Cell,
Vol. 12,1007-1020,
December
The Structure Molecules
1977, Copyright
8 1977 by MIT
of Replicating
Robert L. Lechner and Thomas J. Kelly, Jr. Department of Microbiology Johns Hopkins University School of Medicine Baltimore, Maryland 21205
Adenovirus 2 (AdP)-Infected KB cells were exposed to a 2.5 min pulse of 3H-thymidine at 19 hr after infection. The labeled DNA molecules were separated from cell DNA and mature Ad2 DNA by sucrose gradient sedimentation and CsCl equllibrium centrlfugatlon under conditions designed to minimize branch migration and hybridization of single strands. Electron microscopy’of fractions containing radioactivity revealed two basic types of putative replicating molecules: Ad2 length duplex DNA molecules with one or more singlestranded branches (type I) and Ad2 length linear DNA molecules with a single-stranded region extending a variable distance from one end (type II). Length measurements, partial denaturation studies and 3’ terminal labeling experiments were consistent with the following model for Ad2 DNA replication. Initiation of DNA synthesis occurs at or near an end of the Ad2 duplex. Following initiation, a daughter strand is synthesized in the 5’ to 3’ direction, displacing the parental strand with the same polarity. This results in the formation of a branched replicating molecule (type I). Initiations at the right and left molecular ends are approximately equal in frequency, and multiple initiations on the same replicating molecule are common. At any given displacement fork in a type I molecule, only one of the two parental strands is replicated. Two nonexclusive mechanisms are proposed to account for the replication of the other parental strand. In some cases, before completion of a round of displacement synthesis initiated at one end of the Ad2 duplex, a second initiation will occur at the opposite end. In these doubly initiated molecules, both parental strands serve as templates for displacement synthesis. Two type II molecules are generated when the oppositely moving displacement forks meet. Alternatively, displacement synthesis may proceed to the end of the Ad2 duplex, resulting in the formation of a daughter duplex and a parental single strand. Replication of the displaced parental strand is then initiated at or near its 3’ terminus, producing a type II molecule. Daughter strand synthesis proceeds in the 5’ to 3’ direction in type II molecules generated by either mechanism, and completion of synthesis results in the formation of a daughter duplex.
Adenovirus
2 DNA
Introduction The genome of the human adenovirus type 2 (Ad2) is a nonpermuted, linear, double-stranded DNA molecule with a molecular weight of about 23 million daltons (Green et al., 1967; Doerfler and Kleinschmidt, 1970; Murray and Green, 1973). The genome of Ad2, like that of other adenoviruses, shows two novel features. First, the nucleotide sequence at one terminus of each strand of the Ad2 DNA molecule is complementary to the nucleotide sequence at the other terminus (Wolfson and Dressler, 1972; Garon, Berry and Rose, 1972; Roberts, Arrand and Keller, 1974). The length of this “inverted terminal repetition” is 102 nucleotides (J. R. Arrand and R. J. Roberts, personal communication). Second, the 5’ end of each strand is covalently linked to a protein with a molecular weight of about 5 X lo4 daltons (Robinson and Bellett, 1974; Sharp, Moore and Haverty, 1976; Padmanabhan and Padmanabhan, 1977; Rekosh et al., 1977). The role of these structural features in the virus life cycle is not known. Adenovirus DNA replication is semiconservative (Pearson and Hanawalt, 1971; van der Vliet and Sussenbach, 1972; van der Eb, 1973). The properties of replicative intermediates of Ad2 (and the closely related Ad5) have been studied in several laboratories. These molecules are characterized by a greater sedimentation rate in neutral sucrose gradients and a higher buoyant density in CsCl gradients than mature adenovirus DNA molecules (Pearson and Hanawalt, 1971; Sussenbach and van der Vliet, 1972; Pettersson, 1973; Robin, BourgauxRamoisy and Bourgaux, 1973; Pearson, 1975). The nascent daughter strands in replicative intermediates are heterogeneous in length, but never longer than the strands of mature adenovirus DNA (Horwitz, 1971; Sussenbach and van der Vliet, 1972; van der Eb, 1973; Pearson, 1975). The synthesis of both daughter strands terminates at or near their 3’ ends (Horwitz, 1976; Weingartner et al., 1976). Several lines of evidence indicate that Ad2 and Ad5 replicative intermediates contain extensive stretches of single-stranded DNA (Sussenbach et al., 1972; Pettersson, 1973; Robin et al., 1973; Sussenbach, Ellens and Jansz, 1973; van der Eb, 1973; Pearson, 1975). Based on this property and on an electron microscopic study of putative Ad5 replicative intermediates, a tentative model for adenovirus DNA replication has been proposed in which viral DNA synthesis is initiated exclusively at the right end of the genome (Ellens, Sussenbach and Jansz, 1974). Synthesis of a daughter r strand then proceeds in the 5’ to 3’ direction with concomitant displacement of the parental r strand. In the model, initiation of synthesis of the daughter I
Cell 1006
strand on the displaced parental strand template is considerably delayed, thus accounting for the single-stranded regions in replicative intermediates. An important prediction of this model is that the single-stranded DNA in adenovirus replicative intermediates should consist exclusively of r strand sequences. Recent data indicate, however, that DNA isolated from adenovirus-infected cells contains single-stranded DNA sequences derived from both the I and r strands (Tolun and Pettersson, 1975; Lavelle et al., 1975; Flint, Berget and Sharp, 1976; Sussenbach, Tolun and Pettersson, 1976). In this paper, we present the results of an electron microscopic analysis of putative Ad2 replicating molecules isolated by a method designed to minimize branch migration and hybridization of single strands. Our data indicate that replication is initiated at or near an end of the Ad2 duplex, and that initiations at the right and left ends are approximately equal in frequency. Following initiation, a daughter strand is synthesized in the 5’ to 3’ direction, displacing the parental strand with the same polarity. Synthesis of the complementary daughter strand is initiated at or near the 3’ end of the displaced parental strand and also proceeds in the 5’ to 3’ direction. A model for Ad2 DNA replication based on these findings is presented. Results Isolation of Ad2 Replicating Molecules Ad2-infected KB cells were exposed to a 2.5 min pulse of 3H-thymidine at 19-20 hr after infection. At the conclusion of the labeling period, nuclei were isolated from the infected cells and gently lysed with SDS (sodium dodecylsulfate). The lysate was immediately sedimented through a sucrose gradient in 1.0 mM HEPES, 0.2 mM EDTA and 0.1% SDS at neutral pH. A typical example of such a gradient is shown in Figure 1. A peak of radioactivity was observed in the region of the gradient where mature Ad2 DNA would be expected to sediment, however, a significant fraction of the pulse-labeled DNA sedimented more rapidly. It was verified in control experiments that >90% of the radioactivity incorporated at 19 hr after infection was in viral sequences (Ginsberg, Belle and Levine, 1967). In other control experiments, the sedimentation behavior of cell DNA which had been labeled prior to infection was examined. It was found that >95% of prelabeled cell DNA sedimented to the bottom of the gradient. The sucrose gradient fractions containing significant radioactivity were pooled and incubated for 1 hr at 37°C in the presence of 2.6% glyoxal. The glyoxal was removed by dialysis. Glyoxal appears to form an adduct with exposed guanylic acid residues in single-stranded nucleic acids which
prevents subsequent base pairing (Brude and Budowski, 1971; Hsu, Kung and Davidson, 1973; Brown and Vinograd, 1974). In control experiments, we found that partially melted Ad2 DNA molecules treated with glyoxal could be put through the remainder of the isolation procedure (see below) and stored at 4°C for periods of at least 10 days without loss of denaturation loops. After glyoxal treatment, the pool of pulse-labeled Ad2 DNA molecules was deproteinized and then sedimented to equilibrium in CsCl (Figure 2). The pulse-labeled Ad2 DNA was found in a broad band with an average buoyant density greater than the mature Ad2 DNA marker. The increased buoyant density of Ad2 replicating molecules has previously been shown to be due to their partial single-strand character (Sussenbach et al., 1972, 1973; van der Eb, 1973; Pettersson, 1973). The fractions with buoyant densities greater than the marker, containing about 60% of the total radioactivity in the gradient, were pooled and examined by electron microscopy. Electron Microscopy of Ad2 Replicating Molecules Two basic types of putative Ad2 replicating molecules was observed. Type I molecules consisted of linear duplexes with one or more single-stranded branches. Figures 3A and 3B show typical type I molecules with one and two single-stranded
0’0 -Q Figure
I
10 fMC,iO”
1. Sedimentation
of Pulse-Labeled
I 18
to bo++om
Ad2 DNA
AdZ-infected KB cells were pulse-labeled with 3H-thymidine (330 &i/ml) for 2.5 min at 19.5 hr after infection. Immediately following the pulse, nuclei were isolated from the infected cells and lysed with SDS (see Experimental Procedures). The lysate was layered on a 520% sucrose gradient [in 1 mM HEPES (pH 7). 0.2 mM EDTA. 0.1% SDS] and centrifuged at 40,000 rpm for 6 hr at 14°C in the Beckman SW41 rotor. Fractions were collected from the top of the gradient and assayed for acid-insoluble radioactivity. The fractions that contained significant radioactivity (fractions 6-l 5 in this example) were pooled.
Adenovirus 1009
2 DNA Replication
single-stranded molecules were observed. Both these populations were heterogeneous in length. The duplex population included a significant number of molecules that were equal in length to mature Ad2 DNA molecules. The majority of the single-stranded DNA molecules were shorter than unit length Ad2 strands.
Length Measurements Molecules
Figure 2. Cesium Chloride Equilibrium ugation of Pulse-Labeled Ad2 DNA
Density
Gradient
Centrif-
The pool of pulse-labeled Ad2 DNA obtained following sucrose gradient sedimentation (see Figure 1) was treated with glyoxal and deproteinized as described in Experimental Procedures. The resulting preparation was mixed with marker 3*P-Ad2 DNA, adjusted to a density of 1.72 with CsCl and centrifuged at 40,000 rpm for 60 hr in the Beckman Ti50 rotor. 32P marker Ad2 DNA (0); 3H pulse-labeled Ad2 DNA (0).
branches, respectively. Molecules with up to five branches were observed. The second type of putative Ad2 replicating molecule (type II) was an unbranched linear molecule that was partially duplex and partially single-stranded (Figure 3C). The duplex region extended from one end to a variable point along the molecule, and the remainder of the molecule was single-stranded-that is, type II molecules contained only one point of transition from double-stranded to single-stranded DNA. A small fraction of the molecules had the characteristics of both type I and type II molecules (Figure 3D). These type l/II molecules contained a linear DNA segment with one double- to single-stranded transition like type II molecules; there was also, however, a single-stranded branch emerging from the duplex portion of the molecule, like type I molecules. Type I, type II and type l/II molecules accounted for 85-90% of the molecules that contained both singleand double-stranded DNA (excluding obvious fragments less than half the length of the Ad2 genome). The remainder consisted largely of linear or branched molecules with single-stranded gaps within duplex segments (2% of total) and partially duplex linear molecules with singlestranded regions at both termini (9%). Branched molecules whose branches contained duplex regions were rare (
Vol. 12,1007-1020,
December
The Structure Molecules
1977, Copyright
8 1977 by MIT
of Replicating
Robert L. Lechner and Thomas J. Kelly, Jr. Department of Microbiology Johns Hopkins University School of Medicine Baltimore, Maryland 21205
Adenovirus 2 (AdP)-Infected KB cells were exposed to a 2.5 min pulse of 3H-thymidine at 19 hr after infection. The labeled DNA molecules were separated from cell DNA and mature Ad2 DNA by sucrose gradient sedimentation and CsCl equllibrium centrlfugatlon under conditions designed to minimize branch migration and hybridization of single strands. Electron microscopy’of fractions containing radioactivity revealed two basic types of putative replicating molecules: Ad2 length duplex DNA molecules with one or more singlestranded branches (type I) and Ad2 length linear DNA molecules with a single-stranded region extending a variable distance from one end (type II). Length measurements, partial denaturation studies and 3’ terminal labeling experiments were consistent with the following model for Ad2 DNA replication. Initiation of DNA synthesis occurs at or near an end of the Ad2 duplex. Following initiation, a daughter strand is synthesized in the 5’ to 3’ direction, displacing the parental strand with the same polarity. This results in the formation of a branched replicating molecule (type I). Initiations at the right and left molecular ends are approximately equal in frequency, and multiple initiations on the same replicating molecule are common. At any given displacement fork in a type I molecule, only one of the two parental strands is replicated. Two nonexclusive mechanisms are proposed to account for the replication of the other parental strand. In some cases, before completion of a round of displacement synthesis initiated at one end of the Ad2 duplex, a second initiation will occur at the opposite end. In these doubly initiated molecules, both parental strands serve as templates for displacement synthesis. Two type II molecules are generated when the oppositely moving displacement forks meet. Alternatively, displacement synthesis may proceed to the end of the Ad2 duplex, resulting in the formation of a daughter duplex and a parental single strand. Replication of the displaced parental strand is then initiated at or near its 3’ terminus, producing a type II molecule. Daughter strand synthesis proceeds in the 5’ to 3’ direction in type II molecules generated by either mechanism, and completion of synthesis results in the formation of a daughter duplex.
Adenovirus
2 DNA
Introduction The genome of the human adenovirus type 2 (Ad2) is a nonpermuted, linear, double-stranded DNA molecule with a molecular weight of about 23 million daltons (Green et al., 1967; Doerfler and Kleinschmidt, 1970; Murray and Green, 1973). The genome of Ad2, like that of other adenoviruses, shows two novel features. First, the nucleotide sequence at one terminus of each strand of the Ad2 DNA molecule is complementary to the nucleotide sequence at the other terminus (Wolfson and Dressler, 1972; Garon, Berry and Rose, 1972; Roberts, Arrand and Keller, 1974). The length of this “inverted terminal repetition” is 102 nucleotides (J. R. Arrand and R. J. Roberts, personal communication). Second, the 5’ end of each strand is covalently linked to a protein with a molecular weight of about 5 X lo4 daltons (Robinson and Bellett, 1974; Sharp, Moore and Haverty, 1976; Padmanabhan and Padmanabhan, 1977; Rekosh et al., 1977). The role of these structural features in the virus life cycle is not known. Adenovirus DNA replication is semiconservative (Pearson and Hanawalt, 1971; van der Vliet and Sussenbach, 1972; van der Eb, 1973). The properties of replicative intermediates of Ad2 (and the closely related Ad5) have been studied in several laboratories. These molecules are characterized by a greater sedimentation rate in neutral sucrose gradients and a higher buoyant density in CsCl gradients than mature adenovirus DNA molecules (Pearson and Hanawalt, 1971; Sussenbach and van der Vliet, 1972; Pettersson, 1973; Robin, BourgauxRamoisy and Bourgaux, 1973; Pearson, 1975). The nascent daughter strands in replicative intermediates are heterogeneous in length, but never longer than the strands of mature adenovirus DNA (Horwitz, 1971; Sussenbach and van der Vliet, 1972; van der Eb, 1973; Pearson, 1975). The synthesis of both daughter strands terminates at or near their 3’ ends (Horwitz, 1976; Weingartner et al., 1976). Several lines of evidence indicate that Ad2 and Ad5 replicative intermediates contain extensive stretches of single-stranded DNA (Sussenbach et al., 1972; Pettersson, 1973; Robin et al., 1973; Sussenbach, Ellens and Jansz, 1973; van der Eb, 1973; Pearson, 1975). Based on this property and on an electron microscopic study of putative Ad5 replicative intermediates, a tentative model for adenovirus DNA replication has been proposed in which viral DNA synthesis is initiated exclusively at the right end of the genome (Ellens, Sussenbach and Jansz, 1974). Synthesis of a daughter r strand then proceeds in the 5’ to 3’ direction with concomitant displacement of the parental r strand. In the model, initiation of synthesis of the daughter I
Cell 1006
strand on the displaced parental strand template is considerably delayed, thus accounting for the single-stranded regions in replicative intermediates. An important prediction of this model is that the single-stranded DNA in adenovirus replicative intermediates should consist exclusively of r strand sequences. Recent data indicate, however, that DNA isolated from adenovirus-infected cells contains single-stranded DNA sequences derived from both the I and r strands (Tolun and Pettersson, 1975; Lavelle et al., 1975; Flint, Berget and Sharp, 1976; Sussenbach, Tolun and Pettersson, 1976). In this paper, we present the results of an electron microscopic analysis of putative Ad2 replicating molecules isolated by a method designed to minimize branch migration and hybridization of single strands. Our data indicate that replication is initiated at or near an end of the Ad2 duplex, and that initiations at the right and left ends are approximately equal in frequency. Following initiation, a daughter strand is synthesized in the 5’ to 3’ direction, displacing the parental strand with the same polarity. Synthesis of the complementary daughter strand is initiated at or near the 3’ end of the displaced parental strand and also proceeds in the 5’ to 3’ direction. A model for Ad2 DNA replication based on these findings is presented. Results Isolation of Ad2 Replicating Molecules Ad2-infected KB cells were exposed to a 2.5 min pulse of 3H-thymidine at 19-20 hr after infection. At the conclusion of the labeling period, nuclei were isolated from the infected cells and gently lysed with SDS (sodium dodecylsulfate). The lysate was immediately sedimented through a sucrose gradient in 1.0 mM HEPES, 0.2 mM EDTA and 0.1% SDS at neutral pH. A typical example of such a gradient is shown in Figure 1. A peak of radioactivity was observed in the region of the gradient where mature Ad2 DNA would be expected to sediment, however, a significant fraction of the pulse-labeled DNA sedimented more rapidly. It was verified in control experiments that >90% of the radioactivity incorporated at 19 hr after infection was in viral sequences (Ginsberg, Belle and Levine, 1967). In other control experiments, the sedimentation behavior of cell DNA which had been labeled prior to infection was examined. It was found that >95% of prelabeled cell DNA sedimented to the bottom of the gradient. The sucrose gradient fractions containing significant radioactivity were pooled and incubated for 1 hr at 37°C in the presence of 2.6% glyoxal. The glyoxal was removed by dialysis. Glyoxal appears to form an adduct with exposed guanylic acid residues in single-stranded nucleic acids which
prevents subsequent base pairing (Brude and Budowski, 1971; Hsu, Kung and Davidson, 1973; Brown and Vinograd, 1974). In control experiments, we found that partially melted Ad2 DNA molecules treated with glyoxal could be put through the remainder of the isolation procedure (see below) and stored at 4°C for periods of at least 10 days without loss of denaturation loops. After glyoxal treatment, the pool of pulse-labeled Ad2 DNA molecules was deproteinized and then sedimented to equilibrium in CsCl (Figure 2). The pulse-labeled Ad2 DNA was found in a broad band with an average buoyant density greater than the mature Ad2 DNA marker. The increased buoyant density of Ad2 replicating molecules has previously been shown to be due to their partial single-strand character (Sussenbach et al., 1972, 1973; van der Eb, 1973; Pettersson, 1973). The fractions with buoyant densities greater than the marker, containing about 60% of the total radioactivity in the gradient, were pooled and examined by electron microscopy. Electron Microscopy of Ad2 Replicating Molecules Two basic types of putative Ad2 replicating molecules was observed. Type I molecules consisted of linear duplexes with one or more single-stranded branches. Figures 3A and 3B show typical type I molecules with one and two single-stranded
0’0 -Q Figure
I
10 fMC,iO”
1. Sedimentation
of Pulse-Labeled
I 18
to bo++om
Ad2 DNA
AdZ-infected KB cells were pulse-labeled with 3H-thymidine (330 &i/ml) for 2.5 min at 19.5 hr after infection. Immediately following the pulse, nuclei were isolated from the infected cells and lysed with SDS (see Experimental Procedures). The lysate was layered on a 520% sucrose gradient [in 1 mM HEPES (pH 7). 0.2 mM EDTA. 0.1% SDS] and centrifuged at 40,000 rpm for 6 hr at 14°C in the Beckman SW41 rotor. Fractions were collected from the top of the gradient and assayed for acid-insoluble radioactivity. The fractions that contained significant radioactivity (fractions 6-l 5 in this example) were pooled.
Adenovirus 1009
2 DNA Replication
single-stranded molecules were observed. Both these populations were heterogeneous in length. The duplex population included a significant number of molecules that were equal in length to mature Ad2 DNA molecules. The majority of the single-stranded DNA molecules were shorter than unit length Ad2 strands.
Length Measurements Molecules
Figure 2. Cesium Chloride Equilibrium ugation of Pulse-Labeled Ad2 DNA
Density
Gradient
Centrif-
The pool of pulse-labeled Ad2 DNA obtained following sucrose gradient sedimentation (see Figure 1) was treated with glyoxal and deproteinized as described in Experimental Procedures. The resulting preparation was mixed with marker 3*P-Ad2 DNA, adjusted to a density of 1.72 with CsCl and centrifuged at 40,000 rpm for 60 hr in the Beckman Ti50 rotor. 32P marker Ad2 DNA (0); 3H pulse-labeled Ad2 DNA (0).
branches, respectively. Molecules with up to five branches were observed. The second type of putative Ad2 replicating molecule (type II) was an unbranched linear molecule that was partially duplex and partially single-stranded (Figure 3C). The duplex region extended from one end to a variable point along the molecule, and the remainder of the molecule was single-stranded-that is, type II molecules contained only one point of transition from double-stranded to single-stranded DNA. A small fraction of the molecules had the characteristics of both type I and type II molecules (Figure 3D). These type l/II molecules contained a linear DNA segment with one double- to single-stranded transition like type II molecules; there was also, however, a single-stranded branch emerging from the duplex portion of the molecule, like type I molecules. Type I, type II and type l/II molecules accounted for 85-90% of the molecules that contained both singleand double-stranded DNA (excluding obvious fragments less than half the length of the Ad2 genome). The remainder consisted largely of linear or branched molecules with single-stranded gaps within duplex segments (2% of total) and partially duplex linear molecules with singlestranded regions at both termini (9%). Branched molecules whose branches contained duplex regions were rare (
