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.
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 asequence 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 thea sequence (5, 15, 28). The asequence 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).
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 asequence 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 thea 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.
Nucleotide sequences of and around the asequence of HSV-1 strain K41. (a) Inverted repeat of the L component. The nucleotide numbered −1 is that adjacent to the asequence. 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 thea 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.
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 exciseda 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).
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 asequence was prepared from hybrid plasmid pUK340 containing theDraI fragment corresponding to a unit-length asequence 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 thermophileBacillus cardotenax and functions best at 65 to 70°C.
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 asequence (37).
Detection of excised DNA fragments hybridizing with thea 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 a32P-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 φX174 phage DNA. Sizes of fragments are given in base pairs.
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 asequence, 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 asequence), 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 thea 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). OneDraI site is present in the a sequence (Fig. 2b), and a pair of DraI fragments corresponding to thea sequence were expected to be present when linear HSV-1 DNAs were analyzed: one is the fragment of one unit of the asequence (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.
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 a32P-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 φX174 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).
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 asequence (b) are shown. ApaI, DraI, andEco47I 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 thea sequence with each of these restriction endonucleases are indicated by double lines. The lengths of these DNA fragments are given in base pairs.
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). TwoDraI 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 endonucleasesApaI, 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).
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 a32P-labeled 0.175-kb SmaI fragment of pUK340 (37). Lane M is a marker mixture of HaeIII digests of φX174 phage DNA, and sizes of fragments are shown in base pairs.
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 thea sequence, is expected to be generated by digestion of two units of the a sequence with DraI andEco47I. 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 withDraI 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 withDraI (Fig. 6, lane 7). After digestion withEco47I, 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 toEco47I 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 andEco47I. 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 and2. The derivation of these insert DNAs is summarized in Fig. 7.
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
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
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.
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.
DISCUSSION
The structures of DNA fragments related to two novel bands, the lengths of which correspond to one and two units of thea 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 asequence 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 thea sequence, in agreement with the report concerning GN28 (Table 1; Fig. 6) (37).
For the site-specific excision of one unit of the asequence, 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 asequence, 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 asequence) is flanked by both Ub and Uc and is affected by bothpac1 and pac2 (Fig. 5b and8) (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 frompac1). 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).
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 thea 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 thea 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 asequence.Although the site-specific excision of two units of thea 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 asequence. 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 asequence 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
- Received 25 July 2000.
- Accepted 10 April 2001.
↵* Mailing address: Department of Virology, Faculty of Medicine, Kyushu University, Fukuoka 812-8582, Japan. Phone: 81-92-642-6136. Fax: 81-92-642-6140.
- Copyright © 2001 American Society for Microbiology
