T4 chromosome shape and rate of replication

Multiple origins of replication contribute to a discontinuous
pattern of DNA synthesis across the T4 genome during infection
J. Rodney Brister and Nancy G. Nossal
Laboratory of Molecular and Cellular Biological, National Institute of Diabetes, Digestive and Kidney
Diseases, National Institutes of Health, Department of Health and Human Services, Bethesda,
Maryland, 20892-1770
Summary
Chromosomes provide a template for a number of DNA transactions, including replication and
transcription, but the dynamic interplay between these activities is poorly understood at the genomic
level. The bacteriophage T4 has long served as a model for the study of DNA replication,
transcription, and recombination, and should be an excellent model organism in which to integrate
in vitro biochemistry into a chromosomal context. As a first step in characterizing the dynamics of
chromosomal transactions during T4 infection, we have employed a unique set of macro array
strategies to identify the origins of viral DNA synthesis and monitor the actual accumulation of
nascent DNA across the genome in real time. We show that T4 DNA synthesis originates from at
least five discrete loci within a single population of infected cells, near oriA, oriC, oriE, oriF, and
oriG, the first direct evidence of multiple, active origins within a single population of infected cells.
Although early T4 DNA replication is initiated at defined origins, continued synthesis requires viral
recombination. The relationship between these two modes of replication during infection has not
been well understood, but we observe that the switch between origin and recombination-mediated
replication is dependent on the number of infecting viruses. Finally, we demonstrate that the nascent
DNAs produced from origin loci are spatially and temporally regulated, leading to the accumulation
of multiple, short DNAs near the origins, which are presumably used to prime subsequent
recombination-mediated replication. These results provide the foundation for the future
characterization of the molecular dynamics that contribute to T4 genome function and evolution and
may provide insights into the replication of other multi origin chromosomes.
Introduction
All organisms share a common need to replicate their genomes. This process is required to
distribute genetic material to progeny during reproduction and to create daughter cells during
development of multi-cellular organisms. Often replication occurs concurrently with
transcription and other DNA transactions on a genomic template cluttered with bound proteins,
presumably creating an obstacle course of molecular machines, performing the various tasks
involved in the maintenance of chromosomes. Such a scenario would imply that DNA
replication, transcription, and other genomic events are orchestrated to prevent one transaction
from impeding another, creating a complex molecular system, which provides a context for
chromosome utility and arbitrates genome evolution.
Corresponding author: J. Rodney Brister, Laboratory of Molecular and Cellular Biology, NIDDK, National Institutes of Health, Building
8, Room 2A-22, Bethesda, MD 20892-0830, USA, PHONE [phone], FAX [phone], vog.hin.kddin|rbsemaj#vog.hin.kddin|rbsemaj
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J Mol Biol. Author manuscript; available in PMC 2008 April 27.
Published in final edited form as:
J Mol Biol. 2007 April 27; 368(2): 336–348.
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The dynamics of DNA replication and other chromosomal transactions remain poorly
understood on a genomic level, and, even in the well-defined Escherichia coli system, where
the mechanics of replication initiation and termination have been determined (reviewed in 1;
2), there is still some controversy surrounding the progression of DNA synthesis across the
genome 3; 4. The situation in eukaryotic systems is even less clear, as many origins contribute
to chromosomal replication, raising a number of questions regarding the regulation of origin
activity and the resolution of the multiple nascent DNAs arising from the various origins into
a single, genomic polymer. Several groups have employed DNA micro arrays to address these
questions 5; 6, and these studies have helped to identify the origins and timing of replication
across entire genomes.
It is not a simple task to map the dynamic accumulation of DNA synthesis products in most
systems. Typically, the chromosome is only replicated a single time, highlighting background
and noise issues, and it can be difficult to maintain synchrony within a population of replicating
cells throughout the entire cycle. We sought to avoid these problems by using the bacteriophage
T4 as a model system in which to study the mechanics and dynamics of chromosomal
replication. Like the chromosomes of eukaryotic cells, the linear, 172 kb, T4 chromosome
contains multiple origins of replication, yet, in contrast, T4 is replicated several hundred times
during a single, 30 to 60 minute infection. This rapid genome synthesis is accomplished by a
viral replisome that polymerizes DNA at a rate of 30–45 kb per minute 7, a spectacular feat
given that transcription is concurrent with replication during infection.
The T4 chromosome is terminally redundant and about 3 kb of DNA sequence is randomly
repeated at the ends of the chromosome, creating a circularly permutated genetic map. The
lack of defined chromosomal telomeres and the rapid production of progeny virions
presumably contribute to a unique T4 replication strategy that includes both origin dependent
and recombination dependent replication. Initial T4 DNA synthesis is thought to originate de
novo from one of several loci, and it has been assumed that nascent DNAs initiated at these
origins are extended bi-directionally until the resultant duplex polymer reaches the ends of the
parental genome 8. Although this origin-mediated replication is sufficient to produce some
full-length, infectious virus, the vast majority of T4 DNA synthesis is dependent on the viral
recombination machinery 8. The transition from origin- to recombination mediated replication
is not well understood, but once the parental viral genome has been copied, there should be
two possible ways in which homologous recombination can be initiated: Nascent daughter
chromosomes could recombine with the homologous parental sequences, or the terminally
redundant, circularly permutated viral chromosome could recombine with itself, in either case
producing a primer for continued DNA synthesis.
Both origin-mediated and recombination-mediated DNA replication use an overlapping set of
T4 gene products, which include the gene 43 DNA polymerase, 45 polymerase clamp, and
44/62 clamp loader, as well as the 41 helicase and the 59 helicase loader 8; 9; 1. The 59 protein
binds several DNA structures that mimic recombination intermediates used to prime
recombination-mediated replication and targets 41 helicase loading to these substrates,
allowing formation of a processive DNA replisome 11. The role of 59 protein during origin
mediated replication is less clear. It stimulates replication on R-loop substrates designed to
mimic RNA primers presumed to initiate origin-mediated replication in vitro 12; 13 but is not
absolutely required for viral replication during infection 8; 14; 15.
Like other T4 recombination genes, including the recA homologue uvsX, mutation of the
helicase loader gene 59 causes viral DNA synthesis to halt, prematurely, giving rise to the socalled
DNA arrest phenotype (reviewed in 8; 15; 16). Although the timing of this arrest is
thought to mark the transition from origin to recombination-mediated replication, little is
known about the switch between the two modes of replication, and the null recombination
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deficient phenotype is poorly defined. Many of the T4 recombination genes contribute to
multiple biological activities, making it difficult to ascribe their replication phenotypes to a
particular defect, and the actual amount of origin-mediated DNA synthesis occurring in either
mutant or normal infections remains unclear.
Historically T4 DNA synthesis in vivo has been measured by supplementing infected cultures
with radioactive thymidine and measuring the incorporation of this pulsed nucleotide into
growing DNA polymers. Unfortunately, using this methodology, the actual amount of DNA
synthesized is not readily apparent, as there is no way to correlate the incorporation of label
with the number of genomes synthesized. Rather, genome replication can only be estimated
by infectious virion production, a method that may not account for all DNA synthesis.
Moreover, early during T4 infection, exogenous label is not efficiently incorporated into
nascent viral DNA, as nucleotides are scavenged from the degraded host chromosome
(reviewed in 17), so pulsed nucleotides may not provide an accurate measurement of initial
DNA synthesis.
The experimental difficulties presented by the bipartite T4 replication strategy are not limited
to shared protein requirements, as is aptly demonstrated by earlier attempts to identify the viral
origins of replication 18. In one of the most exhaustive studies, Kreuzer and Alberts restricted
and shotgun cloned the T4 genome and simply asked if any of the resultant clones could be
maintained in trans and packaged as concatemers during an otherwise wild type infection. After
several rounds of selection, a number of putative origins were recovered. Several of these
mapped to regions of the T4 genome previously shown to be hotspots for recombination 18,
and at least one putative origin supports recombination-mediated replication 19, implying that
origin and recombination mediated replication are not only biochemically linked, but also
physically linked to the same regions of the T4 genome.
In total, at least 7 putative origins have been identified by a number of groups, oriA, oriB,
oriC, oriD, oriE, oriF, and oriG (see figure 1, reviewed in 8; 20; 21). Several of these loci were
identified as restriction fragments enriched for nascent T4 DNA early during infection,
including oriA 22, oriB 23, oriC 23, oriE 22; 24; 25, and oriF 22. Some, including oriD 26, were
observed using electron microscopy, while others, oriF and oriG, were recovered by Kreuzer
and Alberts as autonomously replicating sequences 18; 27. With the exception of oriF and
oriG, most of these putative origins remain uncharacterized, and neither the cis elements
necessary for origin function nor the actual start sites of DNA synthesis have been defined.
Despite previous efforts, it has been unclear to what extent any of the putative origins are used
during the course of a normal T4 infection, as incomplete representation of the T4 genome in
the restriction fragment libraries used to map nascent DNA, disparate experimental conditions
between studies, and incongruent experimental results have prevented a consensus. Without
such basic information, it is impossible to determine how exactly DNA synthesis proceeds
across the T4 genome, not to mention how the interplay between origin- and recombinationmediated
replication and other DNA transactions contribute to the dynamics of replication and
the fruition of full length genomes. Hence, in this study, we have mapped the origins of
replication used during T4 infections and monitored the DNA synthesis originating from these
loci, in real time, across the viral chromosome.
Results
The switch between origin and recombination mediated replication during infection
To begin our characterization T4 DNA synthesis during infection, we empirically defined the
switch between origin and recombination-mediated synthesis during infection using viral
recombination mutants. Needing an accurate way to measure viral DNA synthesis early during
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infection, we developed a simple dot blot assay, wherein DNA was harvested at various times
post infection, blotted to nylon, and hybridized to a random-primed, 32P labeled probe derived
from full-length virus. The amount of T4 DNA associated with each time point was then
measured with a phosophoimager, and accumulation over time was calculated as the foldincrease
in viral DNA over that present at 2 minutes post infection, prior to the onset of
replication. This allowed us to then estimate the average number of T4 genome equivalents in
each infected cell at various times post infection.
We used a straightforward infection protocol, and E. coli was grown in LB at 37°C and directly
infected with T4 at an MOI (multiplicity of infection) of 5 viruses to each cell with no additional
manipulation of cultures. One advantage of the T4 system is that a population of viruses can
be effectively synchronized during infection, and, under these conditions, host cells absorb
more than 90% of the virus within 4 minutes of infection (data not shown). To control for
experimental variances, multiple, independent infections were repeated on several different
days for all viruses, and two replicates of each independent infection were blotted, probed, and
averaged.
We chose to monitor the onset of recombination-mediated DNA synthesis using a virus
harboring a mutation in uvsX, a recombinase gene. Many of the previously characterized
uvsX mutants contain amber stop codons, which can be leaky, though the small amount of fulllength
protein expressed seems to have little affect on the much-reduced virus production 28.
In our hands, the uvsX mutant used in this study produced an average of 15 infectious virions
per cell after a 1 hour infection, compared to 260 for wt (data not shown), essentially the same
amount of virus produced by verified uvsX null mutants 28.
As expected, the uvsX mutation caused an arrest in DNA synthesis, and mutant DNA
accumulation was much reduced compared to wt (Figure 2A). However, this reduction began
much earlier than anticipated and was noticeable as early as 7 minutes post infection, as is
evident in the expanded graph (Figure 2B). This observation was confirmed with a second
recombination mutant, containing a nearly complete deletion within gene 59, constructed as
described in Materials and Methods. At an MOI of 5, gene 59 mutant infections showed similar
amounts of DNA synthesis as uvsX mutant infections: The accumulation of viral DNA was
clearly reduced compared to wt, and this reduction in DNA synthesis was apparent from at
least 7 minutes post infection (Figures 2A and B).
To determine whether our experimental conditions contributed to the early onset of DNA arrest
observed with the recombination mutants, we repeated the experiment with the MOI reduced
to 0.5 viruses per cell, so that each infected cell contained on average a single virus. As can be
seen in Figures 2C and D, this change in the MOI had a profound effect on viral DNA synthesis.
Under otherwise identical conditions, at an MOI of 0.5, the uvsX mutation has little affect on
synthesis, compared to wt, until 15 minutes post infection. Thus the number of infecting viruses
appears to influence the timing at which recombination begins to contribute to T4 DNA
synthesis. Notably, replication of the gene 59 mutant at the lower MOI of 0.5 arrested earlier
than the uvsX mutant, implying that 59 protein is required prior to the onset of recombinationmediated
replication.
Mapping early nascent T4 DNA synthesis
Having defined the time course of origin dependent replication under our conditions, we next
wanted to map the nascent DNA produced during T4 infection at an MOI of 0.5. The
hybridization strategy used in Figure 1 proved to be quite useful in quantifying the amount of
nascent DNA made over time, and we wanted to apply a similar strategy to the mapping of
these synthesis products to discrete regions within the T4 genome. Hence we created an array
of PCR products that provided equal representation of the entire T4 genome and could be used
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for mapping studies. This array is comprised of discrete 2.5 to 3.0 kb PCR products amplified
from 30 loci, spaced evenly across the phage genome, and includes products positioned near
seven putative origins of DNA replication, oriA, oriB, oriC, oriD, oriE, oriF, and oriG,
identified in previous studies (see Figure 1).
Although the T4 replisome progresses at a rapid rate of 30–45 kb per minute 7, at the onset of
replication in a population of infected cells, there should be some number of origin initiated,
leading strands less than 10 kb, but longer than the 2 kb average Okazaki fragments 29. To
map these nascent DNAs, we harvested DNA from infections and fragmented contaminating
E. coli chromosomal DNA with the restriction enzyme HaeIII and resolved the undigested T4
DNA on alkaline agarose gels. HaeIII cleavage is blocked by methylation of the internal
cytosines within the recognition sequence 5′-GGCC-3′ 30, and it does not appear to cut the
hydroxymethylated cytosine containing T4 DNA produced early during our infections (data
not shown).
The shorter, nascent viral DNAs were then excised from the gels, purified, random primed
labeled, and used to probe the T4 genomic macro array. The enrichment of these size selected
DNA’s at specific loci, and hence their origin, was determined using a relative abundance
score. This score allows the abundance of DNA at a locus during infection to be compared to
the abundance of DNA at that same locus in the packaged virus, before infection and subsequent
replication, and is calculated as the ratio between the percentage signal at a given locus
compared to all loci on the array and the same percentage obtained with a control probe derived
from packaged viral DNA. Thus a relative abundance score at a particular locus greater than
one indicates that this locus is enriched in nascent DNA as compared to the packaged, full
length T4 genome.
A large enrichment at all three time points was observed in the nascent 3 to 6 kb wt DNA at a
PCR locus near the putative T4 origin, oriE22; 24; 25 (Figure 3A). A similar enrichment pattern
was also seen in the 6 to 10 kb size selected wt DNA, with the greatest relatives abundances
observed near oriE (Figure 3B). There was also a less pronounced enrichment at another locus
which overlaps a second previously identified T4 origin, oriF 18; 31. Although the sequence
elements required for oriE activity have not been delineated, oriE and oriF are thought to be
mechanistically distinct 32. Thus, it appears that two functional classes of origins are active
within a single population ofT4 infected cells.
To determine if the synthesis observed in Figures 3A and B was true origin-dependent rather
than recombination-dependent synthesis, size selected nascent DNA from uvsX mutant
infections was prepared and hybridized to the array. Similar to wt, the greatest relative
abundance in both size selected populations of mutant DNA was observed near oriE and
oriF, indicating that DNA synthesis originating from these loci during the time monitored is
not dependent on uvsX protein (Figures 3C and D). Thus it appears that there are at least two
origins of de novo synthesis active in a population of infected cells under our conditions. Of
these, oriE appears to be the most active.
Analysis of T4 replication dynamics
Though the previous analysis gives some indication as to the origins of phage DNA synthesis,
our size selection strategies preferentially enriched for nascent DNA derived from origins at
which 3–10 kb nascent DNA polymers accumulate. This could be problematic if, as one would
expect, once initiated, nascent DNA polymers continue to grow at 30–45 kb per minute, until
the entire genome has been replicated. By size selecting nascent DNAs, most of these predicted,
growing polymers would be excluded from analysis, and with them, the ability to follow T4
DNA synthesis across the genome and illuminate the dynamics replication.
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A second mapping strategy was employed to address these issues. Herein a time course of total
T4 DNA harvested early during infection was blotted onto nylon strips, and these strips were
independently probed with each of the PCR loci from the genomic array. This allowed the
amount of DNA synthesized at a particular locus, at a particular time point to be calculated as
the fold increase over the amount at the same locus present at 2 minutes post infection, before
significant replication of the parental genome. An advantage of this methodology is that the
fold-increase in DNA at a given locus should reflect the number of times that locus is replicated,
providing a direct measurement of DNA accumulation. Hence this approach allows one to plot
the synthesis of DNA across the genome in real time and to visualize the dynamics of viral
replication.
Similar to our total viral DNA analysis, multiple, independent infections were repeated on
several different days, and the results from each were averaged. During the first 8 minutes of
wt infections a two to three fold increase in the amount of DNA was observed near oriE (Figure
4A), an expected result, given that the greatest enrichment of size selected nascent DNAs was
also observed at the same PCR locus. As the infection progressed, the DNA accumulation near
oriE continued, and by twelve minutes a nearly 10-fold increase was observed at this locus
(Figure 4B), implying that on average replication was initiated from this region nearly 10 times
per infecting virus during this time.
DNA also accumulated at several other loci with the T4 genome during the first 8 minutes of
infection, including those near oriA, oriC, oriF, and oriG, as well as a discrete locus located
between the latter two origins (Figure 4A). Moreover, the amount of DNA continues to increase
near oriA, oriC, oriF, and oriG, and by 12 minutes post infection a ~5-fold increase in
accumulation is observed near all of these loci (Figure 4B). Thus multiple T4 origins are active
within a single population of infected cells, and these origins continue to fire, producing
multiple nascent DNAs, as the infection proceeds.
Replication dynamics in the uvsX recombination mutant
Our data indicate that at an MOI of 0.5 both wt and uvsX mutant viruses produce essentially
the same amount of DNA during the first 15 minutes of infection (Figure 2C and D). After that
point, DNA synthesis in the uvsX mutant plateaus, and the virus exhibits the DNA arrest
phenotype associated with T4 recombination mutants. The obvious conclusion is that T4
recombination-mediated DNA synthesis does not contribute significantly to phage DNA
replication at the lower MOI until 15 minutes post infection. This conclusion is supported by
the fact that short nascent DNA recovered early during uvsX infections maps to the same loci
as wt (Figure 3). So it is not surprising that the pattern of total DNA accumulation in the
uvsX mutant is also similar to that of wt virus, at least during the first 12 minutes of infection,
with most synthesis mapping near oriE, and less mapping near oriA, oriF and oriG (Figure 4C
and D). Yet, as the time course progresses to 15 minutes post infection, some distinct
differences are observed between the two viruses, most notably that DNA accumulation near
oriA is reduced during uvsX mutant infections, as compared to wt. This could indicate that the
T4 genome contains at least two functionally distinct classes of origins, one more dependent
on recombination proteins for activity under our experimental conditions.
Short nascent DNAs accumulate early during infection
If nascent DNA polymers grew continuously from their origin to the ends of the genome, one
would expect to see the amount of DNA sequentially increase across the genome over time.
So, as a single replication fork moved from an origin to adjacent loci, the amount of DNA
should increase by a factor of two, initially at the origin and subsequently at adjacent loci. This
dynamic should be easily measured by our assay, and, given a physiological replisome rate of
30–45 kb per minute 7, one would expect the two fold increase in DNA associated with each
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nascent replication fork to travel across the T4 genomic array at the rate of four to six adjacent
loci per minute.
In contrast to the expected DNA synthesis dynamic, the fold increases initially observed at the
origin loci do not move evenly across the genome with time (Figure 4). Instead, the pattern of
T4 DNA synthesis early during infection is one of peaks and valleys, wherein 5 to10 fold
increases in the amount of DNA are observed near the origins during the first 12 minutes of
infection, but much smaller increases are observed in the regions adjacent to these loci. So it
appears that nascent replication forks are moving slower than anticipated through some regions
of the T4 genome and, as a result, short nascent DNAs are accumulating near the origins.
To directly determine the size distribution of nascent DNAs, viral DNA harvested early during
infection was size fractionated on alkaline agarose gels and blotted to nylon membranes and
hybridized to random primed probes derived from full length T4 DNA and a PCR product near
oriE (Figure 5). To simplify the analysis, the fraction of DNA less than 27 kb was graphed for
each time point, and, as expected from the replication dynamics in Figure 3, most of the DNA
detected with either full length T4 (Figure 5B) or oriE probes (Figure 5C) was less than 27 kb,
until 12 minutes post infection. After this time most of the DNA detected with both probes was
greater than 27 kb.
It is worth noting that the smaller DNAs observed early during the wt infection do not simply
disappear, as would be expected if a certain number of nascent polymers were initially made,
then elongated over time. Rather a certain abundance of short DNAs are apparent throughout
the first 20 minutes of wt infection, suggesting that they are produced at a more or less constant
rate during this time. This does not occur in uvsX mutant infections, where the abundance of
short DNAs increases throughout the time studied (Figure 5A), as if short polymers were still
being made, but not efficiently elongated, culminating in a larger percentage of polymers less
than 27 kb, compared to wt. This observation is consistent with a previous study 33, and
indicates that uvsX influences the normal size distribution of nascent DNA.
Discussion
The chromosome is the fundamental unit of heredity, a dynamic macro molecular structure
that serves as a template for transcription of genes, replication of genetic material, and the
various DNA transactions required for genome maintenance. As a first step in characterizing
the interplay between these activities, we have developed a unique set of experimental
strategies that allowed us to measure the actual accumulation of nascent DNA, across the T4
genome, over the course of infection. Our data indicate that early T4 DNA synthesis is
discontinuous, with several origins contributing to a punctate replication pattern, comprised of
multiple, short, origin derived polymers, an observation that raises a number of questions
regarding the regulation of T4 DNA replication.
Transition from origin- to recombination-mediated replication
The maintenance of genome integrity during multiple replication cycles is a key priority in the
evolution of chromosomes. Lacking defined telomeres, the linear T4 chromosome has evolved
a unique replication strategy that includes both origin- and recombination-mediated DNA
synthesis. This bipartite strategy ensures the production of full length genomes and allows the
virus to rapidly synthesize several hundred progeny genomes during the short time course of
a single infection. Although the individual components of this replication strategy have been
studied in vitro, little is known about the interplay between these two modes of replication and
the dynamics of resultant DNA synthesis during actual infection.
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The switch between these two modes of replication was previously thought to depend on the
synthesis of proteins made at later times 15; 16. However, we found that the interval prior to
the onset of recombination-mediated T4 replication is not a fixed duration. Rather, it varies
with the number of viruses per infected cell. In singly infected cells the recA homologue
uvsX does not contribute to the total amount of viral DNA produced during the first 15 minutes
of infection. In contrast, when multiple viruses infect the same cell, under otherwise identical
conditions, the transition to recombination-mediated replication occurs much earlier, and both
uvsX and gene59 are required for viral DNA synthesis as early as 7 minutes post infection.
It is not clear how increasing numbers of infecting viruses influence the T4 replication strategy
such that recombination is favored, but scarcity of required host factors such as RNA
polymerase or altered expression of specific viral genes could cause an imbalance in protein
levels, creating an environment wherein origin activation proteins are limited or that
recombination proteins are overly abundant. Of course, increasing the number of viral copies
may also increase the substrates available for recombination and, thus, may in itself be a factor
in the switch to recombination-mediated replication.
It is also not clear why replication slows earlier in the gene 59 mutant compared to the uvsX
mutant in singly infected cells. Taken at face value, this observation implies that gene 59 is
required prior to the onset of recombination-mediated replication. However, it is not likely that
59 protein is absolutely required during the initial steps of origin- mediated replication, since
one would expect no DNA synthesis in gene59 mutants if this was the case. Rather, our
observations are consistent with secondary role for 59 protein during origin-mediated
replication, as asserted in a previous study, where mutation of gene 59 had no effect on initial
leading strand synthesis from oriF but disrupted subsequent retrograde origin-mediated
synthesis 14. Such a role at the T4 origins would presumably cause DNA synthesis to arrest
earlier in gene 59 mutants than in uvsX mutants.
Multiple origins of T4 DNA replication
There are clearly multiple origins of T4 DNA replication, including a major origin near oriE
and at least four secondary origins located near oriA, oriC, oriF, and oriG. Unlike previous
studies 22; 24; 25, we observed synthesis near these origins concomitantly, within a single
population of infected cells, raising the possibility that some or all of these origins are
simultaneously active within a single viral genome. However, this issue is still unresolved, and
it is entirely possible that micro-environmental differences between infected cells influence
origin activity such that particular origins are used discretely within individual infected cells.
There is some reason to suspect that environmental factors may influence origin usage, as the
characterized T4 origins do not appear to be mechanistically similar. Of the five, the best
characterized is oriF. This origin was initially recovered by Kreuzer and Alberts and is able to
support replication of episomal plasmids during T4 infection 18; 27. Normal origin activity
from this locus is dependent on several cis elements, including a T4 middle mode promoter
34; 35, a downstream unwinding element or DUE 31, and a sequence element thought to form
a hairpin structure 35. The current model predicts that the T4 middle mode uvsY promoter
upstream from oriF produces an RNA, which is then used as primer for DNA synthesis by the
T4 replisome. Both oriA and oriG are also located near middle mode promoters, and each is
thought to operate in a similar manner to oriF 21.
The region near oriE appears to be quite different than that near oriF. There is no middle mode
promoter in the region, which lead Gisela Mosig’s group to suggest that DNA replication is
primed by an RNA produced from an early promoter downstream from six copies of a directly
repeated sequence element, 5′-AT(T/C)(T/A)CC(A/T)T(T/C)(A/G)AC-3′ 32. These so called
iterons are each separated by 12 bp of non-repeated sequence and are maintained in the anti-
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sense direction of a large open reading frame encoding the gene 5 base plate lysozyme, a protein
necessary for viral entry into host cells. Similar repeats are found in the syntenic gene 5 region
regions of other T4-like bacteriophages 36, but it is not clear if these iterons are functionally
linked either to gene 5 or to origin activity from oriE.
Thus T4 may have mechanistically distinct classes of origins, and the difference between these
classes could contribute to their functional usage during infection. This would make sense on
at least two levels. First, as genomes evolve, one would expect them to encounter multiple
selective pressures over time, which may lead to the maintenance of several types of origins,
allowing the organism to reproduce under a variety of environmental conditions, and, hence,
increasing fitness. Second, the individual origins may fulfill specific functions during the
replication of a single viral chromosome. Yet, it remains entirely unclear how T4 origin activity
is regulated, and how several origin loci, with distinct genetic elements, function during the
course of infection.
Punctate patterns of T4 DNA synthesis
Early during infection, short, nascent DNAs accumulate near the T4 origins, suggesting that
these loci function as amplicons, producing a number of sub genomic length DNAs that can
be subsequently used during recombination-mediated synthesis (Figures 3 and 4). However,
the physical state of these origin-derived DNAs is not clear. Perhaps, initially, each nascent
DNA is displaced from the origin and later recombines with the parental template where it is
used to prime subsequent DNA synthesis. This might explain why the proportion of short
nascent DNA remained high in the absence of the RecA homologue uvsX, a protein involved
in both recombination and the restart of stalled replication forks 37. Alternatively, the nascent
polymers may remain associated with the template, creating an “onion skin” structure wherein
the multiple nascent strands are held in place inside the parental duplex. These structures have
been seen before in electron micrographs of replicating T4 DNA 38, though not the 5 to 10
layers implied by the data presented here.
It is not clear why replication forks do not efficiently progress into the regions adjacent to the
T4 origins early during infection. Fork progression is regulated by both active and passive
mechanisms in other systems. Terminator sequences impede E. coli DNA synthesis in a defined
polarity through a mechanism mediated by tus proteins 1; 39, but there is reason to suspect
that other mechanisms contribute to discontinuous DNA synthesis observed throughout the E
coli genome 3; 4. Both replication pause sites and initiation rates influence DNA synthesis
patterns in Epstein-Barr virus 40. In eukaryotic cells DNA synthesis rates differ from one
genomic region to another 41, and several factors, including local and global transcription
patterns appear to influence replication dynamics 5; 42.
No replication terminators have been identified in T4, and though it has been suggested that
transcription may impede T4 DNA synthesis 43, the relationship between viral transcription
and replication is not established. There has been a genomic study of T4 transcription during
infection using a microarray, but, unfortunately, the conditions used in this study were different
than ours, preventing any direct comparisons of replication and transcription timing 44. Yet,
it is worth noting that all of the origins of replication identified in Figure 4 are near T4 genes
expressed during late transcription (see Figure 1), suggesting that further investigation into the
interplay between T4 transcription and replication is warranted.
Certainly many aspects of the T4 replication dynamics observed in this study await further
experimental exploration. Yet, the assays developed here should allow us to directly determine
the genetic factors required for the normal pattern of DNA synthesis, and further development
of these assays should provide the experimental tools necessary to define the mechanisms of
origin function. The amenability of T4 to both the genomic investigations as demonstrated here
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and biochemical characterization demonstrated by a number of groups should provide a unique
system where genomic phenomena observed in vivo can be quickly correlated with biochemical
mechanisms defined in vitro. Ultimately, such studies should shed light on the dynamics and
underlying mechanics of genomic transactions, and how these contribute to the evolution of
chromosomes.
Materials and Methods
Strains
E. coli BL21(DE3) and SCS110 were obtained from Stratagene. E. coli B and CR63, as well
as wt T4D, have been maintained in this laboratory. The T4 recombination mutant
T4uvsXam11 was a gift from John W. Drake 45.
Growth of bacteria and phages
All bacteria were grown in LB broth at 37°C. All T4 infections were done at 37°C. T4 isolates
were plaque purified from stock and expanded in either E. coli CR63 or BL21(DE3) cells.
Phage stocks were stored at 4°C in 10mM Tris (pH7.4), 150mM NaCl, 0.03% gelatin.
Construction of T4 phage harboring a deletion of gene 59
The T4 gene 59 deletion was made by site-directed mutagenesis of the plasmid pNN2002 46,
which contains wild type gene 59 and flanking T4 DNA. The method of Kunkel et al. 47 was
modified to include the use of T4 DNA polymerase, T4 44/62 clamp loader, and 45 clamp to
copy the ssDNA template as previously described 48. Two primers, 59del1 (5′-
GCATGCGGAGTTTGATCATAGTAGAAAATC-3′) and 59del2 (5′-
CAATACTTGCAAGTGATCACAGTTTCAATG-3′), were used to introduce BclI restriction
sites into pNN2002 at the termini of the gene 59 open reading frame. Mutated plasmids grown
in the dam methylation deficient host E. coli SCS110, were digested with BclI. The larger
fragment was self ligated to give a circular plasmid, pJRB1113, in which all but six codons,
of the gene 59 open reading frame had been deleted. The gene 59 deletion thus contains a very
small open reading frame that codes for the amino acid sequence MITCKY.
The gene 59 deletion was introduced into the T4D viral background by homologous
recombination as described 49. Briefly, E. coli B (pJRB1113) was grown in LB supplemented
with carbencillin (50 ug/ml) to an OD600 of 0.28 and infected with T4D at a multiplicity of
infection of five viruses per cell. Infected cultures were incubated at 37°C with vigorous
shaking for 2 hours, and then lysed with chloroform. Resultant phage stocks were titered and
plated on E. coli BL21(DE3) cells harboring the gene 59 expression plasmid pNN2859 46.
Plaques were lifted onto nitrocellulose, and probed with an oligonucleotide that spans the gene
59 deletion, 59del3 (5′-TAGATTTTCTACTATGATCACTTG-3′), in 6x SSPE (3M NaCl, 0.2
M NaH2PO4, 0.05 M EDTA), 10% dextran, 1% SDS at 50°C. Filters were washed twice in 2x
SSPE, 1% SDS at 50°C. There was no hybridization of the gene 59 deletion probe with wt
T4D plaques under these conditions.
Six plaques harboring gene 59 deleted phage were identified and isolated in this first screen.
These plaques were resuspended in LB, titered, and plated again on E. coli BL21(DE3) cells
harboring pNN2859. A secondary plaque hybridization screen was then done using the gene
59 deletion oligonucleotide as above, and several plaques were isolated. A stock designated
T4gene59Δ was expanded from one of these plaques. The mutant phage yields pin point plaques
on E. coli BL21(DE3) cells and normal sized plaques when complimented with pNN2859.
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Isolation of T4 DNA from Infected Cells
E. coli BL21(DE3) host cells were grown to a density of 3 × 108 cells per ml at 37°C. and
infected in parallel with pre-warmed wt and mutant phage. After addition of phage to bacterial
cultures at 37°C and through mixing for 1 min, phages were allowed to absorb for an additional
45 seconds without mixing. Samples were then mixed again for 15 seconds, then mixing was
stopped and two minute samples were withdrawn. Mixing was again started and vigorous
mixing was then maintained, except for brief stops to withdraw samples. Aliquots of infected
cultures were withdrawn and immediately submersed in ½ volume phenol and ½ volume
chloroform. Tubes were inverted 5–7 times to completely lyse the cells. After all aliquots had
been withdrawn, all phenol/chloroform extractions were inverted another 10–14 times and
centrifuged to separate aqueous and organic phases. The recovered aqueous phase was
extracted with 1 volume of chloroform and stored at 4°C overnight.
Quantification of in vivo T4 DNA synthesis
T4 DNA synthesis in vivo was monitored by a dot blot assay. Recovered aqueous phase aliquots
from the chloroform extractions detailed above were digested with 40 units/ml RNAse If (New
England Biolabs) at 37°C for 30 minutes. Digested samples (100 uL) were denatured by the
addition of 1 volume of 0.5M NaOH, 1.5M NaCl at 65°C for 10 minutes and cooled to room
temperature. Denatured aliquots were applied to Hybond-XL membranes (Amersham
Biosciences) using a Minifold-1 dot blot system (Schleicher and Schuell) in accordance with
manufacturer’s instructions. Blots were dried at room temperature for 15–30 minutes,
neutralized in 6x SSPE (3M NaCl, 0.2 M NaH2PO4, 0.05 M EDTA, pH 7.4), and dried
completely.
In preparation for hybridization, blots of viral DNA samples were pretreated in 6x SSPE (pH
7.4), 1% SDS at 62°C for 4 to 6 hours. This buffer was then replaced with fresh hybridization
buffer, 6x SSPE (pH 7.4), 1% SDS, 10% dextran. Blots were hybridized to probes generated
from full length T4 DNA isolated from purified virions or from PCR fragments amplified from
the T4 genome. In either case, probes were made using the Prime-It II random primer labeling
kit (Stratagene), cleaned using ProbeQuant G50 spin columns (GE Healthcare), and denatured
at 95°C for 10–15 minutes prior to hybidization with blots at 62°C for 12 to 16 hours.
Approximately 5 × 105 cpm of probe per ml of hybridization solution was used.
After hybridization, blots were washed 3 times in 2x SSPE (pH7.4), 0.1% SDS at room
temperature for 30 minutes each, and once in 2x SSPE (pH7.4), 0.1% SDS at 62°C for 30
minutes. These hybridization and wash conditions were empirically determined to prevent
cross-hybridization of T4 DNA with E. coli DNA as judged by dot blotting. Washed blots were
exposed to a phosphoimager screen, which was scanned by a FUJIFILM FLA-3000
phosphoimager. Data was analyzed using FUJIFILM Image Guage V3.12 software.
The amount of DNA present at a given time point was calculated as a fold increase over the
amount in the same infection at the 2 minute time point, prior to the onset of viral replication.
In infections done at an MOI of 0.5, each infected cell contains on average a single infecting
virus, and the amount of DNA at 2 minutes post infection is equivalent to one T4 genome per
infected cell. Thus the fold increase in DNA over that at 2 minutes is equal to the average
number of genomes per infected cell. In contrast, at an MOI of 5, on average each cell is infected
with five genomes, and so that the fold increase in DNA is not over a single genome, but five
genomes, and the fold increase must be multiplied by 5 to determine the average number of
genomes per infected cell.
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Amplification of the T4 macro array
The PCR fragments that comprise the T4 macroarray were amplified from full length T4 DNA
isolated from purified virions using the Expand Long Template PCR kit (Roche) with buffer
2 as directed by the manufacturers instructions using 150 ng of T4 DNA in each 50 uL reaction.
The PCR primers are listed in Table 1 and were designed such that all hybridized to the T4
genomic template at the same temperature, allowing all the reactions to be run concurrently in
the same thermal cycler. The thermal cycling was done in a GeneAmp PCR system 9700
(Applied Biosystems) under the following conditions: 10 cycles of 92°C for 2 minutes, 55°C
for 30 seconds, and 68°C for 4 minutes followed by 25 cycles of 10 cycles of 92°C, 2 minutes,
55°C for 30 seconds, and 68°C for 4 minutes plus an additional 20 seconds each additional
cycle. Under these conditions each primer pair produced a single band when visualized on EtBr
stained agarose gels.
The blotting of PCR products to Hybond-XL membranes was done essentially as described
above. A total of 50 ng of each PCR product was blotted in 0.5M NaOH, 1.5M NaCl. Blots
were hybridized to probes generated from T4 DNA harvested from infections as above. A
control probe generated from full length T4 DNA isolated from purified virions was also used
in duplicate. The blots were washed and scanned as described above. The relative abundance
of DNA at a particular locus was then calculated 24; 25. First, the percent abundance of a given
locus in each DNA sample was determined by dividing the number of phosphoimager units
(PSLs) associated with an individual PCR fragment probed with that DNA by the sum of PSLs
at all PCR fragments in the array when probed with the same DNA. Second, the percent
abundance of each experimental data point was standardized by dividing this quotient by the
percent abundance of the same PCR locus, when probed by control DNA purified from T4
virions in parallel hybridizations.
Size selection of nascent T4 DNA
DNA recovered from infections as described previously in this section was precipitated with
the addition of isopropanol. This DNA was resuspended in 10mM Tris-HCL, 10mM MgCl2,
50mM NaCl, 1mM EDTA (pH 7.9) and digested for 1 hour at 37°C with Hae III, to fragment
bacterial DNA, and Rnase If (New England Biolabs). DNA was then ethanol precipitated at
room temperature for 15 minutes and loaded onto a 1% alkaline agarose gel in 50mM NaOH,
1mM EDTA. New England Biolabs 1kb ladder was loaded as a size marker. The DNA was
resolved at 25 volts (160mA) for 16 hours, and the gel was neutralized and stained in 1x TBE
(0.4ug/mL ETBr) for 1hr. DNA was visualized with UV light, excised from the gel, and purified
using the Qiagen Qiaex II kit.
T4 DNA replication dynamics
T4 DNA synthesis in vivo at the loci along the T4 macroarray was monitored by a dot blot
assay. Recovered aliquots of DNA from infections were processed and blotted to nylon
membranes as described above. These blots were then hybridized with random primed probes
generated from each of the 30 PCR fragments that comprise the T4 genomic macroarray. The
amount of DNA present at a given locus at a given time point was then calculated as a fold
increase over the amount at the same locus in the same infection at the 2 minute time point.
Southern blots of DNA from infected cells
DNA for Southern blotting was prepared from infections as described above. Hae III, RNAse
If (New England Biolabs) digested DNA samples were then resolved on a 0.7% alkaline
agarose gel in 50mM NaOH, 1mM EDTA, at 30 volts (200 mA) for 21 hours. A lane containing
Pac I digested T4 DNA was included as a size standard. The gel was equilibrated in 0.5N
NaOH, 1.5M NaCl for 1hr, and blotted to Hybond-N+ (Amersham) in the same buffer overnight
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by capillary action. Southern blots were probed as described previously in this section and
stripped according to manufacturer’s instructions.
Acknowledgements
This research was supported by the Intramural Research Program of the NIH, NIDDK.
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Figure 1.
The T4 genomic macroarray. Although the terminally redundant, 172 kb T4 chromosome is
linear, the ends are is circularly permutated, and there are no fixed telomeres. This results in a
circular genetic map as depicted here. The location and position of PCR fragments included
in the genomic array are indicated with yellow tabs with identifying numbers that correspond
to the PCR loci (LP numbers) listed in Table 1. Putative T4 origins of DNA replication have
been placed on the map as reported previously 8, 21 and are indicated with green lollipops and
identifiers (a, b, ect.). Major open reading frames (>100 amino acids) are indicated with arrows.
These were placed on the genetic map using pDRAW32 and were color coded to indicate the
timing of transcription, green, early; yellow, middle; and red, late transcripts based on Luke et
al 44. Additionally, the position of two smaller T4 late genes soc and rI.-1 are indicated with
red arrows near oriA and oriC, respectively.
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Figure 2.
T4 DNA synthesis during wt and recombination deficient infections. The increase in T4
genomes over the course of infection was monitored by hybridization to viral T4 DNA as
described in Materials and Methods. (A) E. coli BL21(DE3) cells were infected at a multiplicity
of five viruses per cell with either wt T4D (n=6), uvsX mutant (recA homologue, n=2), or
gene59 mutant (helicase loader, n=5), where n is the number of independent experiments. (B)
The first 15 minutes of the infection in (A) are plotted on an expanded scale. (C) E. coli BL21
(DE3) cells were infected at a multiplicity of 0.5 viruses per cell with either wt T4 (n=6),
uvsX mutant (n=5), or gene59 mutant (n=3). Thus, on average, most infected cells contain a
single virus. (D) The first 15 minutes of the infection in (C) are plotted. The symbols used in
graphs are as follows: wt, open squares; uvsX mutant, open circles; gene59 mutant, open
triangles. Error bars indicate standard error.
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Figure 3.
Relative abundance of size-fractionated nascent T4 DNA synthesized early during infection.
Wild type T4D and the recombination uvsX mutant were used to infect E. coli BL21(DE3)
cells at a multiplicity of 0.5 viruses per cell. Total viral DNA harvested from infections at 6,
7, and 8 minutes post infection was size fractionated on alkaline agarose gels and used to
generate random primed probes as described in Materials and Methods. These labeled DNAs
were then used to probe the T4 genomic macroarray, immobilized on nylon membranes, and
the relative abundance of viral DNA at each locus along the array was calculated as described
in Materials and Methods. (A) Wild type nascent T4D DNA, 3–6 kb. (B) Wild type nascent
T4D DNA, 6–10 kb. The filled circles used in (A) and (B) are 6 minutes, black: 7 minutes,
orange: 8 minutes, blue. (C) Mutant uvsX nascent DNA, 3–6 kb. (D) Mutant uvsX nascent
DNA, 6–10 kb. The open circles used in (C) and (D) are 6 minutes, black: 7 minutes, orange:
8 minutes, blue. The position along the 168 kb T4 genome is identified in kilobases along the
x-axis.
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Figure 4.
T4 DNA replication dynamics in wt and recombination deficient infections. DNA synthesis
was monitored across the viral genome using labeled PCR fragments from the T4 macroarray
to probe blotted DNA from infections. The amount of viral DNA present at a given time point
at a given locus was plotted as a fold increase over the DNA in the same infection at the same
locus at 2 minutes post infection, as described in Materials and Methods. (A) E. coli BL21
(DE3) cells were infected with wt T4D (n=3) at a multiplicity of 0.5 viruses per cell, where n
is the number of independent experiments. (B) The same wt T4D infections at later time points.
The filled circles used in (A) and (B) are 7 minutes, orange: 8 minutes, blue: 10 minutes, red;
12 minutes, purple; 15 minutes, green. (C) E. coli BL21(DE3) cells were infected with mutant
uvsX (n=4) phage at a multiplicity of 0.5 viruses per cell. (D) The same uvsX infections at later
time points. The open circles used in (C) and (D) are 7 minutes, orange: 8 minutes, blue: 10
minutes, red; 12 minutes, purple; 15 minutes, green. The position along the 168 kb T4 genome
is identified in kilobases along the x-axis. To maintain graphic clarity only the upper extent of
the standard error at each data point is indicated with error bars.
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Figure 5.
Size distribution of nascent T4 DNA synthesized early during infection. (A) Wild type T4D
and the recombination mutant uvsX were used to infect E. coli BL21(DE3) cells at a multiplicity
of 0.5 viruses per cell. Total viral DNA, harvested from wt and uvsX infections at 2, 6, 8, 10,
12, 15, and 20 minutes post infection, was size fractionated on an alkaline agarose gel,
transferred to nylon and probed with full length T4 DNA as described in Materials and
Methods. Numbers above lanes indicate minutes post infection and M identifies the PacI
digested viral DNA molecular weight standard. (B) The blot in (A) was exposed to a
phosphoimager screen and scanned. To determine the fraction of DNA shorter than 27 kb, the
PSL (detected radiation) in a given lane associated with 2.7 to 27 kb DNA was divided by the
PSL present in the total DNA in the same lane, from 2.7 kb to the top of the well, as described
in Materials and Methods. Results from wt infections are graphed with filled bars and uvsX
infections with open bars. (C) The blot in (A) was stripped and reprobed with a PCR fragment
that overlaps the putative position of oriE, LP16 from Table 1. The fraction of DNA less than
27 kb was then calculated as in (B).
Brister and Nossal Page 20
J Mol Biol. Author manuscript; available in PMC 2008 April 27.
NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript
NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript
Brister and Nossal Page 21
Table 1
Oligonucleotides used to construct the T4 macro array
PCR Locus Map Position Oligonucleotide sequences
LP1 168102
1413
5′-TTACAACTCGGCTGGTTGAACTACC-3′
5′-ACCGTAACTGGCTAAGCATTCGC-3′
LP2 4033
6512
5′-TACCACGCAATGGATAACCACCG-3′
5′-GGTGTTGATACTCTTCTGAAACGTATCGC-3′
LP3 9559
11721
5′-GCGCTTTATGGAATGTTGATGCAGG-3′
5′-CGTTTAGAGCATACTCAGGCATCTGC-3′
LP4 14745
16978
5′-AAGAACTTCCACATGAATCACCTGCC-3′
5′-GGGCAGTCGAATGTTTTAGAACACCAC-3′
LP5 19392
21773
5′-CACCGACATACTCTGTGCTGCG-3′
5′-TCTCCGGAAGACCACAGCTGG-3′
LP6 24549
27103
5′-CTCATTGACTCTATCAATGAGTTCAGCACG-3′
5′-AGATGATTCTGGACCCTTTGGATTCC-3′
LP7 29048
31963
5′-GCTGCTAAATTTGAAGGTGAACACGC-3′
5′-CAACTGATTCCAAAGAGAATGACGGC-3′
LP8 34525
37124
5′-GCAGGTCTTCCACAAGCTTTCTTCG-3′
5′-TGGTAAATCCACTGTAATGGAAGGTCTGG-3′
LP9 39786
42055
5′-CACGGCTGTCATAGACATTGTGAACG-3′
5′-CCAGATGAAGTAGGTCGTTGTCCTGG-3′
LP10 44749
46729
5′-AAGCTAATCACCTCGACCATGACC-3′
5′-TCATTCGTTCAGTCCAGTCAGTTCC-3′
LP11 49197
52446
5′-TCGTATTATCATCAAAGGATTCAACTACGTCC-3′
5′-GGTGCTCGATTGGCTAAAGGTCTAG-3′
LP12 55230
57809
5′-TCTCCTTGTGCATCTACCGGAGC-3′
5′-TGTAATGATGAGCGAGGCGAAACG-3′
LP13 60217
62007
5′-GAGAAACGACTTCACAGACAGAATCGC-3′
5′-GCATCACGCCTACGGAATGTTCC-3′
LP14 65319
67876
5′-CGATATTCAGCCATTAAGTGTTGGTCAGC-3′
5′-TGATAGAGTTGATTGGAATGGTTGTTCCG-3′
LP15 71150
74155
5′-GAACCATCAGTCCGACGACTTACC-3′
5′-CAAGATGGATCGACTCTTGTACTTGTGC-3′
LP16 76349
79504
5′-CTGACGAGCTTTAGGTGGAATATAGTGC-3′
5′-TTCCTTCAACTAAAGTGGTAGCATCTCC-3′
LP17 82520
85510
5′-GATCATTCTGTCATTGGTTCATCAGCG-3′
5′-ACTTCTTGACCAGCAATTAATCGTCCC-3′
LP18 88424
91406
5′-CCAGTTACACTAGTAGCTGCTTCCG-3′
5′-TGACCACCTCTTTGCTGGATAACG-3′
LP19 94422
96976
5′-CCATTCCTCAACCATTTCACGAGTCC-3′
5′-CGGTCAAATTTTAGCACGCCACC-3′
LP20 100895
103871
5′-TGCTCGTCAGAAGTTCGTTGATTGG-3′
5′-TTGTGTCAGTCAATGAACCTAATCCACG-3′
LP21 107170
110021
5′-TCGTCAAGAGTAGCATGAGCTCCG-3′
5′-TCTGGAAAGTCATTCATTGAAATTGCTCG-3′
LP22 112743
115578
5′-CATCGATTGTGAAGAGAGCACGTTCC-3′
5′-CTTACTGCTGACACGGTTGAACGC-3′
LP23 119325
122020
5′-GTTGAGGATGATACTGCTGAATCTGTGG-3′
5′-GAACTGTACGTTGACTCATGGCACC-3′
LP24 125094
128001
5′-TAAGAAGGACGTATCTACCACCTGAACG-3′
5′-TGTTACAGCATTGGGTGATTCTATTGACG-3′
LP25 131764
134628
5′-GGCATAAAGTCTGCATCACGAGTACC-3′
5′-TAACCTGAATCCTGAACGTCGCC-3′
LP26 138347
141120
5′-CCATCAGTACCGGATTGAAGTTGACG-3′
5′-GTTACCAACTATGCAGCTTGGCTGG-3′
LP27 144009
146978
5′-ACTTATGGGTTTCTGGATTCCAACGC-3′
5′-GGTAGTTCATCTAGTGCTGATGACACG-3′
LP28 150898
153249
5′-ATCAACGTAGCTTTAGCTGATCGTACC-3′
5′-AGTAGGTTCACTCTGCGCAATCC-3′
LP29 156094
159006
5′-TGGCATTCAAGTCCCATATGCTCC-3′
5′-AATCCAGCAGGAACTGAATCACTCG-3′
LP30 162235
165005
5′-CCACGTTCAATACTTACTTGTCCAGCG-3′
5′-CACAAACTCGCTTCTATTGCAGGTGG-3′
J Mol Biol. Author manuscript; available in PMC 2008 April 27.