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Journal of Virology, July 2001, p. 5870-5878, Vol. 75, No. 13
0022-538X/01/$04.00+0 DOI: 10.1128/JVI.75.13.5870-5878.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Cleavage in and around the DR1 Element of the a
Sequence of Herpes Simplex Virus Type 1 Relevant to the Excision of
DNA Fragments with Length Corresponding to One and Two Units of
the a Sequence
Kenichi
Umene*
Department of Virology, Faculty of Medicine,
Kyushu University, Fukuoka 812-8582, Japan
Received 25 July 2000/Accepted 10 April 2001
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ABSTRACT |
The a sequence of herpes simplex virus type 1 (HSV-1)
is a region bracketed by two direct repeats named DR1. Concatemeric HSV-1 DNA, the product of DNA replication, is cleaved at a specific site on the second DR1 distal from the S component (authentic cleavage)
to yield unit-length linear HSV-1 DNA prior to or during packaging of
HSV-1 DNA. The presence of two DNA bands, of 0.25 kb (shorter band) and
0.5 kb (longer band), the lengths of which correspond to one and two
units of the a sequence, was identified using acrylamide
gel electrophoresis of HSV-1 DNA preparations extracted by the method
of Hirt. Twelve DNA fragments from each band were molecularly cloned,
and nucleotide sequences were determined. Both termini of eight (67%)
DNA clones from the shorter band corresponded to the specific cleavage
site on DR1. Five (41%) DNA clones from the longer band had a terminus
corresponding to the specific cleavage site on DR1 on one side, but not
on the opposite side. Thirteen (54%) of 24 termini of 12 analyzed DNA
clones from the longer band were in and around DR1. Thus, cleavage
events of DR1 can be classified into three categories: (i) authentic
cleavage; (ii) site-specific cleavage on the third DR1 distal from the
S component (secondary site-specific cleavage), which is related to the
generation of the shorter DNA band in combination with authentic
cleavage; and (iii) less-specific cleavage events in and around other
DR1 elements which relate to the generation of the longer DNA band.
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INTRODUCTION |
The herpes simplex virus type 1 (HSV-1) genome is a 152-kb linear duplex DNA molecule composed of two
covalently linked components, L and S (Fig.
1a) (16, 25, 38). Each
component consists of unique sequences (UL and
US) flanked by inverted repeat sequences (TRL,
IRL, IRS, and TRS). The short
sequence a is repeated directly at both ends of the genome
and is present in inverse orientation at the L-S junction (Fig. 1b)
(6, 23, 24, 39). One to several copies of the a
sequence are present at the end of the L component and at the L-S
junction, but only one copy is present at the end of the S component.
The a sequence encodes several cis-acting sites
involved in (i) cleavage of the unit-length HSV-1 DNA from concatemeric
forms generated by rolling-circle replication and encapsidation of the
excised molecule (cleavage-packaging system) (3, 7, 26,
40), (ii) the circularization of viral DNA after infection
(10, 24, 25, 41, 49), (iii) recombination relating to L-S
inversion (1, 2, 4, 8, 9, 12, 14, 17, 22, 27, 29, 30, 34, 44,
46-48), and (iv) the expression of an mRNA extending from the
a sequence (5, 15, 28). The a
sequence contains the unique (U) and directly repeated (DR) sequence
elements DR1 and Ub, a DR2 array, a DR4 stretch, and Uc and is flanked
by another DR1 element (Fig. 2b)
(1, 6, 21, 23, 35-39, 45). The DR2 array consists of a
number of DR2 elements, and the copy number of DR2 is variable among
HSV-1 strains (16, 21, 23, 33, 35, 42).
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FIG. 1.
Maps of HSV-1 DNA. (a) Structure of the HSV-1 genome
(16, 25, 38, 39). HSV-1 DNA is 152 kb long and consists of
two covalently linked components, L and S, that constitute 82 and 18%
of the genome, respectively. The L component consists of a unique
sequence (UL) flanked by a pair of inverted repeat
sequences (termed TRL for terminal copies or
IRL for internal copies, indicated by boxes with slanting
lines). Likewise, the S component consists of a unique sequence
(US) flanked by a pair of inverted repeat sequences (termed
TRS for terminal copies and IRS for internal
copies, indicated by closed boxes). The a sequence is
indicated by an open box. (b) Arrangement of the a sequence
(indicated by a single line) (6, 24, 25). The a
sequence is flanked by DR1 elements (shown as open boxes). One to
several copies of the a sequence (one and two copies are
depicted) are present at the end of the L component and at the L-S
junction, while only one copy is present at the end of the S component.
The a sequence is repeated directly at both ends of the
HSV-1 genome but is present in inverse orientation at the L-S junction
(orientation of the a sequence is indicated by horizontal
arrows). (c) Generation of HSV-1 genomic termini from the L-S junction
with two copies of the a sequence (the simplest
configuration producing a genomic terminus with one copy of the
a sequence at both ends of the L and S components). The
orientation of the a sequence (indicated by horizontal
arrows) is the same as that at both genomic termini shown in panel a
but is the inverse of that at the L-S junction shown in panel a. A DR1
element shared by two adjacent a sequences is cleaved site
specifically (as indicated by a vertical arrow and a broken line),
generating genomic termini with 3' single-base extensions. This
explains why the terminal boxes in panel b are shown as being stepped.
(d) Generation of HSV-1 genomic termini from the L-S junction with
three copies of the a sequence. HSV-1 DNA with one copy of
the a sequence at the end of the S component and two copies
of the a sequence at the end of the L component is produced
by authentic site-specific cleavage of the second DR1 distal from the S
component.
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FIG. 2.
Nucleotide sequences of and around the a
sequence of HSV-1 strain K41. (a) Inverted repeat of the L component.
The nucleotide numbered  1 is that adjacent to the a
sequence. The IR2 defined in strain F is underlined (23).
Nucleotide sequences put in parentheses (187 to 210) are from
strain 17 (6). (b) The a sequence
(35). The left end of each component (DR1, Ub, DR2, DR4t,
and Uc) of the a sequence is indicated. Nucleotide numbers
start at the left end of the left DR1 and terminate at the right end of
Uc (nt 255). The right DR1 is shown within parentheses (nt 256 to 275).
The cleavage sites of restriction endonucleases ApaI,
DraI, and Eco47I are indicated. (c) Inverted
repeat of the S component. The nucleotide numbered 1 is adjacent to the
a sequence. The DR3 and DR6 defined in strain F
(corresponding to Reiteration II, defined in strain 17) are underlined
(6, 23). The adenine residue at nt 197 (in parentheses) is
from strains F and 17.
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The a sequence is bracketed by two DR1 elements arranged in
the same orientation, and tandemly reiterated a sequences
share the intervening DR1 (Fig. 1c and d). Linear unit-length HSV-1 DNA
present in the viral particle is generated by cleavage at a specific
site on DR1 shared by two neighboring a sequences. Thus, the
free terminus of the L component (with a DR1 of 18 bp plus a 3'
single-base extension of a G residue) and the free terminus of the S component (with a DR1 of 1 bp plus a 3' single-base
extension of a C residue) together form one complete DR1 (Fig. 1c
and d) (24, 38, 39). The pac1 (present in Ub)
and pac2 (present in Uc) sequences, regions of close
homology among the a sequences of diverse herpesvirus
genomes, are candidates for signals that direct site-specific cleavage
(17-20, 29, 45). The cleavage reaction is assumed to
involve two site-specific breaks that are made at defined distances
from pac1 and pac2 signals and normally at the
same position within each DR1. If such a cleavage reaction could occur
on all DR1 sequences, HSV-1 DNA molecules with only partial sequences
of DR1 (but not with subregions of the a sequence other than
DR1) at genomic termini would be generated. However, most of the virion
DNA has one to several copies of the a sequence at the end
of the L component and just one copy at the end of the S component,
suggesting the presence of a mechanism that promotes site-specific
cleavage of the second DR1 distal from the S component (authentic
cleavage) but inhibits cleavage of the other DR1 elements (Fig. 1c and
d) (24, 25, 38, 39).
In a previous study, a novel DNA band composed of an excised
a sequence of one unit (shorter band) was identified on an
acrylamide gel after electrophoresis of HSV-1 DNA extracted by the
method of Hirt, and this band was then analyzed (37). Most
termini of DNAs of the shorter band were at the site-specific cleavage site on DR1, indicating that a site-specific cleavage event can occur
on both of the DR1 elements flanking an a sequence. The question remained, can DR1 elements, except for the second one distal
from the S component, be equally, and site specifically, cleaved? In
the present study, another novel DNA band, the length of which
corresponds to two units of the a sequence (longer band), was analyzed. The termini of several DNAs of the longer band were at
the site-specific cleavage site on DR1 on one side, but not on the
opposite side. Approximately half of the termini of the analyzed DNAs
of the longer band were in and around DR1. Thus, cleavage of DR1 seems
to be topologically different, and cleavage events of DR1 can be
classified into three categories: (i) authentic (most frequent; site
specific) cleavage on the second DR1 distal from the S component; (ii)
less frequent site-specific cleavage on the third DR1 distal from the S
component (secondary site-specific cleavage), which is related to
excision of the a sequence of one unit (corresponding to the
shorter band) in combination with authentic cleavage; and (iii)
less-specific cleavage events in and around other DR1 elements relating
to the excision of two units of the a sequence
(corresponding to the longer band).
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MATERIALS AND METHODS |
Viruses and cells.
The HSV-1 clinical strains GN28, K41,
K56, and K85 were used (36, 43). HSV-1 was propagated on
Vero cells in Eagle minimum essential medium with 2% fetal bovine serum.
Extraction of HSV-1 DNA by the method of Hirt.
A Vero cell
monolayer infected with an HSV-1 stock was collected by low-speed
centrifugation. The pellet was lysed with a solution containing 0.01 M
Tris-HCl (pH 8.0), 0.01 M EDTA, and 0.6% sodium dodecyl sulfate. NaCl
was added to a final concentration of 1.0 M, and the lysate was
maintained overnight at 4°C. The supernatant was separated by
centrifugation and extracted as previously described (37).
Restriction endonuclease digestion, acrylamide gel
electrophoresis, and Southern hybridization.
Restriction
endonucleases were purchased from Toyobo Co. (Osaka, Japan), and the
conditions of digestion used were those recommended by the
manufacturer. DNA was separated in a 5% acrylamide gel as previously
described (33, 42). Southern hybridization was carried out
on a Biodyne B transfer membrane (Pall Biosupport, Port Washington,
N.Y.) as recommended by the manufacturer. The 0.175-kb SmaI
DNA fragment used as a probe for the detection of the a
sequence was prepared from hybrid plasmid pUK340 containing the
DraI fragment corresponding to a unit-length a
sequence of HSV-1 clone TW14 at the SmaI site of pUC18
(37).
DNA sequencing.
A restriction fragment from the hybrid
plasmid was subcloned into both M13mp10 and M13mp11 for sequencing.
Sequences were determined using the BcaBest dideoxy
sequencing kit (Takara Shuzo Co., Kyoto, Japan) (36, 37).
The BcaBest DNA polymerase was obtained from thermophile
Bacillus cardotenax and functions best at 65 to 70°C.
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RESULTS |
Presence of DNA fragments with a length corresponding to two units
of the a sequence in HSV-1 DNA preparations extracted by
the method of Hirt in addition to those with a length of one unit.
In a previous study, a novel DNA band, the length of which corresponds
to one unit of the a sequence (shorter band), was identified in HSV-1 DNA preparations extracted by the method of Hirt (Fig. 3) (37). The structures of
DNA fragments from the shorter band of HSV-1 strain GN28 were analyzed
using molecular cloning and sequencing. The termini of most DNA
fragments corresponded to the site-specific cleavage site on DR1,
indicating site-specific excision of one unit of the a
sequence (37).
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FIG. 3.
Detection of excised DNA fragments hybridizing with the
a sequence in DNA preparations of different HSV-1 strains
using Southern hybridization procedures (33). HSV-1 DNAs
extracted by the method of Hirt were electrophoresed in a 5%
acrylamide gel, transferred to a nylon membrane, and hybridized with a
32P-labeled 0.175-kb SmaI fragment of pUK340
(37). Lanes: 1, HSV-1 strain GN28 (a sequence
of 0.33 kb); 2, K56 (a sequence of 0.37 kb); 3, K85
(a sequence of 0.48 kb); 4, K41 (a sequence of
0.25 kb). Lane M is a marker mixture of HaeIII digests of
 X174 phage DNA. Sizes of fragments are given in base pairs.
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Another novel DNA band, the length of which corresponds to two units of
the
a sequence (longer band), was present in HSV-1
DNA
preparations extracted by the method of Hirt (Fig.
3) and
was analyzed
in the present study. The longer DNA band and the
shorter DNA band were
detected in DNA preparations of different
HSV-1 strains (Fig.
3). Thus,
the generation of two DNA bands,
the lengths of which correspond to one
(the shorter band) and
two (the longer band) units of the
a
sequence, is common to all
HSV-1 strains examined so far and is not
restricted to a particular
strain. HSV-1 strain K41 (having the
shortest
a sequence, of 0.25
kb) was used for further
analyses of these novel DNA bands, as
the shorter sequence is more
manageable.
Besides the two novel DNA bands analyzed in the present study (the
shorter band corresponding to one unit of the
a sequence
and
the longer band corresponding to two units of the
a
sequence),
another novel DNA band, the length of which seems to
correspond
to three units of the
a sequence, was present in
the HSV-1 DNA
extracted by the method of Hirt, albeit at a lower
density (Fig.
3, lanes 1 and 4). A thin DNA band migrating slightly
slower than
the shorter band (corresponding to one unit of the
a sequence)
appeared to also be present in the HSV-1 DNA
extracted by the
method of Hirt (Fig.
3, lanes 1 and
2).
Analyses of the appearance of novel DNA bands in single-step
growth.
To analyze the appearance of novel DNA bands, Vero cells
were infected with K41 and harvested at various times up to 24 h postadsorption (Fig. 4). A single-step
growth curve was constructed (Fig. 4d). HSV-1 DNAs from infected cells
harvested at various times were extracted by the method of Hirt and
analyzed using Southern hybridization (Fig. 4a to c). One
DraI site is present in the a sequence (Fig. 2b),
and a pair of DraI fragments corresponding to the
a sequence were expected to be present when linear HSV-1 DNAs were analyzed: one is the fragment of one unit of the a
sequence (fragment of 255 bp in Fig. 5b) and the other is 16.5 bp
shorter as a result of site-specific cleavage of DR1 (fragment of 238.5 bp in Fig. 5a and b) (35,
37). The shorter DraI fragment generated by
site-specific cleavage of DR1 disappears after infection because linear
HSV-1 termini are absent as a result of the circularization of linear
DNAs.
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FIG. 4.
Detection of excised DNA fragments in the single-step
growth of HSV-1 strain K41. Vero cells were infected with K41 at a
multiplicity of infection of 5 PFU per cell. After adsorption for
2 h at 37°C, the cells were washed three times and overlaid with
Eagle minimum essential medium containing 2% fetal bovine serum.
Incubation at 37°C was continued until the infected cells were
harvested at 0, 3, 6, 9, 15, and 24 h postadsorption. HSV-1 DNAs
were extracted by the method of Hirt, digested with DraI (b
and c) or undigested (a), and electrophoresed in a 5% acrylamide gel.
The DNAs were transferred to a nylon membrane and hybridized with a
32P-labeled 0.175-kb SmaI fragment of pUK340
(37). The autoradiograms after longer (b) and shorter (c)
exposures are shown. Lane M is a marker mixture of HaeIII
digests of X174 phage DNA, and sizes of fragments are given in base
pairs. The infectious virus yields from each sample were titrated in
Vero cells, and a one-step virus growth curve was constructed (d).
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FIG. 5.
Schematic representations of one (a) and two (b) units
of the a sequence of HSV-1 strain K41 (35). The
orientation of the a sequence is indicated by horizontal
arrows as in Fig. 1. A vertical arrow indicates the site-specific
cleavage site on DR1. The structure of excised DNA assumed to be
generated by the site-specific cleavage of two DR1 elements flanking
one unit of the a sequence (a) and that by site-specific
cleavage of two DR1 elements flanking two units of the a
sequence (b) are shown. ApaI, DraI, and
Eco47I restriction endonuclease cleavage sites are indicated
by closed circles. The DNA fragments which are assumed to be generated
by the digestion of an excised one (a) or two (b) units of the
a sequence with each of these restriction endonucleases are
indicated by double lines. The lengths of these DNA fragments are given
in base pairs.
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For up to 3 h postadsorption, one
DraI fragment with a
length of one unit of the
a sequence (255 bp) was detected,
but the
shorter one (238.5 bp) was not evident (Fig.
4b). Two
DraI fragments,
including the shorter one due to
site-specific cleavage, were
detected at and after 6 h
postadsorption (Fig.
4b and c). Both
novel DNA bands, of 0.25 and 0.5 kb, were first detectable at
6 h postadsorption (Fig.
4a). This
simultaneous appearance of
HSV-1 linear termini and novel DNA bands
suggests an association
of the generation of novel DNA bands with
functions of the cleavage-packaging
system.
Analyses of novel DNA bands using restriction endonucleases.
To analyze the novel DNA bands of 0.25 and 0.5 kb detected in a DNA
preparation of HSV-1 strain K41, regions of an acrylamide gel
corresponding to DNA fragments of 0.2 to 0.3 kb (for the 0.25-kb band)
and 0.4 to 0.6 kb (for the 0.5-kb band) were cut out. DNAs recovered
from each region of the gel were cleaved with restriction endonucleases
ApaI, DraI, and Eco47I and were
analyzed using Southern hybridization after electrophoresis in an
acrylamide gel (Fig. 6). DNA fragments
expected to be generated by restriction endonuclease digestion of one
unit of the a sequence with both termini by the site-specific cleavage of DR1 are shown in Fig. 5a. The lengths of the
DNA fragments detected in the Southern hybridization analyses and shown
in lanes 2 to 4 of Fig. 6 corresponded to those expected, as shown in
Fig. 5a. Thus, the majority of the DNA fragments of the shorter band
(0.25 kb in the case of strain K41) were assumed to be generated by
cleavage in and around DR1, as was observed in another HSV-1 strain,
GN28 (37).
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FIG. 6.
Southern hybridization profiles of excised DNA fragments
after digestion with restriction endonucleases. HSV-1 DNA of K41
extracted by the method of Hirt was electrophoresed in a 5% acrylamide
gel, and DNAs were recovered from two regions of the gel corresponding
to 0.2 to 0.3 kb (for the shorter novel band) and 0.4 to 0.6 kb (for
the longer novel band) (37). The recovered DNAs of 0.2 to
0.3 kb (lanes 1 to 4) and 0.4 to 0.6 kb (lanes 5 to 8) were
electrophoresed in a 5% acrylamide gel after no digestion (lanes 1 and
5) or digestion with ApaI (lanes 2 and 6), DraI
(lanes 3 and 7), or Eco47I (lanes 4 and 8). The DNAs were
transferred to a nylon membrane and hybridized with a
32P-labeled 0.175-kb SmaI fragment of pUK340
(37). Lane M is a marker mixture of HaeIII
digests of X174 phage DNA, and sizes of fragments are shown in base
pairs.
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The DNA fragments expected to be generated by restriction endonuclease
digestion of two units of the
a sequence with both
termini
by site-specific cleavage of DR1 are shown in Fig.
5b.
A DNA fragment
of 255 bp, which corresponds to the length of one
unit of the
a sequence, is expected to be generated by digestion
of two
units of the
a sequence with
DraI and
Eco47I. DNA fragments
(a
DraI fragment of 238.5 bp and an
Eco47I fragment of 204 bp;
Fig.
5b), of which one
end is due to restriction endonuclease
digestion and the other end is
due to site-specific cleavage of
DR1, are also expected to be generated
by the digestion of two
units of the
a sequence with
DraI and
Eco47I (Fig.
5b), like digestion
of one
unit of the
a sequence (the shorter band) (Fig.
5a). Two
DNA
fragments which are assumed to be 255 bp (corresponding to
the length
of one unit of the
a sequence) and 238.5 bp (corresponding
to a DNA fragment of which one end is due to
DraI digestion
and
the other end is due to site-specific cleavage of DR1) were
detected
after digestion of DNA from the longer band with
DraI (Fig.
6,
lane 7). After digestion with
Eco47I, two DNA fragments, of 255
bp (corresponding to the
length of one unit of the
a sequence)
and 204 bp
(corresponding to a DNA fragment of which one end is
due to
Eco47I digestion and the other end is due to site-specific
cleavage of DR1), were detected (Fig.
6, lane 8). The expected
DNA
fragments in Fig.
5b were detected during Southern hybridization
analyses of DNA from the longer band, while smear-like DNA fragments
remained in the digested product with
DraI and
Eco47I. Therefore,
the majority of DNA fragments of the
longer band (0.5 kb in the
case of K41) were assumed to be generated by
cleavage in and around
DR1, as was the case for DNA fragments of the
shorter
band.
Structures of the DNA fragments related to novel DNA bands.
To
analyze the structures of DNA fragments related to novel DNA bands, DNA
preparations of K41 extracted by the method of Hirt were treated with
the Klenow fragment and electrophoresed in an acrylamide gel. Regions
of the gel corresponding to DNA fragments of 0.2 to 0.3 kb (for the
shorter band) and 0.4 to 0.6 kb (for the longer band) were cut out.
DNAs were extracted from the gels and cloned into the SmaI
site of pUC18. Twelve hybrid plasmids containing DNAs from the shorter
band (pUK373 series) and 12 containing DNAs from the longer band
(pUK374 series) were constructed. The nucleotide sequences of the
insert DNAs of these hybrid plasmids were determined and are summarized
in Tables 1 and
2. The derivation of these insert DNAs is
summarized in Fig. 7.
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TABLE 1.
Structures of cloned DNA fragments derived from a region
of 0.2 to 0.3 kb (for the 0.25-kb novel band) present in HSV-1 (strain
K41) DNA preparations extracted by the method of Hirt
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TABLE 2.
Structures of cloned DNA fragments derived from a region
of 0.4 to 0.6 kb (for the 0.5-kb novel band) present in HSV-1
(strain K41) DNA preparations extracted by the method of Hirt
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FIG. 7.
Derivation of excised DNA fragments molecularly cloned
into pUC18. Clones were classified into three groups: (a) those
containing an inverted repeat of the S component, (b) those not
containing inverted repeats of the S and L components, and (c) those
containing an inverted repeat of the L component. A closed box
indicates a terminus of an excised DNA fragment which corresponds to a
site-specific cleavage site on DR1. An open box indicates a terminus of
an excised DNA fragment on DR1 other than the site-specific cleavage
site. An open circle indicates a terminus of the excised DNA fragment
located within 10 bp of DR1.
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The genomic termini of the linear HSV-1 DNA molecule are generated by a
single-base-pair staggered cleavage between nucleotides
(nt) 18 and 20 of DR1 with a 3' single-base extension, leaving
1.5 bp of DR1 at the S
terminus and the remaining 18.5 bp of DR1
at the L terminus
(site-specific cleavage by the cleavage-packaging
system)
(
24). Treatment of these termini with the Klenow fragment
removes the 3' extension to leave a flat end (at nt 18 and 20)
(
37). Eight (67%) of 12 cloned DNAs from the shorter
novel band
(pUK373 series) were the same and corresponded to the
excised
a sequence generated by the site-specific cleavage
of two DR1
elements encompassing an
a sequence (Table
1).
Nineteen (79%)
of 24 termini of the pUK373 series were at sites
corresponding
to the site-specific cleavage of DR1. A DNA fragment of
exactly
two units of the
a sequence which could be generated
by site-specific
cleavage of two DR1 elements encompassing two units of
the
a sequence
was not included in the 12 cloned DNAs from
the longer band (pUK374
series) (Table
2). Five (21%) of 24 termini of
the pUK374 series
were at sites corresponding to the site-specific
cleavage of DR1.
Thirteen (54%) of 24 termini of the pUK374
series were in and
around (within 10 nt from)
DR1.
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DISCUSSION |
The structures of DNA fragments related to two novel
bands, the lengths of which correspond to one and two units of the
a sequence and which are present in HSV-1 DNA preparations
extracted by the method of Hirt, were analyzed using HSV-1 strain K41.
DNA fragments of two units of the a sequence (from the
longer band) were analyzed first, while those of one unit (from the
shorter band) of another HSV-1 strain, GN28, had been previously
analyzed (37).
Site-specific excision of one unit of the a sequence
and secondary site-specific cleavage.
Six (86%) of seven cloned
DNAs from the shorter band of GN28 were one unit of the a
sequence generated by the site-specific cleavage of two DR1 elements
encompassing an a sequence (indicating site-specific
excision of one unit of the a sequence), as previously reported (37). Eight (67%) of 12 cloned DNAs from the
shorter band of K41 (pUK373 series) analyzed in the present study were of a structure due to site-specific excision of one unit of the a sequence, in agreement with the report concerning GN28
(Table 1; Fig. 6) (37).
For the site-specific excision of one unit of the
a
sequence, the occurrence of a site-specific cleavage event of two DR1
elements encompassing an
a sequence is required. The second
DR1
distal from the S component, which is authentically cleaved by
the
cleavage-packaging system, is assumed to be one of two DR1
elements
involved in the site-specific excision of the
a sequence.
Two DR1 elements (one is proximal to and the other is the third
one
distal from the S component) which are connected to the second
DR1
distal from the S component through one unit of the
a
sequence,
can be the other DR1 element related to the site-specific
excision
of one unit of the
a sequence. For the following
two reasons,
the third DR1 distal from the S component seems to be more
suitable
as the second DR1 element than the DR1 proximal to the S
component
does. First, the third DR1 distal from the S component (at
the
L-S junction with three or more copies of the
a
sequence) is flanked
by both Ub and Uc and is affected by both
pac1 and
pac2 (Fig.
5b and
8) (
7,
45). However, the DR1
proximal to the S component
is flanked only by Uc (not by Ub) and is
subjected to effects
only from
pac2 (not from
pac1). Second, the product due to site-specific
cleavage at
the DR1 proximal to the S component (corresponding
to the terminus of
the S component having a partial copy of DR1,
of 1.5 bp, but not having
other subregions of the
a sequence)
has not been identified
(
24,
25). Thus, the third DR1 distal
from the S component
is assumed to serve as the secondary site-specific
cleavage site
(albeit less frequently than the authentic DR1,
which is the second one
distal from the S component) and relates
to the site-specific excision
of one unit of the
a sequence (Fig.
7).
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|
FIG. 8.
Hypothesis on the relationship of a cleavage event in
the L-S junction to the excision of the a sequence. L-S
junctions with two (a), three (b), and four (c) copies of the
a sequence are shown. The authentic site-specific cleavage
of the second DR1 distal from the S component is indicated by a large
vertical arrow. Secondary site-specific cleavage of the third DR1
distal from the S component (in L-S junctions with three or more copies
of the a sequence) is indicated by a small vertical arrow.
Derivations of the excised a sequence of one (d) and two (e)
units are indicated. A box with horizontal lines indicates a terminus
on DR1 related to site-specific cleavage. The broken horizontal line
indicates a terminus in and around DR1 (other than that related to
site-specific cleavage). An ellipse indicates a terminus unrelated to
DR1 on the inverted repeat of the L and S components. Excision of one
unit of the a sequence by the cleavage of the second and
third DR1 elements distal from the S component (in the L-S junction
with three or more copies of the a sequence such as in
panels b and c) (d-1), by cleavage of the second DR1 distal from the S
component and in and around the third DR1 distal from the S component
(in the L-S junction with two copies of the a sequence such
as in panel a) (d-2), and by cleavage of the second DR1 distal from the
S component and in and around the DR1 proximal to the S component (d-3)
is shown. Excision of the a sequence of two units in length
by cleavage of the second DR1 distal and in and around the fourth DR1
distal from the S component (in the L-S junction having three or more
copies of the a sequence such as in panels b and c) (e-1) is
shown. Excision of DNA fragments containing sequences other than the
a sequence by cleavage of the second DR1 distal from the S
component and breakage in the inverted repeat of the L component (in
the L-S junction having two copies of the a sequence such as
in panel a) (e-2) and by cleavage of the second DR1 distal from the S
component and breakage in the inverted repeat of the S component (e-3)
is shown.
|
|
Excision of two units of the a sequence with the
site-specific terminus on one side but not on the opposite side.
A
hybrid clone with an insert DNA of which both termini correspond to the
site-specific cleavage site was not present in 12 clones of the pUK374
series analyzed in the present study (Table 2; Fig. 7). Therefore, the
occurrence of site-specific cleavage on two DR1 elements encompassing
two units of the a sequence (the site-specific excision of
two units of the a sequence) is assumed to be unusual, while
that encompassing one unit (the site-specific excision of one unit of
the a sequence) is not unusual.
One terminus of five clones, pUK374-10, -14, -16, -18, and -27, was
located at the site-specific cleavage site of DR1, but
the other
terminus was not located on DR1 (Table
2; Fig.
7).
One terminus of two
clones, pUK374-21 and -32, was at a site on
DR1 which can be related to
site-specific cleavage, but the other
terminus was not located on DR1.
Thus, the insert DNA of 7 (58%)
of 12 clones of the pUK374 series has
a terminus on DR1 related
to site-specific cleavage (probably due to
the authentic cleavage
of the second DR1 distal from the S component)
on one side but
not on the opposite side. The presence of a product due
to site-specific
excision of one unit of the
a sequence
suggested the occurrence
of a secondary site-specific cleavage on the
third DR1 distal
from the S component, in addition to the authentic
site-specific
cleavage of the second DR1 distal from the S component
(Fig.
8).
The absence of a product due to site-specific excision of two
units of the
a sequence suggests that DR1 elements, except
the
second and third ones distal from the S component, infrequently
function as site-specific cleavage
sites.
Involvement of DR1 elements in excision of the a
sequence.
Although the site-specific excision of two units of the
a sequence appears to be unusual, a DNA band with a length
which corresponds to that of the excised a sequence of two
units in length was detected (Fig. 3 and 4a). The presence of a
detectable DNA band suggests the existence of a region(s) that can be
more easily cleaved (or broken) than other regions in the a
sequence. Of 24 termini of 12 insert DNAs of the pUK374 series, 5 (21%) were at the site-specific cleavage site of DR1, 2 (8%) were on
DR1, 6 (25%) were around (within 10 nt from) DR1, and 5 (21%) were on
short repeat sequences (DR3 and DR6 in the inverted repeat of the S
component, DR2 in the a sequence, and IR2 in the inverted
repeat of the L component) (Table 2; Fig. 2 and 7). Repeated sequences
including DR1 are presumed to be less stable, and the DR2 and DR6
repeat arrays were shown to possess an unwound S1 nuclease-sensitive
DNA conformation of non-B DNA (13, 28, 31, 32, 35, 38, 47,
48). Thirteen (54%) of 24 termini of insert DNAs of the pUK374
series were in and around DR1, suggesting a close association of the DR1 element with cleavage (or breakage) events and the generation of a
novel DNA band of the excised a sequence.
A hypothesis on the relationship of a cleavage event in the L-S
junction to the excision of the
a sequence is proposed as
follows (Fig.
8). Cleavage events of DR1 are classified into three
categories: (i) authentic site-specific cleavage of the second
DR1
distal from the S component; (ii) secondary (less frequent)
site-specific cleavage of the third DR1 distal from the S component
(related to the site-specific excision of one unit of the
a
sequence
in combination with authentic cleavage [Fig.
8, d-1]); and
(iii)
(less accurate) cleavage in and around DR1 (related to excision
of the
a sequence of one [Fig.
8a, d-2, and a to c, d-3]
and two
[Fig.
8b and c, e-1] units in length, in combination with
authentic
cleavage). Breakage events on inverted repeats of the L and S
components are assumed to relate to the excision of DNA fragments
containing sequences other than the
a sequence, in
combination
with authentic cleavage (Fig.
8a, e-2, and a to c, e-3).
| |
ACKNOWLEDGMENTS |
Gratitude is extended to M. Ohara for assistance with the
preparation of this report.
Part of this study was supported by grants from the Ministry of
Education, Science, Technology, Sports and Culture of Japan.
| |
FOOTNOTES |
*
Mailing address: Department of Virology, Faculty of
Medicine, Kyushu University, Fukuoka 812-8582, Japan. Phone:
81-92-642-6136. Fax: 81-92-642-6140.
| |
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Journal of Virology, July 2001, p. 5870-5878, Vol. 75, No. 13
0022-538X/01/$04.00+0 DOI: 10.1128/JVI.75.13.5870-5878.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
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Abstract
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