JOURNAL OF VIROLOGY, Feb. 1977, p. 724-731 Copyright © 1977 American Society for Microbiology

Vol. 21, No. 2 Printed in U.S.A.

Replication Process of the Parvovirus H-1 VIII. Partial Denaturation Mapping and Localization of the Replication Origin of H-1 Replicative-Form DNA with Electron Microscopy IRWIN I. SINGER AND SOLON L. RHODE III* Putnam Memorial Hospital Institute for Medical Research, Bennington, Vermont 05201

Received for publication 11 June 1976

Partial denaturation mapping, restriction endonuclease digestion, and electron microscopy were used to determine which end of the linear duplex replicative-form (RF) DNA molecule contains the origin of RF replication for the parvovirus H-1. This origin was localized within approximately 300 base pairs of the arbitrarily designated right end of the RF DNA, in the EcoRI or HaeII-A fragment. Based on denaturation behavior in formamide, the right end was also found to have a relatively high guanine plus cytosine content, whereas the region adjacent to the left terminus of the RF DNA molecule was adenine plus thymine rich. In the previous paper of this series (10), we The tsl mutant of the H-1 parvovirus was grown at presented electron microscopic evidence indi- the restrictive temperature (39.5°C) in serum-syncating that replication of replicative form (RF) chronized hamster embryo fibroblasts; RF DNA repDNA, in the nondefective parvovirus H-1 (tsl lication is enhanced, and progeny DNA synthesis occurss via a double-stranded (ds) lin- shows mutant), this Infected cells wereinhibition labeled in with mutant.a temperature-dependent muant), ear Y-Shape rep licatioventtermedate (RIl, [3H]bromodeoxyuridine (Q3H]BUdR), and the viral 1.55 .tm in length. Replication appeared to hi- DNA was extracted by the Hirt method. Labeled H-1 tiate near (within 15%o of the genome length) RF and RI DNA were purified by velocity sedimenone end of the RF DNA molecule; a single tation in a sucrose gradient followed by isopycnic replication fork proceeded toward the other end centrifugation in a second gradient of Cs2SO4. The at a uniform rate. The purpose of this study is yield of viral DNA from one preparation was suffito determine which end of the H-1 RF DNA cient for all of the experiments described below (exmolecule contains the initiation site of RF repli- cept for one in which the label was [3H]thymidine instead of [3H]BUdR). RF DNA was digested with cation, via partial denaturation mapping of in- EcoRI or HaeII (from Haemophilus aegyptius) retact RF DNA, RI DNA, and purified EcoRI or o striction endonuclease (9; was unpublished data); the tact purified Eo R RFndonua,eRI enA,and completion of the digestion verified by endonuclease-generated fragments of RF analytiNaeII

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cal agarose-gel electrophoresis, and the fragments

We found that the origin of RF replication is were separated via velocity sedimentation through a localized near the external end of the EcoRI or sucrose gradient. Partial denaturation of the viral HaeII "A" RF fragment, which constitutes ap- DNA was achieved using high concentrations of proximately 80% of the H-1 genome length (9). formamide (Matheson Scientific Co.) in the DNAIn addition, this terminus of the A fragment spreading solution; 82% was the formamide concento initiate partial denaturation, hereafter termed the right end of the RF mole- tration expected 90% the formamide to comanticipated cule, has aa relativelywhgh DNA (13).wasThree high guanine plus microliters of a cule, plus cyto-whereas cyto- pletely melt sine (G+C) content, whereas the small EcoRI 0.5-mg/ml cytochrome c solution (horse heart, or HaeII "B" RF fragment (representing about Sigma, type III) in 0.5 M Tris(pH 8.5)-0.05 M EDTA 20% of H-1 genome), at the left end of the RF was thoroughly mixed with 1 ,l of purified (10) H-1 DNA, has an elevated adenine plus thymine DNA (in 0.01 M TrisipH 8.5]-0.001 M EDTA), 41 to (A+T) composition. 45 ,ul of formamide, and 1 to 5 Al of water to produce 50 ,ul of spreading solution possessing the desired

MATERIALS AND METHODS The methods of H-1 virus propagation, RF DNA labeling and purification, and electron microscopy have already been described in detail in the previous papers of this series (9, 10); therefore, only a brief methodological summary will be presented here.

formamide concentration. This was

immediately spread onto a freshly prepared hypophase of 1 mM Tris(pH 8.5)-0.1 mM EDTA containing from 52 to 60% formamide; a 30% difference between the formamide concentrations of the spreading solution and hypophase was always maintained to approximate isodenaturing conditions in both "phases" (3). All 724

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RESULTS Evaluation of restriction endonuclease digestions and purification of H-1 RF DNA fragments. Analytical gel electrophoresis was used to determine whether all of the H-1 RF DNA had been cleaved by EcoRI or HaeII. Figure 1 shows an agarose-gel profile for the EcoRI digestion used in this study. All of the labeled H-1 RF was cleaved into fragment A, representing approximately 78% of the label, fragment B representing about 22% of the label, and a small amount of dimer-length B fragment, presumably derived from the digestion of dimer RF DNAs which are linked "tail-to-tail" at the left ends of their component monomers (9). Similar results were obtained with endonuclease HaeII (unpublished data). The A and B H-1 RF fragments were separated using velocity sedimentation through a sucrose gradient(Fig. 2), or b tation trgauory

preparative gel electrophoresis. Electron microscopic examination of RF fragments prepared by either method showed slight interpeak conI 30

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solutions were adjusted to 23°C before spreading, and were also membrane-filtered (Millipore Corp.) (except for the DNA and formamide). Preparation of grids, DNA spreading, uranyl acetate staining, PtPd rotary shadowing, electron microscopy, and measurement of the DNA contour length were performed as previously detailed (10; 4).

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30 20 Tube Number FIG. 2. Preparative sucrose gradient centrifugation of restriction endonuclease EcoRI fragments A and B of tsl H-1 RF DNA. The EcoRI digest of H-1 RF DNA described in Fig. 1 was subjected to velocity sedimentation in a gradient of 5 to 20% sucrose in 50 1

mM Tris-hydrochloride (pH 8.0), 1 M NaCl, 1 mM EDTA, and 0.2%Sarkosyl, for 7 h at 42,000 rpm in a SW50 rotor at 4°C. Fractions of 150 ,l were collected through the bottom of the tube, and 10-pi aliquots were used to determine the position of radioactivity. Fractions containing the EcoRI-A and EcoRI-B fragments were pooled as illustrated, and the DNA was precipitated with 2.5 volumes ofethanol at -20°C for 16 h. The precipitates were collected by centrifugation, washed once with 70% ethanol-30% 0.3 M NaCl-50 mM Tris(pH 7.5)-i mM EDTA, recentrifuged, and finally dissolved in 10 mM Tris(pH 8.5)-i mM EDTA for electron microscopy. The direction of sedimentation is from right to left. tamination, but this was of little consequence since these fragments are readily distinguishable on the basis of length. These preparations of H-1 RF A and B fragments were utilized in the experiments outlined below. Partial denaturation mapping of H-1 RF, RI, and restriction endonuclease fragments of RF DNA. A highly purified mixture of H-1 ds RF and RI DNA, combined with either 82, 84, or 86% formamide in the presence of cytochrome c and immediately spread onto an isodenaturing hypophase at 23°C, exhibited a unique partial denaturation pattern. One to two loops, presumably formed by the preferential melting of A+T-rich regions of DNA (5-7), were found at an end of the RF molecules (Fig. 3A-H). This A+T-containing region extended from a DNA end (often forming a terminal loop) al e to loop) of the genomlngth genome length into the RF along 20% of molecule in 76% of the partially denatured RFs; the loop traversed 20 to 50% of the RF length in the remaining DNAs (Fig. 4A-C). The extent of

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FIG. 3. Electron micrographs of tsl H-i RF DNA, RI DNA, HaeII-B, and EcoRI-B fragments, partially denatured with formamide. Bar = 0.5 pm. (A) RF DNA partially denatured with 82% formamide; the hypophase contained 52 % formamide. (B-H) As (A), but partially melted with a spreading solution containing 84 % formamide and a hypophase with 54 % formamide. (I-L) Uncleaved RI DNA incompletely melted as in (B -H). The percentage of genome replication increases from left to right: I = 12 %, J = 14 %, K = 36%, and L = 78%. (M-S) HaeII-B fragments labeled with [3H]thymidine and partially denatured as in (B-H). (T-Z) [3H]BUdR-substituted EcoRI-B fragments partially melted in an 86% formamide spreading solution, using a hypophase containing 56% formamide.

intramolecular melting increased with rising formamide concentrations above 82%: 14% of the partially denatured DNAs showed more than 20% melting with 82% formamide, 19% with 84% formamide, and 44% in the presence of 86% formamide. Partially denatured RIs (Fig. 31-L) were also observed, constituting 24% of the total number of partially denatured molecules; a similar frequency of RIs was seen when this DNA was prepared under nondenaturing conditions with 50% formamide. Only the unreplicated regions of these RI DNA molecules exhibited evidence of partial melting. Thus, terminal denaturation loops were localized at the end of the molecule opposite to that containing the origin of RF DNA replication

(Fig. 4F). DNA fragments isolated from restriction endonuclease HaeII digests of the H-i RF described above were also partially denatured with 84% formamide. Incomplete melting was observed in the small B fragments (Fig. 3M-S) and was conspicuously absent from the larger A fragments. Approximately 60% of these partially denatured B fragments apparently were melted close to the left end of the RF DNA, thus exhibiting terminal loops, whereas others appeared to have melted through the HaeII cleavage site, forming Y-shaped fragments (Fig. 4D). Similar results were obtained using restriction endonuclease EcoRI, whose cleavage site is located 0.016 of the genome length to the right of the HaeII site on the physical map of H-

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___________________ _________________ A_ _ _______________

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1, and thus produces B fragments equal to 21.6% of the RF DNA length (9; unpublished data). EcoRI-B fragments also exhibited either terminal loops or forked ends (Fig. 3T-Z, Fig. --____________ 4E), whereas the A fragments appeared unmelted upon exposure to 86% formamide. The initiation site for H-1 RF DNA replication is ________________ therefore located at the right end of the mole~ -______________ cule, within a domain comprised of 6% of the - _______________ genome (the smallest fork size), whereas a _ ______________ unique region of elevated A+T content, localized by reference to the EcoRIIHaeII cleavage E ________________ sites, is at the left end of the RF DNA molecule. -____________ =Localization of a G+C-rich region near the ______________ initiation site of H-1 RF DNA replication. -________ During experiments conducted to verify that ___ _D our preparation of H-1 ds RF DNA was fully - -=________ melted by 90% formamide under the same con________ -ditions employed for partial denaturation, we observed a dramatic increase in RF contour ____ ____, __________ length (loops were no longer found). The mean _ C lengths of H-1 RF DNA decreased with increas-EE ing concentrations of formamide: 1.33 + 0.12 ,um (n = 55. +99% confidence interval) at 82% .9 + 0.04 ,um (n = 154) for 84%, 1.25 ,um formamide, HaI Ecofa and 1.13 ± 0.08 (n = 66) at 86%. We thereFRACTIONAL LENGTH OF GENOME fore expected the RF length to decrease further when fully melted in 90% formamide, especially since the length of single-stranded (ss) H___________________ 1 viral DNA was 33% shorter than that of ds H___________________ 1 RF DNA in 50% formamide (10). However, ________________ the mean contour length of H-1 RF spread in ______________________ 90% formamide was 1.40 + 0.02 ,um (n = 131), ____________________ whereas that of monomeric ss H-1 DNA purified from virions was nearly half that value =___________________ when prepared under the same conditions: X = F 0.71 ± 0.04 Am (n = 141) (Fig. 5A). These ____________________ results suggested that 90% formamide had en-__________________ tirely converted ds H-1 RF to a ssDNA molecule of dimer length. Since treatment of ds H-1 RF __-_-

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63 0:i 07 dI FRACTKNAL LENGTH OF GENOME FIG. 4. Formamide partial denaturation maps of tsl H-1 DNA. (A-C) Intact RF DNA, (D) HaeII endonuclease B fragments of RF DNA, (E) EcoRI endonuclease B fragments ofRF DNA, (F) RIs ofH-1 RFDNA. Contour lengths of the measured molecules were normalized to 1.0 for the RFs and RIs, or to 0.2, the approximate fractional size of the H-1 chromosome for the RF B fragments generated by both endonucleases; arrows indicate the positions of the HaeII and EcoRI cleavage sites on the H-1 genome map. The location of denatured regions (indicated by heavy lines) was oriented to the left end of the RF and RI molecules, and expressed as a fraction of the H-1 genome length. The lengths of these denatured ss regions were not corrected for increased shrinkage, relative to ds areas, which occurs with the formamide concentrations used here. (A) 82% Formamide; (B) 84% formamide; (C) 86% formamide; (D) 6di

[3H]thymidine-labeled HaeII-B fragments, 84% formamide; and (E) [3H]BUdR-substituted EcoRI-B fragments, 86% formamide. In the case of the B fragments, denaturation sites represented by closed loops were placed at the left end of the map, and those melted regions that appeared forked (presumably melted through the nuclease cleavage sites) were oriented to the right. (F) Partially denatured H-1 RI DNAs obtained using 82, 84, or 86% formamide. Branched (replicating) molecules whose lengths (sum oflengths of unreplicated base plus one branch) were within the length range of RF DNAs prepared with corresponding formamide concentrations, and which also contained denaturation loops, were selected for this map. The lengths were normalized and the positions of the replication forks were plotted as a fraction of the total chromosomal length; denaturation loops were positioned as in (A-C). These RIs are displayed in order of their extent of replication.

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Length (m) FIG. 5. Histograms of the contour lengths of intact tsl H-i RF DNA molecules and their endonuclease EcoRI-generated fragments spread after "fully denaturing" (90% formamide, 230C), or fhyperdenaturing" (96%formamide,s300C) incubations. Singlesarrowsidepict mean lengths forssDNA,anddoublearrows denote the mean lengths of dsDNA. (A) H-i RE DNA melted with 90% formamide at 230C (solid lines) has a mean length of 1.40 pm, corresponding to dimer-length ssDNA, determined by measuring ssDNA extracted from purified virions (broken lines; = (0.71 pm), under the same denaturation and spreading conditions. The mean length of ds RE DNA monomers measured under partially melting conditions (86% formamide, 230C) was 1.13 pm (histogram not shown). (B) Distribution of H-i Re DNA lengths obtained by treatment under whypermelting conditions" (96% formamide, 300C) and immediate spreading in 90% formamide at 23mC.The mean length is 0.86 pm, and the major histogram peak corresponds to that ofssDNA monomers (ss viral DNA in [AD spread in the same manner. (C) Endonuclease EcoRI-A and -B fragments of H-i RE DNA prepared in 90% 233C. atssB fragments hada mean lengthof(0.19 m, which is shorter than that observed for formamide = monomer ds B fragments prepared under partially denaturing conditions (86% formamidet, s 0f24 23tC; pm, histogram not shown). A fragments exhibited two major peaks in 90% 23[C (solid lines), formamide at with means corresponding to ss dimer length (~ = 1.15 pmn), and ss monomer length (* = 0.61 pm). The mean length of monomer ds EcoRI-A fragments prepared in 86% formamide at 239C (partially denaturing conditions) was 0.86 pm (histogram not shown). (D) Histogram of EcoRI-A fragments of H-i RE '"hyperdenatured" with 96% formamide at 30"C, and spread as in (B). The DNA was completely dissociated into ss monomer A fragments (~ = 0.64 pm), whose contour length distribution coincides with the ss A fragment monomers observed after denaturation with 90% formamide (shown in [C]).

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with an alkaline sucrose gradient does convert after similar treatment of ds H-1 RF (Fig. 5A). 90% of it into monomer ss H-1 DNA (9), we Incubation of EcoRI-A fragments of H-1 RF decided to further investigate the nature of the DNA in 96% formamide at 30°C for 30 min linkage between the two ss monomers. ds H-1 converted them into ss monomers (£ = 0.64 + RF DNA (in 10 mM Tris[pH 8.5]-1 mM EDTA) 0.03 ,um; n = 107) (Fig. 5D). These data collecwas placed in 96% formamide at 30°C for 30 tively demonstrate that the region of high G+ C min, followed by the addition of sufficient cyto- content is located close to the right end of the chrome c solution (in 0.5 M Tris[pH 8.5]-0.05 M RF DNA map. EDTA) to lower the formamide concentration of DISCUSSION the resulting solution to 90%, and immediately spread onto an isodenaturing hypophase conFeatures of the H-1 RF DNA monomer, as taining 60% formamide at 23°C. We estimated determined in this and the two preceding studthat the melting temperature (Tm) of the 90% ies in this series, are summarized diagramatiformamide spreading solution was approxi- cally in Fig. 6. Our results show that a region mately 300 below the T,, of poly(deoxyguani- susceptible to melting under partial denaturing dylic acid:deoxycytidylic acid)[poly(dG dC), conditions exists adjacent to the endo R- EcoRI and that the Tm of the 96% formamide mixture and HaeII cleavage sites, which are located was 10.40C above that of this homopolymer. approximately 20% of the genome length from These calculations are based on a Tm of 780C the arbitrarily defined left end of the H-1 RF for dG dC in a solution with a cation concen- DNA molecule (9). Partially denatured intertration of 6 mM (3) and a pH of 7.0 (12), as well mediates in H-1 RF DNA replication (RIs) simias a 0.6°C Tm reduction for each 1% increase larly exhibited melting near the left end of the in formamide concentration (1). After exposing molecule, whereas the origin of replication, deds H-1 RF DNA to the 96% formamide solution, termined by the position of early replication the contour length decreased to that of ss H-1 forks, was localized close to the right end of this DNA monomer size (Fig. 5B): X = 0.86 ± 0.04 DNA. Based on the size of the smallest daughum, n = 197, indicating that the region com- ter arms observed, the region containing the prising the intermonomer linkage probably has replication origin is located within 700 base a high G+C content and more stable hydrogen pairs (10), but more probably within 300 base bonding, as opposed to a covalent "turnaround" pairs (this study) of the right end of the H-1 RF of ribonucleotides. We performed the above experiments on Left Right EcoRI restriction endonuclease-generated A A-T Rich G-C Rich and B fragments of ds H-1 RF DNA to deterc R mine which end of this molecule is G+C rich C and responsible for linking the termini of the 'E monomers in the ss dimers generated with 90% RI V HaeEL Eco Origin t formamide. The mean length of the ds A fragI I 1 ment in 86% formamide (distribution not 0 2 3 4 5 shown) was 0.86 ± 0.05 am (n = 72), representKilobase Pairs ing 78.2% of the genome, and that of the ds B FIG. 6. Schematic representation of H-i RF DNA fragment spread under the same conditions was 0.24 + 0.02 ,um (n = 75), equal to 21.8% of the monomer in the foldback configuration (9). The molRF length. These size measurements are in ecule is 4,900 base HaeII pairs inandlength, restricEcoRIwith sitesthe(arrows) tion endonuclease good agreement with data obtained from ana- located 900 and 1,000 base pairs, respectively, from lytical gel electrophoresis of EcoRI-generated the end containing the covalent turnaround (based H-1 RF DNA fragments (9). When the B frag- on molecular weights given in [9D; the large and ments were spread in the presence of 90% form- small RF fragments generated by these endonucleaamide, their mean length decreased to 0.19 ± ses are referred to as A and B (respectively). V, Viral 0.01 ,tm (n = 85), indicating that this fragment strand; C, complementary strand. The zone in which does not contain the G+C-rich linkage group the initiation site forRF DNA replication is located is under study (Fig. 50). Preparation of the A delineated by the open bar and is composed of base (6%content of genome). The B fragment in 90% formamide generated two sub- pairs 4,600 through 4,900A +T (indicated by region is relatively rich in popultn ( e banalysis, crosshatched bar), and the right end has an elevated by a puion (Fig. 50) with(revea meanledlengths Of 0.61 ± 0.033 zm G+C composition (asterisk). More recent experi(n = 94) and 1.15 + 0.03 ,um (n = 108), repre- ments have shown that the structure of the right end senting the ss A fragment monomer and ss A is more complicated than indicated in this diagram fragment dimer, respectively, as was observed (manuscript in preparation).

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DNA molecule. The right terminus of the H-1 chromosome also exhibits a region that is refractory to melting in a solution containing 90% formamide [with an effective melting temperature about 3°C below that of poly(dG dC)], resulting in the formation of ss dimers under these conditions. No knobs or branches were detected at the midpoint of these ss dimers after electron microscopic study of more than 200 molecules, indicating that this presumably ds linkage region is relatively small. The observations that it remains intact under conditions that completely melt the rest of the RF molecule, and that exposing this DNA to an estimated T,,, of 10.4°C above the melting point of dG dC homopolymers converts the ss dimers into ss monomers, strongly suggested that this linkage is due to a G+C-rich terminus rather than to covalent bonding with ribonucleotides. The renaturation properties of the EcoRI-A fragment, which contains the right end, have also ruled out the possibility of a covalent linkage at the right end of H-1 RF DNA (9). Current experiments indicate that the structure of the right end is more complex than indicated in Fig. 6. In addition, the HaeII or EcoRI endonuclease B fragment, containing the left end of the RF DNA, forms denaturation loops under partially denaturing conditions (82, 84, or 86% formamide), and therefore is A+T rich (5-7). Most of the RF molecules observed had partially melted so close to the left end that a terminal loop was generated. Since only 10% of H-1 RF molecules exhibited evidence of a covalently closed turnaround at the left end (9), it seems unlikely that covalent bonds were responsible for stabilizing the apex of this end in the other 90% of the molecules under partially denaturing conditions. It is more probable that this B apex is stabilized by a dG dC enrichment similar to that present at the right terminus, but exhibiting a lower melting temperature, since EcoRI-B fragments (with the left end) were completely dissociated by 90% formamide, whereas the A fragments (with the right end) were not. The expected 10% of the B fragments, which should have been dimer length in 90% formamide due to the presence of a covalently closed turnaround, were not demonstrated with any certainty using electron microscopic methods. We also observed that incomplete melting conditions gave rise to Y-shaped B fragments in addition to loops located at either the natural left terminus, or near the middle of this fragment. We postulate that these Y-shaped fragments resulted from melting through the EcoRI and HaeII cleavage sites, rather than from melting of the natural left end of the RF, be-

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cause many of the undigested RF molecules treated with 86% formamide exhibited melting at these cleavage sites. Furthermore, complete melting of the left terminus of RI molecules subjected to partial denaturation would have generated double Y-shaped molecules (>