THE JOURNAL OF BIOLOGICAL CHEMISTRY 0 1989 by The American Society for Biochemistry and Molecular Biology, Inc.

Val. 264, No. 2, Issue of January 15, pp. 823430, 1959 Printed in U. S. A.

Replication of Kinetoplast DNA in Trypanosoma equiperdum MINICIRCLE H STRAND FRAGMENTS WHICH MAP AT SPECIFIC LOCATIONS*

(Received for publication, August 9, 1988)

Kathleen A. Ryan and Paul T. EnglundS From the Department of Biological Chemistry, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205

The mitochondrial DNA of trypanosomes, kineto- plast DNA, is a network containing thousands of to- pologically interlocked minicircles. Minicircles are replicated as free molecules after being detached from the network. The minicircle L strand appears to be synthesized continuously and the H strand discontin- uously. This paper describes properties of Trypano- soma equiperdum minicircle H strand fragments which could be Okazaki fragments. These fragments consti- tute a family of molecules of discrete sizes (ranging from about 70 to 1000 nucleotides) which map to spe- cific locations. Three of the most prominent fragments, a 73-mer, 83-mer, and 138-mer, map at contiguous or overlapping sites. Based on their position relative to the initiation site for L strand synthesis, the 73-mer may be the first Okazaki fragment to be synthesized and either the 83-mer or the 138-mer may be the second. The 5‘ end of the 73-mer lies within a sequence, GGGCGT, found at a similar location in minicircles of all trypanosomatid species. During the maturation of free minicircles and after their reattachment to the networks there appears to be continued extension and ligation of the H strand fragments. However, the liga- tion of the 73-mer, 83-mer, and 138-mer to the rest of the H strand is delayed; their eventual ligation results in covalent closure of the minicircles.

Kinetoplast DNA (kDNA),’ the mitochondrial DNA of trypanosomes and related parasitic protozoa, is a network of thousands of topologically interlocked DNA minicircles and 50-100 maxicircles (see Refs. 1-3 for reviews of kDNA). Minicircle synthesis involves the topoisomerase-mediated re- lease from the network of covalently closed molecules, which then undergo replication. The products, which contain nicks or small gaps, are then reattached to the network. The nicks and gaps are all eventually closed, and the network, which has doubled in size, is divided in two. At the time of cell division, the two progeny networks segregate into the two daughter cells (see Refs. 4 and 5 for reviews of kDNA repli- cation).

Previous reports have described the structures of several free minicircle replication intermediates in both Crithidia

* This work was supported by National Institutes of Health Grant GM-27608 and by a grant from the MacArthur Foundation. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduer- tisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

.$ To whom correspondence should be addressed Dept. of Biological Chemistry, The Johns Hopkins University, 725 N. Wolfe St., Balti- more, MD 21205. ’ The abbreviations used are: kDNA, kinetoplast DNA kb, kilo- base(s); dNTP, deoxynucleoside triphosphates.

fasciculuta and Trypanosoma equiperdum (6-11). In both parasites one of the minicircle strands, designated L,* appears to be synthesized continuously. L strand synthesis initiates at the universally conserved minicircle sequence GGGGTTGGTGTA (6, 11, 12), and one or two ribonucleo- tides, presumably derived from a replication primer, persist at the 5’ end (6, 11).

Studies in C. fasciculata indicate that synthesis of the H strand is discontinuous (7,8,10). Newly synthesized H strand fragments are found on multiply gapped free minicircles; these molecules form a smear during electrophoresis on an agarose gel, probably because of heterogeneity in gap size and number. The H strand fragments are extended and ligated as the multiply gapped molecules are first converted into nicked free minicircles and then are reattached to the network (7,8).

In this paper we describe in greater detail the newly syn- thesized H strand fragments. We have used T. equiperdum as a model system because of the small size (1 kb) and sequence homogeneity of its minicircles and the presence of a single replication origin. We have found that many of the H strand fragments, which could be Okazaki fragments, are discrete in size and map to unique positions within the minicircle se- quence. Three of the most prominent fragments, a 73-mer, 83-mer, and 138-mer, map to contiguous or overlapping sites in the region where L strand synthesis is initiated. Based on their location, the 73-mer may be the first Okazaki fragment to be synthesized and the 83-mer or the 138-mer may be the second. The 5’ terminus of the 73-mer lies within a sequence GGGCGT which is conserved within minicircles of all species.

MATERIALS AND METHODS

Cultivation of Trypanosomes and Isolation of kDNA-T. equiper- dum (Pasteur strain, BoTat 24) was maintained and grown as de- scribed previously (12). kDNA free minicircles and networks were labeled with [3H]thymidine and isolated also as described previously (6, 9). A map of the 1012-base pair minicircle, derived from its published sequence (13), is shown in Fig. 1.

Gel Electrophoresis-Agarose gel electrophoresis, polyacrylamide gel electrophoresis (nondenaturing, denaturing, and strand separat- ing), electroelution from agarose, and Southern transfer methods have been described previously (6, 9, 14). Radioactive fragments on gels were detected by autoradiography or fluorography (12). DNA fragments were electrotransferred from polyacrylamide denaturing gels by the method of Church and Gilbert (15) using a Bio-Rad Transblot apparatus (60 V, 0.25 mA, 4 h, 4 “C).

DNA Clones and Probes-pJN1 and pJN6 (9) both contain the complete minicircle sequence, and probes were prepared by the ran- dom primer method (16) on electroeluted minicircle insert. pKAR3- 1 and pKAR5-1 have the minicircle insert from pNJ6 in M13mp18 and M18mp19, respectively. pKAR3-1 probe hybridizes to L strand and the pKAR5-1 probe hybridizes to H strand (9); pJNl and pJN6

* To simplify comparison with minicircles from C. fasciculata, the

minicircle sequence GGGGTTGGTGTA is designated the H strand. 2’. equiperdum minicircle strand containing the universally conserved

The complementary strand is designated L.

823

824 Hinf I

(1 )

Kinetoplast Minic

GGGGTTGGTGTA

Bgl I I (345)

(557)

FIG. 1. Map of T. equiperdum minicircle. This map is derived from the nucleotide sequence (13), and numbering begins at the HinfI site. Numbers in parentheses refer to the nucleotide on the 5' side of the restriction enzyme cleavage site in the H strand. GGGGTTGGTGTA (800-811) and GGGCGT (727-732) are on the H strand; see text for discussion. Map positions of synthetic 20-base oligonucleotides, which hybridize with the H strand, are indicated by solid rectangles. The numbers 3, 347, 523, 720, and 796 denote the base at their 5' end.

probes hybridize to both strands. DNA bound to nylon filters (Genescreen, DuPont) was hybridized with 32P-labeled oligonucleo- tides as described previously (9).

Other DNA probes were prepared by restriction enzyme digestion of plasmid or network DNA. Synthetic oligonucleotides, 20 nucleo- tides long, are designated by the position of their 5'-terminal nucleo- tide (Fig. 1; Ref. 13 gives nucleotide numbering system); the prefix H- indicates that they hybridize to the H strand. Restriction frag- ments or synthetic oligonucleotides were 5'-end-labeled with T4 polynucleotide kinase (6) and then separated from [y3'P]ATP by chromatography on Nensorb (Du Pont-New England Nuclear) or by ethanol precipitation in the presence of 2 M ammonium acetate. To prepare DNA probes for S1 nuclease protection assays, 5'-end-labeled minicircle restriction fragments were cut with a second enzyme. The labeled fragments were separated by nondenaturing 5% polyacryl- amide gel electrophoresis and the fragments with the 5'-end-labeled L strand were electroeluted from the gel.

SI Nuclease Protection Assays-For S1 assays (17), free minicircles were lyophilized in the presence of 5'-32P-labeled probe (1-50 X lo6 cpm/pmol). After dissolving in 20 pl of hybridization buffer (0.3 M NaC1,O.l M Tris-HC1 (pH 8.0), 10 mM sodium phosphate (pH 8.0), 2 mM EDTA), samples were heated to 100 "C for 5 min and then hybridized for 8 to 13 h at 64 "C. The samples were chilled on ice for 1 min and then treated with 280 pl of S1 digest mixture (30 mM sodium acetate (pH 4.5), 200 mM NaCl, 5 mM ZnS04, and 3 or 9 units of S1 nuclease (Bethesda Research Laboratories)). After 1 h at 20 "C, the digestion was quenched with 60 pl of stop solution (1 M sodium acetate (pH.7.0), 0.33 M Tris-HC1 (pH 9.5), 67 mM EDTA, 60 pg/ml tRNA) and 0.9 ml of absolute ethanol. The ethanol precipitated DNA was redissolved in 2 pl of denaturing gel sample buffer (14), heated 3 min at 90 "C, and loaded immediately on a 6% polyacrylamide dena- turing gel. Size standards were either 5'-32P-labeled Hue111 and HpaII digests of q5X-174 RF DNA plus HpaII digest of pBR322 DNA, or 5'- 32P-labeled probe which had been cleaved by a modification of the chemical cleavage method of Maxam and Gilbert (18). Roughly 0.01 pmol of both free minicircle DNA and probe were used in each assay, although the probe was in excess as judged by titration with free minicircles. Both strands of the probe were usually present during hybridization although identical results were obtained if the unlabeled complementary strand was first removed from the probe by strand separation gel electrophoresis or by exonuclease I11 digestion (data not shown).

Primer E~tensions-5'-~*P-Labeled oligonucleotide (0.01-0.1 pmol) was annealed to free minicircles (0.001 pmol) or networks (0.004 pmol of minicircle) by heating to 100 "C in 10 mM Tris-HC1 (pH 7.5), 5 mM MgC12,l mM dithiothreitol (15 p1) for 3 min and then cooling on ice for 10 min. The four dNTPs (1 pl each of 1 mM stock

:ircle Replication solutions, final concentration 50 p ~ ) and DNA polymerase I large (Klenow) fragment (1 pl, 1 unit, New England Biolabs or Boehringer Mannheim) were added. After 1 h at room temperature, the DNA was ethanol-precipitated, resuspended in denaturing gel sample buffer, and electrophoresed on a 6% polyacrylamide denaturing gel. Control sequencing ladders were produced by the dideoxy chain termination method (19).

RESULTS

Size Measurements of T. equiperdum Minicircle H Strand Fragments-To measure the sizes of H strand fragments, we subjected free and network minicircles to denaturing electro- phoresis on a polyacrylamide gel, electrotransferred the DNA to a nylon filter, and then probed the filter with an H strand- specific probe (Fig. 2, panel A ) . In both free minicircles (lane 1 ) and networks (lune 2) there is a family of discrete H strand fragments ranging in size from about 70 to 1000 nucleotides. The sizes of these fragments appear to be the same in the free minicircle and the network preparations, although in net- works there is a much greater proportion of larger fragments (200-1000 nucleotides). Oligonucleotide probe H-720 (see map in Fig. 1 for location) detected only a subset of the H strand fragments (Fig. 2, panel B ) ; in free minicircles (lane 1) the major ones were 83 and 138 nucleotides long, whereas in networks (lane 2) there were also a number of larger frag- ments.

FIG. 2. Size measurement of H and L strand fragments in free minicircles and networks. Panel A , unlabeled free minicir- cles, (approximately 1 ng, lane 1 ) and networks (75 ng, lane 2 ) were

transferred to a nylon filter, and hybridized with 3ZP-labeled pKAR5- denatured, resolved on a 6% polyacrylamide denaturing gel, electro-

1 which is complementary to the H strand. Panel B, same as panel A but hybridization was with oligonucleotide H-720 probe. Panel C, kDNA, labeled in vivo for 40 min with [3H]thymidine, denatured, electrophoresed on a 6% polyacrylamide denaturing gel, and analyzed by fluorography. Lane 1, free minicircles (15,000 cpm); lane 2, net- works (15,000 cpm). Panel D, same as Panel B but hybridization was with pKAR3-1 probe which hybridizes with L strand. Scale on the left indicates marker sizes in nucleotides; same sizes also indicated by ticks on left side of panels B, C, and D. Some smaller H strand fragments (60-40 nucleotides) can be observed on longer exposures of probed filters and fluorograph similar to those in panels A and C.

Kinetoplast Minicircle Replication 825

To evaluate whether H strand fragments detected by hy- bridization represent newly synthesized molecules, trypano- somes were labeled in vivo with [3H]thymidine. Isolated free minicircles and networks were then fractionated by denatur- ing electrophoresis and radioactive fragments were detected by fluorography (Fig. 2,panel C ) . There are numerous labeled fragments, in both free minicircles (lane 1) and networks (lane 2) , in the size range of 70 to 1000 nucleotides. Although it is not possible to tell in this experiment if the radioactive fragments are H or L strand, some are the same size as those shown to be H strand by probing (i.e. fragments of 73,83, and 138 nucleotides). Some fragments detectable in Fig. 2C could be H or L strands in the process of elongation. However, L strand fragments detected by probing (Fig. 2, panel D) are generally much longer in both free minicircles (lane 1) and networks (lane 2). This in vivo labeling suggests that some of the H strand fragments may be discontinuously synthesized Okazaki fragments.

Mapping 5’ Ends of H Strand Fragments-Since the H strand fragments in free minicircles and networks are of discrete size, it seemed likely that each fragment maps to a specific location. To map the 5’ ends of these fragments we used primer extension, S1 nuclease protection assays, and indirect end labeling. Fig. 3 shows the results of these methods as applied to the minicircle sequence between nucleotides 720 and 984.

Fig. 3, panel A, shows an example of mapping by primer extension. 5’-32P-labeled oligonucleotide H-3 was annealed to denatured free minicircles and extended with the Klenow fragment of DNA polymerase I. The extended primers were then fractionated by denaturing polyacrylamide gel electro- phoresis and detected by autoradiography. A family of ex- tended primers was produced, and the sizes of these fragments indicated the locations of the 5’ ends of H strand fragments in this region. The numbers at the left of the figure identify the map position of the nucleotide at each major 5’ end. Similar mapping studies were conducted using oligonucleo- tides H-347, H-523, H-720, and H-796 as primers, and the results are summarized in Fig. 4.

Fig. 3, panel B, shows an example of S1 nuclease mapping. The probe was a denatured minicircle fragment in which the L strand had been 5”end-labeled at the HinfI site. After annealing to unlabeled denatured free minicircles, the DNA was treated with S1 nuclease. The S1-resistant fragments were then resolved on a denaturing gel and detected by autoradiography. As shown in lanes 1 and 2, a family of S1- resistant fragments was detected. The sizes of these fragments permit mapping of 5‘ termini of H strand fragments, and comparison with panel A reveals a close agreement. The most significant difference between these mapping methods is that the S1 nuclease method (panel B ) reveals slightly more het- erogeneity in the 5’ ends of the fragments. This heterogeneity could be due to either incomplete or slight over-digestion of the hybrids. Similar mapping studies used probes in which the L strand was 5’-end-labeled at nucleotides 348, 220, 91, 851, 796, 659, or 615. The results are summarized in Fig. 4.

Fig. 3, panel C, shows an example of mapping by indirect end labeling. Free minicircles were cut with HinfI, denatured, and DNA fragments were fractionated on a denaturing gel. The fragments were then detected by probing with 5’-32P- labeled oligonucleotide H-3. This oligonucleotide is comple- mentary to the 3‘ end of the H strand of the HinfI-cut minicircle; therefore it detects 1-kb HinfI -linearized minicir- cles (near the top of the gel) derived from covalently closed minicircles. It also detects smaller molecules which derive from minicircles with nicks or gaps in their H strand. The

A B I 2 C

IO

10

1’ I

3’ I

FIG. 3. Mapping H strand 5‘ ends on free minicircles and networks. Panel A , primer extension mapping. 5’-3ZP-labeled oli- gonucleotide H-3 was annealed to denatured free minicircles, ex- tended with DNA polymerase I Klenow fragment, and the products were resolved on a 6% polyacrylamide denaturing gel. Panel B, S1 nuclease mapping. The probe was a Hinff-EcoRV double-stranded restriction fragment (455 base pairs) with the L strand 5’-32P-labeled at the HinfI site. The probe was annealed to free minicircle DNA, digested with S1 nuclease, and the protected fragments resolved on a 6% polyacrylamide denaturing gel. Lane 1, hybrids digested with 3 units of SI; lane 2, with 9 units. Panel C, indirect end labeling. kDNA free minicircles were digested with Hinff, resolved on a 6% polyacryl- amide denaturing gel, electrotransferred to a nylon filter, and hybrid- ized with 5’-32P-labeled oligonucleotide H-3. Numbers on the left refer to map positions of H strand 5’ ends.

size of each molecule is a measure of the distance between its 5’ terminus and the HinfI site. Similar studies, with oligonu- cleotides H-347, H-523, H-720, and H-796 (see Fig. l), allowed the mapping of the F ends of H strand fragments on all regions of the minicircle. The sites determined by indirect end labeling are identical to those obtained by primer exten- sion and S1 mapping (not shown).

Primer extension and indirect end labeling analysis were also applied to network minicircles and these were found to contain H strand 5’ termini which map at the same sites as those found on free minicircles (data not shown). The only significant difference between free and network minicircles was that termini closest to the oligonucleotide primers were relatively more abundant in free minicircles than in networks, a difference probably due to the smaller average size of H strand fragments on free minicircles relative to those on networks.

Inspection of the sequences flanking the H strand 5’ termini reveals no obvious consensus sequences.

Are Ribonucleotides Present at the 5’ Ends of H Strand

826

FIG. 4. 5’ ends of H strand frag- ments. 5’ ends on free minicircles and network minicircles were mapped by primer extension or S1 nuclease map- ping in experiments like those in Fig. 3. Identical results were obtained by indi- rect end labeling (not shown). Solid boxes indicate oligonucleotide primers. Aster- isks indicate the 5’ end of S1 nuclease probe; the restriction enzymes and cleav- age sites used in preparing these probes are indicated. Vertical bars indicate po- sition of major 5’ ends of H strand frag- ments; abundance of 5’ ends varies a t different positions (e.g. see Fig. 3). Thicker bars represent two closely spaced sites. Mapping with HinfI and oligonucleotide H-3 probes (bold letters) is shown in Fig. 3.

Kinetoplast Minicircle Replication - - 0 - 0

0 0

0 0 0 0

W d In (D r. W m 0 7 0 0

- - 0 0 N 0 0 0 0 0 7

I 1 I I I I I I I I I 1 7

Lu 5?z A H-347

A Bgl II (348) d+ Mae Ill (220)

H-3 -* Hinf I (1) - Spe I (851)

A Mbo II (91)

A H-796

Taq I (796) A. H-720 - Mbo II (659)

Taq I (615) k H-523

Fragments?-To explore this possibility we used primer ex- tension mapping or S1 nuclease protection assays on networks and free minicircles that had been treated with alkali (0.3 M NaOH, 37 “C, 17 h, Ref. 6; data not shown). Removal of 5‘ ribonucleotides with alkali would cause a change in position of 5’ ends. Using oligonucleotide H-796 as a primer there was no indication of 5’ ribonucleotides in either free minicircles or networks in the region between nucleotides 500 and 796. Similar results were obtained using S1 nuclease mapping with a probe labeled at the TaqI site at position 796. Using oligo- nucleotide H-3 as a primer, there was also no suggestion of 5’ ribonucleotides on free minicircles in the region between nucleotides 720 and 996. However, using oligonucleotide H-3 as a primer with network DNA demonstrated several new ends (positions 971, 978, 985, and 990) suggesting that the network H strand may have some internal alkali-sensitive sites. We have not studied these sites further.

The Location of Some Major H Strand Fragments-After determining the positions of the 5’ ends of H strand frag- ments, we then determined the location of three of the most abundant H strand fragments, the 138-mer, 73-mer, and 83- mer. To map the 138-mer, intact networks or networks cleaved with restriction enzymes were denatured, fractionated by denaturing polyacrylamide gel electrophoresis, and trans- ferred to a filter. The filter was probed with oligonucleotide H-720 (Fig. 5, panel A ) . Uncut networks (lane 1 ) contained both the 138-mer and the 83-mer, but of these only the 138- mer was cleaved by TaqI (lane 2 ) . The TaqI digest contained two major new fragments of 180 nucleotides and 115 nucleo- tides. These same fragments are also detected in TaqI digests of networks labeled i n vivo with [3H]thymidine (Fig. 5, panel B, lane 2). The 180-mer in panel A, lane 2, derives from the region between the two TaqI sites (see map in Fig. 5, panel C) and therefore would be formed from all covalently closed network minicircles as well as from any H strand fragment spanning that region. The 115-mer, on the other hand, must derive from the 138-mer and from longer H strand fragments which have the same 3’ terminus as the 138-mer. Since one end of the 115-mer is at the TaqI site at position 615, the other end must be at approximately nucleotide 727. The 115- mer could not have been produced by cutting at the other TaqI site, at position 795 (see map in panel C), as no corre- sponding fragment was produced by RsaI digestion (see lane 3, panels A and B) . The 138-mer must therefore extend (5’ to 3’) between nucleotides 590 and 727.

The 73-mer was mapped by a similar method using oligo- nucleotide H-796 as a probe. Uncut networks examined in this way contained the 73-mer, which was converted to a 67-

mer after TaqI digestion (not shown). The 67-mer was also observed in a TaqI digest of in uiuo labeled DNA (Fig. 5, panel B, lane 2). Therefore, one end of the 67-mer is at the TaqI site (nucleotide 795) and the other end at position 728, indi- cating that the 73-mer must map between nucleotides 728 and 800. These results are confirmed by RsaI digestion, in which the i n uiuo labeled 73-mer fragment was converted to a 63- nucleotide product (Fig. 5, panel B, lane 3); this fragment was not detected on the probed filter (not shown) as RsaI cuts the 73-mer within the sequence complementary to oligonucleotide

The 83-mer also hybridizes with oligonucleotide H-720 (Fig. 5, panel A , lane 1 ) but it is not cleaved by TaqI (lane 2 ) , RsaI (lune 3), or MboII (recognition site between nucleotides 646 and 650; cut site at nucleotide 659; data not shown). There- fore, its precise location on the minicircle cannot be mapped by the methods used for the 138-mer and the 73-mer. How- ever, it is most likely positioned adjacent to the 73-mer with its 5‘ end at nucleotide 645 and its 3‘ end at nucleotide 727; there is a major H strand 5‘ end at nucleotide 645 (Fig. 4).

Further Characterization of H Strand Fragments on Net- work Minicircles-To examine the organization of the other H strand fragments, we used a strategy similar to that shown in Fig. 5. Fig. 6, panel A shows the DNA hybridized with oligonucleotide H-523 (see Fig. 6, panel C, for location of this probe). Uncut networks gave numerous discrete bands which correspond to the many H strand fragments located in this region (lane 1 ). BqlII digestion of the networks (lane 2 ) cleaves many of these fragments and yields several new ones. The most prominent new fragments are 383, 300, and 246 nucle- otides; their sizes and their hybridization with oligonucleotide H-523 indicate that they are located between the BglII site and the 5’ ends of the 73-mer, 83-mer, and 138-mer, respec- tively (Fig. 6, panel C). Comparison of the bands in the BglII digest (lane 2 ) with those in the uncut networks (lane 1 ) indicates that there are no three major fragments in uncut networks which could have given rise to the three major new fragments. Therefore, these new bands must derive from multiple H strand fragments in uncut networks. Although these fragments have variable 5‘ ends, they all must have 3’ ends adjacent either to the 73-mer, the 83-mer, or the 138- mer.

Evidence for this organization is also provided by a similar experiment in which the network DNA was digested with EcoRV and hybridized with oligonucleotide H-347 (see Fig. 6, panel C for the location of this probe). Uncut networks have a number of H strand fragments detected by this oligonucle- otide (Fig. 6, panel B, lane I ) . EcoRV cleaved many of these

H-796.

Kinetoplast Minicircle Replication a27

A 1 2 3 1078 --

310

194

118

C

.m - - -m

FIG. 5. Mapping of the major H strand fragments. Panel A, unlabeled networks (75 ng) were digested with Tag1 or RsaI and fractionated on a 6% polyacrylamide denaturing gel. Fragments were electrotransferred to nylon filters and detected by hybridization with oligonucleotide H-720 probe. Lane 1, undigested networks; lane 2, networks digested with TaqI; lane 3, networks digested with RsaI. Size markers are indicated on lejt. Sizes of kDNA fragments are either boxed (uncut by restriction enzymes) or underlined (produced by restriction enzyme cutting). Panel B, networks labeled in vivo with [3H]thymidine for 40 min (16 ng; 15,000 cpm) were digested with TaqI or RsaI, and then resolved on a 6% polyacrylamide denaturing gel which was then fluorographed. Lane 1, undigested networks; lane 2, networks digested with TaqI; lane 3, networks digested with RsaI. Ticks on the left indicate position of size markers identical to those in Panel A. Panel C, map positions of 73-mer, 83-mer, and 138-mer. Exact position of 83-mer is tentative (see text). Map also shows fragments produced by digestion with TaqI and RsaI (see panels A and B ) . Solid boxes indicate oligonucleotide probes. The size of the 115-mer was determined by size markers; its true size is 113 nucleotides.

FIG. 6. Mapping fragments which C flank the 138-mer, 83-mer, and 73- 1078 mer. Panel A , unlabeled networks (75 ng) were digested with BglII; the digests 603 - were fractionated on a 6% polyacryl- amide denaturing gel, electrotransferred to a nylon filter, and hybridized with oligonucleotide H-523 probe. Lane 1, un- digested networks; lane 2, networks di- gested with BglII. Scale on left shows size markers. Bands indicated by arrows are 31 major fragments produced by BglII digestion. Panel B, same as panel A ex- cept networks were digested with EcoRV 14.347 B H 4 2 3 I

and hybridized with oligonucleotide H- 347 probe. Lane I , undigested networks; lane 2, networks digested with EcoRV. - Ticks on lejt shows same size markers as in panel A. Arrows indicate major frag- c ments produced by EcoRV. Panel C, dia- gram of the H strand fragments flanking 194 - the 5’ ends of the 138-mer, 83-mer, and L 1 ’

73-mer. Fragments shown as boxes below .-r the map (246-mer, 300-mer, and 383- mer) were identified in panel A. Frag- ments shown as lines above the map are the ones indicated by arrows in panel B. Solid rectangles indicate probes (at po- sitions 347 and 523). i ,

p

p f - 0 z & s E 1 0

01 I I 1 1 1 1 1 I I I I

I 246 1 13em.r

300 1m-r I731.4

.L -*

- 4

fragments and produced various new ones, marked by arrows schematically at the top of Fig. 6, panel C. Since none of the (Fig. 6, panel B, lane 2). These new fragments have 3’ ends new fragments are dominant, there must be many distinct at the EcoRV site and 5’ ends at variable sites; they are shown fragments in this region. However, the experiments in the

828 Kinetoplast Minicircle Replication

previous paragraph indicate that most of them have 3’ ends which abut either the 73-mer, 83-mer, or 138-mer.

Similarly, at the 3’ ends of the 73-mer, 83-mer, and 138- mer, we have found that there are many H strand fragments which have 5’ ends at positions 728 or 801 and variable 3’ ends. (This conclusion is based on cleavage with HinfI or SpeI (at nucleotide 851) and probing the products with oligonucle- otide H-3 (Fig. 3, panel C; data not shown).) Thus, on either side of the 73-mer, 83-mer, and 138-mer, we find multiple fragments which extend variable distances away.

Effect of T4 DNA Polymerase and T4 DNA Ligase on H Strand Fragments-Since many of the H strand fragments (especially the 73-mer, 83-mer, and 138-mer) persist on min- icircles even after reattachment to networks, experiments were performed to determine whether these fragments are susceptible to ligation in vitro. After treatment of isolated free minicircles with T4 DNA ligase and/or T4 DNA polymerase, the DNA was denatured, resolved on a denaturing polyacryl- amide gel, transferred to filters, and hybridized with a probe specific for total H strand (Fig. 7, panel A ) , with oligonucle- otide H-796 (recognizing the 73-mer, Fig. 7, panel B ) , or with oligonucleotide H-720 (recognizing the 83-mer and the 138- mer, Fig. 7, panel C) . Polymerase alone had little effect on

A 1 2 3 M 4 5 B 1 2 3 M 4 5 C 1 2 3 M 4 5 - bw

* -194

FIG. 7. Treatment of free minicircles with T4 DNA polym- erase and T4 DNA ligase. T4 DNA polymerase (Pharmacia LKB Biotechnology Inc., 5 units) and/or T4 DNA ligase (Pharmacia, 5 units) reactions were conducted in 66 mM Tris-HC1 (pH 7.7), 6.6 mM MgC12, 10 mM dithiothreitol, 500 pM dNTPs, 1 mM ATP, 150 ng of DNA/20 pl for 4 h at 23 “C. After phenol extraction and ethanol precipitation, the DNA was fractionated on a 6% polyacrylamide denaturing gel. The fragments were then electrotransferred to nylon filters. Panel A, DNA hybridized with pKAR5-1 probe, which recog- nizes total H strand sequences. Panel B, same as panel A but hybrid- ized with oligonucleotide H-796 probe which recognizes the 73-mer. Panel C, same as panel A except hybridized with oligonucleotide H- 720 probe which recognizes the 83-mer and the 138-mer. Lanes I and 5, untreated free minicircles; lane 2, same DNA treated with T4 DNA polymerase; lane 3, same DNA, with T4 DNA polymerase and T4 DNA ligase; lane 4, same DNA, with T4 DNA ligase. Lane M, Hue111 and HpaII digests of @X-174 RF DNA, sizes of selected fragments shown on right. The 73-mer, 83-mer, and 138-mer are indicated by arrows on left.

most of the free minicircle H strand fragments (Fig. 7, panel A , lane 2 ) although some of the 73-mer and 83-mer, and also some other fragments, appeared to be extended by one or a few nucleotides (Fig. 7,panels A, B, and C; lane 2) . This result implies that a small fraction of H strand fragments have small gaps at their 3’ ends. However the majority of the fragments must be bordered by nicks as treatment with T4 DNA ligase gave rise to larger fragments (Fig. 7, panels A, B, and C; lane 4 ) . When both polymerase and ligase were used together, an even larger fraction of the H strands became at least 200 nucleotides long (lane 3 ) . The results in Fig. 7 show that most of the free minicircle H strand fragments, in the 60-200 nucleotides size range, have at least one ligase-sensitive 3’ hydroxyl or 5’ phosphate group, although they are sometimes flanked by small gaps.

T4 DNA polymerase and DNA ligase have similar effects on the H strand fragments of minicircles which have been reattached to networks (not shown), but most of the network H strand fragments are sensitive to DNA ligase in vitro without polymerase repair. Therefore, the gaps which occa- sionally flank H strand fragments on free minicircles are even less frequent on network minicircles.

Which Free Minicircle Components Contain H Strand Frug- merits?-Since the H strand fragments have been mapped either on networks or on the total free minicircle population, we wanted to establish which type of free minicircle molecules contained most of the H strand fragments. Agarose gel elec- trophoresis separates free minicircles into several species (Fig. 8, panel A, lane 1 ), the most prominent of which are covalently closed circles (I), nicked circles (ZZ), linearized circles (ZZI), and a smear of multiply gapped circles (ZZg) (7-9). The mini- circles designated as “nicked” (11) actually constitute a doub- let; they are a mixture of nicked molecules (IIa) and minicir- cles with a small gap at a unique position in the newly synthesized L strand (IIb) (6,9,12). All of these components except covalently closed circles are labeled in uiuo with [3H] thymidine (9). To characterize the H strand fragments in each of these components, the nicked (Form 11), linearized (Form 111), and multiply gapped circles (Form IIg) were eluted from a gel such as the one in lane 1. Electrophoresis of the purified nicked (11) and multiply gapped (11,) molecule is shown in lanes 2 and 3.

Samples of these eluted DNAs, and also samples of network DNA, were then denatured and the strands separated on a denaturing gel. The H strand fragments were detected by hybridization with 32P-labeled oligonucleotides. Fig. 8B shows the H strand fragments which hybridize with oligonucleotide H-523. Form 11, contains H strand fragments in the size range of about 50-1000 nucleotides, with a peak centering at about 100-300 nucleotides (lane 1 ). Form 111, which contains some of the slower migrating Form 11, molecules, also has H strands that range from 50 to 1000 nucleotides with an average length of 500 nucleotides (not shown). Form 11, in contrast, contains larger fragments, with the majority greater than 200 nucleo- tides in size (lane 2). Networks contain H strand fragments which appear to be the same or larger in average size than those of Form I1 (lane 3) . Comparable size distributions of H strand fragments were detected when the same DNA was hybridized with 32P-oligonucleotide H-3 (not shown).

A very different distribution of H strand fragments was detected when the same filter was hybridized with oligonucle- otide H-796. Here, the major H strand fragment detected in Form 11, (Fig. 8, panel C, lane I ) , Form I1 (lane 2) , and networks (lane 3 ) was the 73-mer. Except for the 1-kb DNA molecule in lanes 2 and 3 there were very few larger fragments. Form 11, also has some smaller fragments (65-72 nucleotides)

Kinetoplast Minicircle Replication 829

A 1 2 3 8 1 2 3 c 1 2 3 1078 - - 603 - -7 310 - -

I L ” 118

I.

72

I FIG. 8. Characterization of H strands associated with spe-

cific free minicircle molecules. Panel A, unlabeled free minicircles were resolved on 1.5% agarose in TBE buffer containing 1 rg/ml ethidium bromide (lane 1 ). Form I1 and the faster migrating compo- nents of Form 11, minicircles were electroeluted from a gel like that in lane 1 as described under “Materials and Methods.” Lane 2, electroeluted Form I1 minicircle; lane 3, electroeluted Form 11, mini- circles. Band at the bottom of the smear in lane 3 consists of knotted minicircles (9). Minicircles detected by pJN6 probe. Panel B, elec- troeluted free minicircle components and network minicircles were resolved on a 6% polyacrylamide denaturing gel, electrotransferred to nylon membrane, and hybridized with oligonucleotide H-523 probe. Lane 1, gel purified Form 11,; lane 2, gel purified Form II; lane 3, networks. Panel C, same filter as panel B except hybridized with oligonucleotide H-796 probe.

which hybridize with oligonucleotide H-796. These may be growing molecules which will ultimately elongate to the 73- mer. When the same gel was probed with oligonucleotide H- 720, the only major fragments detected in free minicircles and networks were the 83-mer and the 138-mer (data not shown).

DISCUSSION

kDNA minicircles replicate as free molecules after being released from the network (20). The minicircle L strand replicates continuously and unidirectionally starting at a unique position, and the H strand replicates discontinuously (7, 8, 10-12, 22). This paper describes the characteristics of the H strand fragments on T. equiperdum minicircles. These fragments are discrete in size and range from about 70 nucle- otides to 1 kb (Fig. 2). Fragments of the same size are present in both free and network minicircles although those on net- works are larger in average size (Fig. 2). At least some of these fragments are labeled in vivo with [3H]thymidine (Fig. 2, panel C). We therefore infer they are discontinuously synthe- sized Okazaki fragments. We have not, however, ruled out the possibility that some of the H strand fragments are produced by specific endonucleases or by topoisomerase-mediated cleavage during isolation of the DNA.

A

‘ 6-MER

B 138-mer

””__)

5 ’ ... TATGGGCG 4-g;;: : : : . . q-j. . .3 ’ H-Strand 3‘...AT CCCGC . . TT CCCCAACCACA TAT. . .5 ‘ L-Strand *-- x*

L-strand

83-mer 73-mer b

FIG. 9. Map of 73-mer, 83-mer, and 138-mer H strand frag- ments near the L strand initiation site. Panel A, possible early 0 structure showing first Okazaki fragment, the 73-mer. 12-mer and 6- mer sequences are boxed in panel B. Panel B, sequence in region of L strand initiation site. Leftward pointing arrow below sequence indi- cates newly synthesized L strand. * indicates ribonucleotides at the 5’ end (6). Dashes at the end of the arrow indicate that strand growth continues to left. Rightward pointing arrows above sequence are newly synthesized H strand fragments. 6-mer is between nucleotides 727 and 732 and the 12-mer sequence is between 800 and 811.

Oligonucleotide probes hybridize only with a subset of the H strand fragments indicating that they may derive from specific sites on the minicircle sequence (Fig. 2, panel B ) . Mapping studies revealed that their 5’ ends are distributed throughout the molecule at specific sites spaced 10-70 nucle- otides apart. This close spacing indicates that different min- icircles must have different sets of H strand fragments. Three of the most prominent H strand fragments are the 73-mer, 83-mer, and 138-mer. These map at contiguous or overlapping positions in the region near the site of initiation of the L strand. The 73-mer spans nucleotides 728-800, while the 83- mer and 138-mer overlap and have a common 3‘ terminus at nucleotide 727 (Figs. 5, panel C , and 9). Thus the 5‘ ends of the 73-mer and the 3’ ends of the 83-mer and the 138-mer lie within a sequence, GGGCGT, which is conserved in minicir- cles of all species. These fragments and this sequence will be discussed further below.

There are several distinct molecular species in the free minicircle population, the most abundant being Form I1 min- icircles (a mixture of nicked (Form IIa) and uniquely gapped minicircles (Form IIb)), linearized minicircles (Form 111), multiply gapped minicircles (Form II,), and covalently closed minicircles (Form I) (Fig. 8). Form IIb minicircles contain newly synthesized L strand (6,9). Most of the newly synthe- sized H strand fragments are found on nicked (Form IIa) and multiply gapped minicircles (Form 11,) (7, 8, 22). These H strand fragments, based on their sensitivity to T4 DNA po- lymerase and T4 DNA ligase, appear to be flanked by nicks and occasionally by small gaps (Fig. 7). The H strand frag- ments of Form 11, minicircles are the smallest in average size, those on Form I1 minicircles are larger, and those on network minicircles are slightly larger still (Fig. 8, panel B). Thus, it is likely that most H strand fragments are extended and ligated during the maturation of multiply gapped minicircles to nicked minicircles and finally to network minicircles. How-

830 Kinetoplast Minicircle Replication

ever, the 73-mer, 83-mer, and 138-mer, persist unligated for some time even after the minicircles have been reattached to the networks (Fig. 8). Random ligation in the other regions of the minicircle could account for the fact that many H strand fragments, with variable 5’ ends, have 3’ ends which abut the 5’ ends of the 73-mer, 83-mer, or 138-mer (Fig. 6). Similarly, other fragments with variable 3’ ends have 5’ ends adjacent to the 3‘ ends of the 73-mer, 83-mer, and 138-mer (data not shown).

We do not know why the 73-mer, 83-mer, and 138-mer temporarily resist ligation in vivo after the minicircles are reattached to networks. They must have appropriate termini for ligation as they are efficiently ligated to neighboring fragments in vitro by T4 DNA ligase. The sister molecules, with a newly synthesized L strand, also have a gap or a nick in the newly synthesized strand at the site of initiation of synthesis. This discontinuity also resists ligation in vivo (6, 12, 22). These findings are reminiscent of a report that C. fasciculata free minicircles have nicks or gaps which persist in the L strand complementary to the GGGGTTGGTGTA sequence and nicks which persist in the H strand within the sequence GGGCGT (11).

The sequence GGGCGT is conserved in minicircles from all species which have been sequenced (out of 40 which have been sequenced, there is one Leishmania tarentolae minicircle, pLT26, which has GGGCTC at this position (21)). The GGGCGT sequence is always 73 nucleotides (in T. equiper- dum and Trypanosoma brucei) or 92 nucleotides (in all other species) away from the universally conserved GGGGTTGGTGTA sequence, where the L strand synthesis is initiated. Because of its location, we hypothesize that the GGGCGT sequence is part of the initiation site for the syn- thesis of the H strand 73-mer which would be the first Okazaki fragment synthesized after the L strand is initiated at the GGGGTTGGTGTA sequence (see diagram in Fig. 9, panel A). The second Okazaki fragment would be either the 83-mer or the 138-mer. In contrast to the newly synthesized L strand (6), no residual RNA primer was found associated with the H strand fragments.

More study is needed to determine the mechanism by which synthesis of the two minicircle strands initiates at the GGGGTTGGTGTA and GGGCGT sequences.

Acknowledgments-We thank Kary Thompson for help in prepar- ing this manuscript and Viiu Klein for excellent technical assistance with the S1 nuclease mapping. We thank Barbara Sollner-Webb, Abram Gabriel, Sangram Sisoda, Stephen Desiderio, David Garboczi, Carol Rauch, and Theresa Shapiro for many helpful discussions.

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