducible to about
1.5-fold (25). The data in Table 4 reveal no
rnh mutator activity on the rI gene.
Epistasis between rnh and rII
mutations.
Before undertaking the experiments detailed in Tables 3
and 4, we sought to determine the frequencies of r plaque morphology mutants in stocks plated on E. coli B cells. On B cells,
roughly 70% of spontaneous r mutants carry typical rII
mutations; about 25% carry rI mutations; and the remainder
carry leaky or rapidly reverting rII mutations,
rIII mutations, or r mutations at other less well
characterized loci. Because the rII locus comprises about
3,135 bp, the frequency of r mutants is usually much higher on B cells
than on BB cells, even though missense mutations are more efficiently
detected in rI than in rII. Thus, an r-mutant screen on B cells was expected to be a simple, sensitive test for
mutator activity in rnh mutants. The results appear in
Table 5. Contrary to our expectations,
frequencies of r mutants are clearly reduced severalfold in a
rnh background. (The data suggest that this effect may
differ in magnitude between the two different rnh
alleles, but this possibility was not further explored.)
Because our results with the
ac and
rI systems
gave no hint of an antimutator effect, we suspected that the reduced
frequencies
of r mutants in Table
5 reflected some special property of
rnh rII double mutants such as synthetic lethality or
epistasis. This
suspicion was supported by the results of crosses of
the form
rnh ×
r where
r =
rII versus
rI or
rV. If recombinant
rnh r double mutants were lethal or indetectable, then
the frequency
of r mutants should fall in the cross progeny compared to
the
parental mix. We observed no change in
r frequencies in
rnh crosses
against
rI or
rV
(average progeny/parental ratio = 1.02) but a
marked decrease in
rnh crosses against
rII mutants (average
progeny/parental
ratio = 0.48) with both
rnh
mutations. In addition, because the
rnh allele causes
small plaques, we collected numerous progeny
with a small-r
(sharp-edged) phenotype and tested them for the
rnh
allele by PCR; we found
none.
We next performed crosses between
rnh mutants and five
different
rII amber mutants with B40
suI+ host cells for the cross and for plating
the progeny (so that
all progeny necessarily displayed the
r
+ plaque morphology). Ten small-plaque progeny were
isolated from
each cross. These were tested for inability to grow
on
E. coli su K(
) cells, a characteristic
of most
rII mutations; among the
nongrowers, we screened for
the presence of the
rnh allele by
PCR. Each of the
crosses readily yielded progeny that appeared
to bear both mutant
alleles. These progeny were backcrossed against
the original
rII parent to confirm the identity of the
rII
allele.
Thus,
rII mutations (including the frameshift
mutation
rIIUV58 described previously) display an
r
+ plaque morphology in the presence of an
rnh
null allele but do
not regain the ability to grow on K(
)
cells.
| |
DISCUSSION |
Apparent lack of an rnh effect on mutation
rates.
The evidence as a whole indicates that bacteriophage T4
rnh null mutations do not affect mutation rates to an
appreciable extent. No change was detected with the rI
system, and the increase in mutant frequency reported previously with
the Acr system is unlikely to reflect a corresponding
increase in mutation rate for three reasons. First, we did not find a
reliable increase in the frequency of Acr mutants by the
reference protocol (10), although an alternative protocol
(32) appears to be more robust. (We used a different strain
of B cells, namely, BB or B Berkeley, but we have no reason to suspect
that B and BB would behave differently in rnh phage infections.) Second, while most Acr mutants in an
rnh+ background contain ac mutations,
many in an rnh background do not. Third, there was no
discernible difference in the kinds of ac mutants produced
in the two backgrounds, whereas most mutator mutants display
specificity. Therefore, either the host DNA polymerase I 5'-exonuclease
activity that moderately complements the loss of Rnh or the host RNase
H that weakly complements the loss (10) seems to provide any
fidelity components that might be missing in an rnh mutant.
Note, however, that changes in mutational specificity can occur without
changes in mutation rate (
6,
32). The data in
Table
2 can
exclude only major changes in mutational specificity.
However, they do
appear to exclude any strong slippage-like duplication
bias in an
rnh background.
Epistasis between rnh and rII
mutations.
Just as a mutator effect was shown to be an unlikely
explanation for an increased Acr mutant frequency in an
rnh background, an antimutator effect was shown not to be
the cause of a decreased r mutant frequency in an rnh
background. Instead, the severalfold decrease in the median r mutant
frequency in stocks plated on B cells reflected the unanticipated
r+ plaque morphology of rII rnh double
mutants, although such double mutants retain the typical inability of
rII mutants to grow on a lysogen.
The
rII locus interacts with several other loci important in
DNA metabolism (
20,
23,
28).
rII mutations
produce the
r
+ plaque morphology when accompanied by a
49tsC9 (Holliday resolvase)
mutation or by the
gene-
32 (single-stranded-DNA-binding protein)
mutation
32tsL171. Conversely,
rII mutations partly
suppress
32tsL171 and also suppress gene-
30 (DNA
ligase) mutations. Thus,
rII function
is extensively
connected to DNA metabolism, albeit in poorly understood
ways. Indeed,
the two
rII-encoded proteins not only are associated
with
the cell membrane but also cosediment with the huge multiprotein
DNA
replication complex (
9). The failure of
rII
mutations to
express the r plaque morphology on BB and K-12 host cells
suggests
that the
rII locus has only an indirect role in
lysis inhibition
(
23). However, this pattern of interactions
with DNA metabolism
highlights the need for caution when using the
rII system as a
mutational reporter gene (
7,
8).
The ac mutational spectrum.
Because our spectra
are based on only 91 mutations distributed among many sites and
classes, it is premature to analyze the ac mutational
spectrum in detail. In any case, this is not the purpose of the present
report, and we leave such analysis to our colleague and ac
champion Lynn Ripley (32). Note, however, that the pooled
North Carolina and 43+ New Jersey ac
mutations may differ. The two sets were obtained by somewhat different
selection procedures that may have affected the efficiency of
recovering missense mutations, which comprised 17 of 34 of the North
Carolina base pair substitutions but 27 of 33 of the New Jersey base
pair substitutions. The distribution of mutations among classes may be
different in the two collections (df = 13, 2 = 22.4, P = 0.050), but if they are, much of the
difference is due to five GC (GlyArg) mutations at position 112 that are unique to the New Jersey collection; if those five mutations
are not considered, df = 12, 2 = 15.7, and P = 0.21.
Optimal T4 targets for forward mutation.
The sensitivity of a
mutation test reflects the magnitude of changes produced by physical or
chemical treatments or genetic modifiers. The specificity of a mutation
test reflects the information that it provides about what kinds of
mutations arise and where. Reversion tests tend to maximize sensitivity
and specificity but may provide uncharacteristic responses conditioned
by the local DNA sequence in which the reverting base pairs reside;
some potential responses may escape detection altogether, and little or
no information is generated concerning the effects of local DNA
sequence. In contrast, forward-mutation tests may display reduced
sensitivity because only certain kinds of mutations tend to be
increased experimentally, but such tests may display high sensitivity
with regard to effects of local sequence on mutation. Currently, there
are three useful T4 targets for forward mutation, rII,
ac, and rI, each with its particular advantages
and disadvantages (Table 6).
Until recently, the most frequently used T4 mutation system employed
the powerful
rII locus, which can be used to measure
both
forward mutation and reversion. A large amount of information
is
available about both spontaneous and induced mutation in the
rII locus. However, this system has several drawbacks. One
is
lack of information about the biochemical roles of the
rII-encoded
proteins (reviewed in reference
28 and largely unchanged since
then), including
their equivocal role in lysis inhibition (
23).
Another is
the involvement of
rII function in DNA metabolism,
as
detailed above. A third is the large size of
rII, so that
very
large numbers of mutants must be isolated when it is desirable
to
identify preferentially mutable sites, and mutations must be
mapped
before they can be sequenced efficiently. A fourth is the
inefficiency
with which missense mutations are
detected.
The
ac and
rI systems share the advantages of
small size and relatively efficient detection of missense mutations
(
1,
23,
32). (We will describe several aspects of the
rI system in more
detail in a later article.) Small size has
two advantages, ease
of sequencing and ease of detecting differential
mutability from
site to site. The
ac system is the easiest
for collecting mutants
of independent origin but may be difficult to
quantitate, and
the Ac
r screen may perturb DNA metabolism.
The
rI system seems to be
totally independent of DNA
metabolism (although crucial for lysis
inhibition, which leads to more,
apparently quite normal DNA synthesis).
The main drawback of the
rI system is the low frequency of spontaneous
mutants, which
renders quantitation and mutant collection tedious;
however,
this problem disappears under conditions that increase
mutant
frequencies by 10-fold or
more.
We also investigated the
alc system, in which forward
mutations can be selected by plating on a specific host
(
18). Unfortunately,
the resulting
alc plaques
are small under a variety of plating
conditions, so that quantitation
is difficult. In addition,
alc mutations cause T4 DNA to
contain ordinary cytosine instead of
5-hydroxymethylcytosine, a
difference that perturbs DNA
metabolism.
| |
ACKNOWLEDGMENTS |
We thank Nancy Nossal and Ken Kreuzer for providing their
rnh mutants, Nancy Nossal for helpful discussions during
the course of the investigation, and Joe Haseman and Judi Fleming for
assistance in statistical testing. Lynn Ripley, Dmitry Gordenin, and
Roel Schaaper provided invaluable critical comments at various stages of the evolution of this article.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Laboratory of
Molecular Genetics E3-01, National Institute of Environmental Health Sciences, P.O. Box 12233, Research Triangle Park, NC 27709. E-mail: drake{at}niehs.nih.gov.
| |
REFERENCES |
| 1.
| Bebenek, A., H. K. Dressman, and J. W. Drake. Unpublished results.
|
| 2.
|
Bhagwat, M.,
L. J. Hobbs, and N. G. Nossal.
1997.
The 5'-endonuclease activity of bacteriophage T4 RNase H is stimulated by the T4 gene 32 single-stranded DNA-binding protein, but its flap-endonuclease is inhibited.
J. Biol. Chem.
272:28523-28530[Abstract/Free Full Text].
|
| 3.
| Bhagwat, M., and N. G. Nossal. Unpublished
results.
|
| 4.
|
Conkling, M. A., and J. W. Drake.
1984.
Isolation and characterization of conditional alleles of bacteriophage T4 genes uvsX and uvsY.
Genetics
107:505-523[Abstract/Free Full Text].
|
| 5.
|
Drake, J. W.
1963.
Properties of ultraviolet-induced rII mutants of bacteriophage T4.
J. Mol. Biol.
6:268-283.
|
| 6.
|
Drake, J. W.
1993.
General antimutators are improbable.
J. Mol. Biol.
229:8-13[Medline].
|
| 7.
|
Drake, J. W., and L. S. Ripley.
1983.
The analysis of mutation in bacteriophage T4: delights, dilemmas, and disasters, p. 312-320.
In
C. K. Mathews, E. M. Kutter, G. Mosig, and P. B. Berget (ed.), Bacteriophage T4. American Society for Microbiology, Washington, D.C.
|
| 8.
|
Drake, J. W., and L. S. Ripley.
1994.
Mutagenesis, p. 98-124.
In
J. D. Karam (ed.), The molecular biology of bacteriophage T4. American Society for Microbiology, Washington, D. C.
|
| 9.
|
Greenberg, G. R.,
P. He,
J. Hilfinger, and M.-J. Tseng.
1994.
Deoxyribonucleoside triphosphate synthesis and phage T4 DNA, p. 14-27.
In
J. D. Karam (ed.), The molecular biology of bacteriophage T4. American Society for Microbiology, Washington, D. C.
|
| 10.
|
Hobbs, L. J., and N. J. Nossal.
1996.
Either bacteriophage T4 RNase H or Escherichia coli DNA polymerase I is essential for phage replication.
J. Bacteriol.
178:6772-6777[Abstract/Free Full Text].
|
| 11.
|
Hollingsworth, H. C., and N. G. Nossal.
1991.
Bacteriophage T4 encodes an RNase H which removes RNA primers made by the T4 DNA replication system in vitro.
J. Biol. Chem.
266:1888-1897[Abstract/Free Full Text].
|
| 12.
|
Hosfield, D. J.,
C. D. Mol,
B. Shen, and J. A. Tainer.
1998.
Structure of the DNA repair and replication endonuclease and exonuclease FEN-1: coupling DNA and PCNA binding to FEN-1 activity.
Cell
95:135-146[Medline].
|
| 13.
|
Hwang, K. Y.,
K. Baek,
H.-Y. Kim, and Y. Cho.
1998.
The crystal structure of flap endonuclease-1 from Methanococcus jannaschii.
Nat. Struct. Biol.
5:707-713[Medline].
|
| 14.
|
Johnson, R. E.,
G. K. Kovali,
L. Prakash, and S. Prakash.
1995.
Requirement of the yeast RTH1 5' to 3' exonuclease for the stability of simple repetitive DNA.
Science
269:238-240[Abstract/Free Full Text].
|
| 15.
|
Koch, R. E., and J. W. Drake.
1970.
Cryptic mutants of bacteriophage T4.
Genetics
65:379-390[Free Full Text].
|
| 16.
|
Kokoska, R. J.,
L. Stefanovic,
H. T. Tran,
M. A. Resnick,
D. A. Gordenin, and T. D. Petes.
1998.
Destabilization of yeast micro- and minisatellite DNA sequences by mutations affecting a nuclease involved in Okazaki fragment processing (rad27) and DNA polymerase  (pol3-t).
Mol. Cell. Biol.
18:2779-2788[Abstract/Free Full Text].
|
| 17.
|
Kutter, E.,
T. Stidham,
B. Guttman,
E. Kutter,
D. Batts,
S. Peterson,
T. Djavakhishvili,
F. Arisaka,
V. Mesyanzhinov,
W. Rüger, and G. Mosig.
1994.
Genomic map of bacteriophage T4, p. 491-518.
In
J. D. Karam (ed.), The molecular biology of bacteriophage T4. American Society for Microbiology, Washington, D. C.
|
| 18.
|
Kutter, E.,
T. White,
M. Kashlev,
M. Uzan,
J. McKinney, and B. Guttman.
1994.
Effects on host genome structure and expression, p. 357-368.
In
J. D. Karam (ed.), The molecular biology of bacteriophage T4. American Society for Microbiology, Washington, D. C.
|
| 19.
|
Masurekar, M.,
K. N. Kreuzer, and L. S. Ripley.
1991.
The specificity of topoisomerase-mediated DNA cleavage defines acridine-induced frameshift within a hotspot in bacteriophage T4.
Genetics
127:453-462[Abstract].
|
| 20.
|
Mosig, G.
1994.
Homologous recombination, p. 54-84.
In
J. D. Karam (ed.), The molecular biology of bacteriophage T4. American Society for Microbiology, Washington, D. C.
|
| 21.
|
Mueser, T. C.,
N. G. Nossal, and C. C. Hyde.
1996.
Structure of bacteriophage T4 RNase H, a 5' to 3' RNA-DNA and DNA-DNA exonuclease with sequence similarity to the RAD2 family of eukaryotic proteins.
Cell
85:1101-1112[Medline].
|
| 22.
|
Orgel, A., and S. Brenner.
1961.
Mutagenesis of bacteriophage T4 by acridines.
J. Mol. Biol.
3:762-768[Medline].
|
| 23.
|
Paddison, P.,
S. T. Abedon,
H. K. Dressman,
K. Gailbreath,
J. Tracy,
E. Mosser,
J. Neitzer,
B. Guttman, and E. Kutter.
1998.
The roles of the bacteriophage T4 r genes in lysis inhibition and fine-structure genetics: a new perspective.
Genetics
148:1539-1550[Abstract/Free Full Text].
|
| 24.
|
Ripley, L. S.,
J. S. Dubins,
J. G. deBoer,
D. M. DeMarini,
A. M. Bogerd, and K. N. Kreuzer.
1988.
Hotspot sites for acridine-induced frameshift mutations in bacteriophage T4 correspond to sites of action of the T4 type II topoisomerase.
J. Mol. Biol.
200:665-680[Medline].
|
| 25.
|
Santos, M. E., and J. W. Drake.
1994.
Rates of spontaneous mutation in bacteriophage T4 are independent of host fidelity determinants.
Genetics
138:553-564[Abstract].
|
| 26.
|
Shen, B.,
Z. Qiu,
D. Hosfield, and J. A. Tainer.
1998.
Flap endonuclease homologs in archaebacteria exist as independent proteins.
Trends Biochem. Sci.
23:171-173[Medline].
|
| 27.
|
Silver, S.
1965.
Acriflavin resistance: a bacteriophage mutation affecting the uptake of dye by the infected bacterial cells.
Proc. Natl. Acad. Sci. USA
53:24-30[Free Full Text].
|
| 28.
|
Singer, B. S.,
S. T. Shinedling, and L. Gold.
1983.
The rII genes: a history and a prospectus, p. 327-333.
In
C. K. Mathews, E. M. Kutter, G. Mosig, and P. B. Berget (ed.), Bacteriophage T4. American Society for Microbiology, Washington, D. C.
|
| 29.
|
Streisinger, G.,
Y. Okada,
J. Emrich,
J. Newton,
A. Tsugita,
E. Terzaghi, and M. Inouye.
1966.
Frameshift mutations and the genetic code.
Cold Spring Harbor Symp. Quant. Biol.
31:77-84[Abstract/Free Full Text].
|
| 30.
|
Symington, L. S.
1998.
Homologous recombination is required for the viability of rad27 mutants.
Nucleic Acids Res.
26:5589-5595[Abstract/Free Full Text].
|
| 31.
|
Tishkoff, D. X.,
N. Filosi,
G. M. Gaida, and R. D. Kolodner.
1997.
A novel mutation avoidance mechanism dependent on S. cerevisiae RAD27 is distinct from DNA mismatch repair.
Cell
88:253-263[Medline].
|
| 32.
|
Wang, F. J., and L. S. Ripley.
1998.
The spectrum of acridine resistant mutants of bacteriophage T4 reveals cryptic effects of the tsL141 DNA polymerase allele on spontaneous mutagenesis.
Genetics
148:1655-1665[Abstract/Free Full Text].
|
| 33.
|
Woodworth, D. L., and K. N. Kreuzer.
1996.
Bacteriophage T4 mutants hypersensitive to an antitumor agent that induces topoisomerase-DNA cleavage complexes.
Genetics
143:1081-1090[Abstract].
|
Journal of Bacteriology, May 1999, p. 3123-3128, Vol. 181, No. 10
0021-9193/99/$04.00+0
This article has been cited by other articles:
-
Miller, E. S., Kutter, E., Mosig, G., Arisaka, F., Kunisawa, T., Ruger, W.
(2003). Bacteriophage T4 Genome. Microbiol. Mol. Biol. Rev.
67: 86-156
[Abstract]
[Full Text]
-
Bebenek, A., Dressman, H. K., Carver, G. T., Ng, S.-s., Petrov, V., Yang, G., Konigsberg, W. H., Karam, J. D., Drake, J. W.
(2001). Interacting Fidelity Defects in the Replicative DNA Polymerase of Bacteriophage RB69. J. Biol. Chem.
276: 10387-10397
[Abstract]
[Full Text]
Top
Abstract
Introduction
