ray 1987 si.pdf

PLASMID 17, 177-190 (1987)

REVIEW

Kinetoplast DNA Minicircles: High-Copy-Number Mitochondrial Plasmids’

DAN S. RAY

Molecular Biology Institute and Department o/Biology, University of California. Los Angeles. Calfornia 90024

Received January IS, 1987; revised March 30, 1987

The kinetoplast DNA of trypanosomes is a highly unusual network of catenated DNA circles of two kinds: maxicircles, the equivalent of conventional mitochondrial DNA, and minicircles, high-copy-number mitochondrial plasmids with no known function. Kinetoplast minicircles share many features with bacterial plasmids and represent a novel model system for the study of the mechanisms and regulation of DNA replication in eukaryotic organisms. e 1987 Academic

The relative simplicity of bacterial plasmids has made them popular systems for the study of the regulation of DNA replication. Plasmids often can be sequenced in their en- tirety and can be obtained in large quantities for biochemical analysis. Replication origins and regulatory genes can be defined and studied by both biochemical and genetic means. Even the complex replication appa- ratus of the bacterial chromosome can now be analyzed through the use of small oriC plasmids (Fuller ef al., 1981). A minimal functional origin has been defined by dele- tion analysis and many of the proteins that participate in the replication process have been identified and purified. The earliest steps in the initiation of a round of replication currently are subjects of intensive investigation (Zyskind and Smith, 1986).

The numerous bacterial plasmids have provided a variety of systems with which to probe the various mechanisms of control of DNA replication and partitioning in pro- karyotic cells. Analagous model systems for dissecting the mechanisms regulating the DNA replication cycle and coordinating it with the cell cycle are not as readily available for eukaryotic cells. While viral systems such as adenovirus and SV40 have provided important in vitro replication systems that have allowed the identification of cellular replica-

’ Work in the author’s laboratory was supported by Grant A120080 from the National Institutes of Health.

tion proteins, their replication free of the constraints of the cell cycle limits their use- fulness as a model of normal cell cycle regulation of DNA replication (Challberg and Kelly, 1982; Campbell, 1986).

Two model systems currently are available for the study of the molecular mechanisms coordinating the initiation of DNA replication with the eukaryotic cell cycle. The first is the yeast 2 cc plasmid which replicates under the control of the yeast cell cycle. This system has been reviewed recently (Campbell, 1986) and is not discussed here. Rather, our purpose here is to review our knowledge of a less well-known eukaryotic plasmid that also replicates under the control of the cell cycle. This system is the kinetoplast DNA minicircles of trypanosomes (for earlier reviews see Borst and Hoeijmakers, 1979; Englund et al.. 1982; Stuart, 1983; Simpson, 1986).

Trypanosomes are flagellated protozoa which have a single multilobed mitochon- drion per cell. Within this single mitochon- drion near the base of the flagellum is a disk- shaped body termed a kinetoplast. The kinetoplast contains an enormous network of catenated circular DNA molecules that account for up to about 20% of the total cellular DNA (Fig. 1). The kinetoplast DNA network (kDNA)2 consists of some

r Abbreviations used: LDNA, kinetoplast DNA; UMS, universal minicircle sequence; ORF, open reading frame.

177 0147-619X/87 $3.00 Copyright 8 1987 by Academic Prcs. Inc. All rigJm of reprcduclh in any form -cd.

DAN S. RAY

FIG. 1. Electron micrograph showing the organization of the kDNA network from C. fasciculuta. The micrograph was kindly provided by Christian Sheline.

5000- 1 O,ooO copies of a small circular DNA species (minicircles) and about 20-40 copies of a much larger circular DNA species (maxicircles). Minicircles account for approximately 95% of the kDNA. The maxicircles appear to be homogeneous within a single organism and contain mitochondrial structural genes analagous to those found in other organisms. Minicircles, on the other hand, are only about 1-2 kb in size and different species of trypanosomes vary in regard to their degree of minicircle sequence homo- geneity within a single organism. The unusual organization of kDNA and the intriguing topological problems associated with the replication and segregation of daughter networks have drawn considerable attention over the past 10 to 15 years. In light of the replication of kinetoplast DNA in approximate synchrony with the nuclear S phase

(Cosgrove and Skeen, 1970; Simpson and Braly, 1970) and the replication of minicircles free from the topological constraints of the kDNA network (Englund, 1979) attention has turned to the detailed mechanism of minicircle replication. It is hoped that such studies will lead to experimental approaches to understanding the mechanisms that coordinate DNA synthesis in this organelle with that in the nucleus. In this review I focus on the kDNA minicircles since their sequence organization and mechanisms of replication are understood in far greater detail than those of maxicircles.

CELL CYCLE CONTROL

Tritium thymidine incorporation and autoradiographic analysis of both exponentially growing (Cosgrove and Skeen, 1970)

KINETOPLAST DNA MINICIRCLES 179

and synchronized cells (Simpson and Braly, 1970) indicate that nuclear and kinetoplast S phases occur in approximate synchrony. In Leishmania tarentolae cells synchronized by treatment with hydroxyurea, DNA synthesis begins soon after removal of the drug, reaches a maximum by 1 h, and then de- creases to a low level by 4 h at which time dividing forms start appearing. The most common dividing form contains two kinetoplasts and two nuclei, although in some cases kinetoplast division is completed first, yield- ing cells containing two kinetoplasts and only one nucleus. A second cycle of DNA synthesis occurs at about 7 h followed by a second cycle of cell division at about 9 h. Autoradiographic analysis of individual cells shows clearly a synchronization of kinetoplast and nuclear DNA synthesis during each wave of replication. Similarly, autoradiographic studies of exponentially growing Crithidia fasciculata indicated that the nuclear and kinetoplast S phases are of similar length and occur in approximate synchrony. It is honed that the identification of specific factors involved in the turn on of kinetoplast DNA synthesis, and of minicircle replication in particular, will shed some light on the cell cycle control of DNA synthesis in general.

MINICIRCLE REPLICATION FREE OF THE kDNA NETWORK

Early autoradiographic studies on kDNA replication showed that a short [3H]thymidine pulse label was incorporated primarily into the periphery of networks (Simpson et al., 1974; Simpson and Simpson, 1976). In the shortest pulses the label was localized to two sites at the periphery of the networks and separated by 180”. In longer pulses the silver grains were found around the entire periphery and following a chase the labeled DNA appeared to remain in place as further synthesis expanded the size of the network. After a chase which exceeded one generation time, the silver grains were found to be distributed randomly.

Sedimentation and electron microscopic studies by Englung (1979) suggested that the

silver grains observed in the autoradiographic experiments probably do not represent sites of synthesis but rather mark sites of reattachment of progeny minicircles replicated free of the network. It was found that about 0.4% of the total kDNA is in the form of free minicircles, and that the free minicircles are labeled at very high specific activity after pulse labeling with [3H]thymidine. Es- sentially all of the pulse label could be chased out of the free minicircles. These results together with the observation that the free minicircle population turns over during steady-state labeling suggested that minicircles are replicated free of the network and are then rejoined to the network. Support for this interpretation was provided by the electron microscopic observation that a small fraction of the free minicircles have a Cairns-type structure (Englund, 1979). Some of the structures appeared to have one single-stranded branch similar to the expanded D-loop structures which are intermediates in animal mitochondrial DNA replication. Free minicircles having a Cairns-type structure have also been observed in DNA from kinetoplasts of Trypanosoma cruzi (Brack et al., 1972).

The peripheral labeling of kDNA networks also correlates with a peripheral location of nicked and gapped minicircles in par- tially replicated networks (Englund, 1978). Dye-buoyant density centrifugation frac- tionates networks into form I networks containing about 5000 covalently closed minicircles and form II networks containing about 10,000 minicircles which are nicked or gapped. When cells are pulse labeled with [3H]thymidine, radioactivity is preferentially incorporated into networks having a density intermediate between form I and form II. Electron microscopic examination of these replicating networks revealed a central region of covalently closed minicircles and a peripheral zone of nicked or gapped minicircles. These studies have led to a model of kDNA replication in which covalently closed minicircles are released from the interior of the network, replicated free of the network,

180 DAN S. RAY

and then rejoined to the periphery of the network while still in a nicked or gapped form (Englund et al., 1982). This model invokes a topoisomerase that would randomly release unreplicated (covalently closed) molecules from the network by a breakage and rejoining mechanism. The nicked or gapped progeny minicircles are proposed to be rejoined specifically to the periphery of the network and to be refractory to release by the topoisomerase. This model is shown in Fig. 2 (redrawn from Englund et al., 1982). In addition to accounting for the autoradiographic and electron microscopic results, the model has the feature of providing a mechanism that ensures that each minicircle replicates only once during the cell cycle.

A type II DNA topoisomerase detected in extracts of C. fZz.sciculata appears to have a substrate preference and catalytic properties

FIG. 2. Proposed scheme for minicircle release, replication, and reattachment to the kDNA network. Mini- circles are decatenated (a) from regions (gray) of the network in which ail of the minicircles arc covalently closed. The free minicircles are replicated to give progeny molecules containing nicks and gaps (b). The progeny minicircles are then catenated (c) to the periphery of the network. The area of the network occupied by replicated minicircles is indicated by stippling and in- creases with the extent of replication. Many of the Oka- z&i-like fmgments are not joined together until after reattachment of the minicircles to the network. Nicks and gaps in the origin regions of minicircles are not repaired until the entire network has been replicated. The double-size network contains approximately 10,000 nicked and gapped minicircles and is termed a form II network. Concomitant with division of the network (by unknown mechanisms) or immediately thereaRer, all of the minicircles are completely repaired to give a form 1 network with approximately 5000 covalently closed minicircles. Redrawn from Englund et al. (1982).

consistent with a possible role in minicircle replication (Shlomai and Zadok, 1983). This enzyme was found to efficiently decatenate form I networks while no significant decatenation was observed for networks nicked by a C. fasciculata nicking enzyme (Shlomai and Linial, 1986). In the presence of spermi- dine or a C. Jasciculata double-stranded DNA-binding protein, the same enzyme fractions catalyzed the catenation of pBR322 plasmid DNA. Purification of this enzyme, further characterization of the catenation- decatenation reactions, and immunocyto- logical localization of the enzyme will be required to firmly establish its role in kDNA replication.

An intriguing aspect of the regulation of kDNA replication is the observation that the progeny minicircles appear to remain nicked or gapped up until the division of the network (Englund, 1978). When alI of the minicircles have been replicated, the form II network is double the size of the form I network. The mechanisms by which the network is cleaved into two progeny networks and the minicircles covalently closed are entirely unknown.

SEQUENCE ORGANIZATION OF MINICIRCLES

Minicircles of most trypanosome species are heterogeneous in nucleotide sequence. At one extreme the minicircles of Trypanusoma brucei are about 1 kb in size but show a ki- netic complexity of at least 300 kb (Chen and Donelson, 1980; Jasmer and Stuart, 1986). At the other extreme, the minicircles of Try- panosoma equiperdum are sufficiently homogeneous to allow direct sequencing of network-derived minicircles by the Maxam- Gilbert method (Barrois et al., 1981). Similarly, the major sequence class of minicircles of C. jhsciculata is nearly homogeneous in sequence (Birkenmeyer et al., 1985). Analysis of multiple clones of half- length minicircle fragments showed that sequence variations primarily are the result of base substitutions which occur at a frequency


of approximately 0.3% (Sugisaki and Ray, 1987). Hybridization studies and electron microscopic heteroduplex analysis of the total minicircle populations from strains of C. fasciculata and C. luciliae also indicated the occurrence of extensive segmental rear- rangements of the minicircle sequence (Hoeijmakers and Borst, 1982; Hoeijmakers et al., 1982).

Minicircles from different trypanosome species show very little cross-hybridization (Steiner-t et al., 1976). Sequence analysis of more than a dozen minicircles from six different species (Barrois et al., 198 1; Chen and Donelson, 1980; Ponzi et al., 1984; Kidane et al., 1984; Jasmer and Stuart, 1986; Ma- cina et al., 1986; Sugisaki and Ray, 1987) indicates that the largest sequence conserved among these species is a 1Znucleotide sequence which has been termed the universal minicircle sequence (UMS). As is discussed later, this sequence coincides with the site of RNA-primed initiation of one of the minicircle strands.

A common feature in the sequence organization of minicircles is the presence of a conserved sequence region of 100-200 bp which is present in all minicircles of a given species. This sequence contains the UMS and is found in each minicircle of all sequence classes. Several lines of evidence indicate that this conserved sequence contains origins of replication of both strands of the minicircle. In the case of C. fasciculata, the origin of the L-strand is associated with the UMS while the origin of the H-strand is associated with a 29-bp sequence located about 100 bp away from the UMS (Birkenmeyer and Ray, 1986; Birkenmeyer et al., 1987). A similar sequence in the L. tarentolae minicircle shows conservation of the sequence at 26 of 29 nucleotides (Sugisaki and Ray, 1987). This latter result suggests that the strand of the L. tarentolae minicircle corresponding to the H-strand of C. fasciculata may be initiated by a mechanism similar to that of the C. fasciculata H-strand.

The diversity of minicircle sequences and the extreme complexity of minicircles in

species such as T. brucei has suggested that minicircles do not encode protein products. This view is consistent with the fact that termination codons are frequent in all possible reading frames in all sequenced minicircles. Except for a single result in the literature, the search for minicircle transcripts has been negative. Fouts and Wolstenholme (1979) reported finding a 240nucleotide transcript from the minicircles of Crithidia acanthocephali. However, several laboratories have failed to detect minicircle transcripts by probing Northern blots with high specific activity minicircle probes (Simpson, 1986).

The only other indication that minicircles might encode a protein product was reported by Shlomai and Zadok (1984). They found that fragments from C. fasciculata minicircles could be expressed as tribrid fusion proteins in Escherichia coli in open reading frame (ORF) cloning vectors. Antisera raised against one such protein appeared to react with C. fasciculata antigens both in a West- em blot and in immunofluorescent microscopic localization. This interesting result needs to be confirmed and the cellular antigens purified. The C. fasciculata minicircle sequence (2.5 kb) has been analyzed for ORFs (Sugisaki and Ray, 1987) assuming that TGA codes for tryptophan as has been found for the maxicircles of T. brucei and L. tarentolae (de la Cruz et al., 1984; Hensgens et al., 1984; Payne et al., 1985). Five of the six possible reading frames contain 20 or more termination codons while the remain- ing reading frame contains only 11 termination codons. The largest proteins that could be encoded by these ORFs would contain 176, 104, and 84 amino acids. However, by appropriate RNA splicing, still larger proteins could potentially be encoded by the minicircles. It will be of considerable interest to purify the C. fasciculata antigens identified by Shlomai and Zadok and to compare their amino acid sequences with those pre- dicted from the minicircle ORFs.

An unusual feature of minicircle DNA from most species of trypanosomes is the presence of a sharp bend in the DNA causing

182 DAN S. RAY

it to migrate anamolously slowly in acryl- amide gels (Simpson, 1979; Marini et al., 1982, 1984; Kidane et al., 1984). The presence of a static bend in the DNA is thought to interfere with the snaking of linear DNA fragments through the pores of the gel. Other physical studies including rotational diffu- sion measurements (Marini et al., 1982) and

direct visualization by electron microscopy (Griffith et al., 1986; Ray et al., 1986) sup port a bent structure for these minicircle fragments. The location of the bent helix can be determined relative to specific restriction sites by electron microscopic observation of minicircles cut with various restriction enzymes as shown in Figs. 3a and b. Analysis of

C 2290 2300 2310 2320 2330 CRCRCTCTRR RGCRGRTCCG TRCRCGTTGR CATTTTGRTT TTTGRCTGCG

GTCTGRGRTT TCGTCTRCGC RTCTGCRRCT CTRRRRCTRR RRRCTCRCGC -- 001 I

9 40 2350 2360 2370 2380

TTTTTGG CR TTTTTTGCCC RTTTTTCCCT ToTTCRR TmTTGcG MCCGCT -CGGG TmGGGR RTTTTRRCTT RTTTTRRCGC

Toq I 2390 2400 + 2410 2420 2430

GERTTTTTTR CCRTTTTTGT CGRTTTTTGG GGTRTTTTCG CTGTTTTTTG

CCTRRRRRRT CCTRAARACR GCTECC CCRTBGC GRCRRRRRRC 801 I

2440 b 2450 2460 2470 2480 GCRTTTTTTG CCCRTTTTTC CTTGRTTTTG GGCRCTTTTC CGGCTCCR~

CETRRRRRRC CCtT~t GRRCTRAAAC CCGTCaG CCCGRGGTTT

stu1 2490 2500 2510 4 5

~GTRRcCT CGCCRTTTTC GCCTGCRRTT TTRGGCCTCC TGGCRGGI$~

TTTCRTTGGR CCGCT~G CGGRCCTT~TCCGGRGG RCCGTCCCCC

FIG. 3. Electron micrograph and DNA sequence of the bent helical region of the C.f~c~icu~uta minicircle. Free minicircles isolated from purified kinetoplasts were digested with either Sac11 (a) or XhoI (b) and prepared for microscopy by the aqueous Kleinschmidt procedure. In the unit-length minicircles (2.5 kb) cut by Sacll, the bent helical region mapped at 0.4 1 kb from one end of the molecule. In molecules cleaved with X/WI, the bend was located at I. 12 kb from one end. The DNA sequence of the bent helical region is shown in (c). Tracts of 4-6 dA residues are indicated by solid lines The upper strand is the H-strand and is written in the 5’ to 3’ direction. Modified from Ray ef al. ( 1986).


such fragments from minicircles derived from various species of trypanosomes indicates that the bend in the DNA is an intrinsic property of the nucleotide sequence (Kitchin ec al., 1986; Ray et al., 1986). This property is retained in minicircle fragments cloned in E. coli. DNA sequence analysis of several such fragments suggests that the bend is the consequence of runs of 4-6 A residues separated by 10-l 1 bp (one turn of the DNA helix). The most anomalous fragment identified so far is from C. fmciculuta minicircles (Ntambi et al., 1984). In the major sequence class of these minicircles, the region of the bend contains 19 homopolymeric dA * dT tracts with 16 of the tracts oriented in the same direction (Ray et al., 1986). The nucleotide sequence of this region is shown in Fig. 3c. A similar result was also found for a C. fasciculata cloned minicircle that appears to represent a minor sequence class (Kitchin et al., 1986).

The biological role of the bent segment of the minicircle is unknown. Minicircles from C. fusciculata contain two replication origins located 180” apart (Fig. 4) but only one copy of the bent region (Birkenmeyer and Ray, 1986; Birkenmeyer et al., 1987; Ray et al., 1986; Sugisaki and Ray, 1987). The presence of only a single bend located midway between the two origins on one side of the ring suggests that the bend is not an intrinsic part of an origin of replication. A possible role in the organization of the kDNA network structure has been proposed (Marini et al., 1982; Silver et al., 1986). Some role in direct- ing minicircles to sites of replication or to sites of reattachment on the network periphery has also been suggested (Ray et al., 1986).

MINICIRCLE REPLICATION INTERMEDIATES

The characterization of replication intermediates has provided significant insights into the mechanism of minicircle replication. Because minicircle replication occurs free of the network, the analysis of replication intermediates has focused primarily on

FIG. 4. Physical map of the major sequence class of C. jiiciculata Cf-CI minicircles. The heavy (H) and light (L) strands and their orientations are indicated by arrows. Nucleotide coordinates of several restriction enzyme cleavage sites are shown in parentheses. The region of the minicircle containing the bent helix is shown in black and the replication origins (A and B), two nearly identical conserved sequence regions, are indicated by cross-hatching. Sites of H- and L-strand discontinuities identified in nascent minicircles are indicated by open and filled triangles, respectively.

the pool of free molecules. However, as is discussed later, some of the final steps in the complete replication of minicircles to yield covalently closed circles occurs after reattachment to the network.

Pulse-labeling experiments both in vivo and in an isolated organelle system indicate that one strand (the H-strand) is replicated discontinuously (Kitchin et al., 1984, 1985; Birkenmeyer and Ray, 1986; Birkenmeyer et al., 1987). A major component of pulse-labeled minicircles is a highly gapped circular molecule. The labeled strand in this species is entirely the H-strand. Alkaline gel analysis demonstrated that most of the radioactivity in these molecules was contained in H- strands of less than 0.2 kb. These fragments could not be joined by T4 DNA ligase, and even after filling in gaps with T4 DNA polymerase, many termini remained unligatable.

In the course of the maturation of the H- strand, a half-minicircle-length fragment is observed. The termini of these fragments occur at specific sites located 180” apart on the minicircle map and are contained within the two conserved sequence regions (Birken- meyer and Ray, 1986; Birkenmeyer et al., 1987). In contrast to discontinuities at other

184 DAN S. RAY

sites on the H-strand, these specific discontinuities remain long after most other nicks and gaps are sealed. The repair of the discontinuities in the H-strand is initiated while the minicircle molecules are free but is not completed until after reattachment to the network (Kitchin et al., 1985). Alkaline gel analysis of newly replicated minicircles after their rejoining to the network shows that the aver- age fragment size of the H-strand is greater than that in nascent free minicircles. How- ever, in both free and network-bound minicircles, the nicks and gaps are sealed rapidly except for the specific discontinuities in the conserved sequence regions (Birkenmeyer and Ray, 1986).

Precise localization and characterization of the specific discontinuities in the H-strand place them within a 29-bp sequence which is highly conserved in major and minor sequence classes of C. fasciculata minicircles (Birkenmeyer et al., 1987; Sugisaki and Ray, 1987). These specific discontinuities appear to be nicks having juxtaposed 3’-hydroxyl and S-phosphoryl termini since they can be joined by ligation. The exact site of each nick can vary by a few nucleotides but always lies at one of the sites indicated by arrows (Fig. 5a). T4 ligase can seal all of the H-strand nicks while E. coli ligase can seal only the nicks indicated by the 3’-most arrow. Since only T4 ligase is capable of joining the S- phosphoryl terminus of RNA to a juxtaposed 3’-hydroxyl terminus of DNA, some of the joinings appear to be DNA-RNA junctions. However, the resistance to alkaline hydrolysis of the bonds formed only by T4 hgase suggests that the terminal nucleotides may be modified ribonucleotides. The fact that the nick that lies furthest 3’ in the sequence is a DNA-DNA junction based on its closure by E. coli ligase suggests that the series of H- strand nicks may be the result of a sequential 5’ to 3’ excision of modified nucleotides involved in priming of H-strand synthesis.

Newly replicated L-strands of the C. fisciculata minicircles are found largely in open circular molecules. Analysis of these molecules on denaturing gels indicates that the

a)

5.-T’CTCGGG’TAGGGGCGTTCTC-H 3’-AGAG CCCATCCCCGCAAGAC-1.

b) t ‘? 2p ?

TCATTCCTOGOTCCOGG- II MoGACCCM3GCCC- I.

C) B

d)

Nick

S.--AA GGC3TTffiK.T TACAC- , ‘ -TT$!rT TGTG- ‘@@*

FIG. 5. DNA sequences at sites of specific discontinui-

ties in newly synthesized minicircles. (a) Sequence at the

sites of H-strand discontinuities in both the A and the B

regions of C. ji~scicula~a minicircles. Sites at which nicks

are found more often are indicated by larger arrows. (b,

c) Sequence at sites of nicks and gaps in the L-strand of

C’. /hscictclufu minicircles. The 12-nucleotide universal

minicircle sequence is enclosed in a box. The L-strand

contains a single nick or gap located with approximately

equal frequency in the A and B regions. Most gaps span

5-6 nucleotides but, for simplicity, only molecules con-

taining a S-nucleotide gap are shown. In molecules con-

taming a nick rather than a gap, the nick site is at the

position shown by the vertical arrow. Nucleotides re-

leased by alkaline treatment are indicated by a bracket.

(d) Sequence at the gap in the nascent strand of T. eqqui-

perdum. One or two Sterminal rA residues have been

detected in some molecules on the 3’ side of the gap.

Data are from Birkenmeyer ef a!. (1987). Ntambi and

Englund ( 1985). and Ntambi et al. ( 1986).

newly synthesized L-strand contains only a single discontinuity (Kitchin et al., 1985; Birkenmeyer and Ray, 1986). Analysis of both free and network-associated nascent minicircles in vivo indicates that the daughter molecules having a newly synthesized L- strand become reattached to the network sig- nificantly faster than do those having a newly synthesized H-strand. By cleavage of these open circular molecules with single-cut restriction enzymes and alkaline gel analysis, the L-strand discontinuities have been mapped. Unexpectedly, the newly synthesized L-strands of C. fasciculata minicircles synthesized in the organelle system were found to contain a single discontinuity at ei-


ther of two sites located 180” apart (Birken- meyer and Ray, 1986). The single discontinuity was found to be present at either site with about the same frequency. In contrast to the H-strand, no molecules were detected in which discontinuities were present at both sites simultaneously. These sites of L-strand discontinuity were found to be located about 100 bp away from the identified H-strand sites of discontinuity as shown in Fig. 4. Both the H-strand and the L-strand sites of discontinuity lie within the two conserved sequence regions on opposite sides of the minicircle.

The C. fusciculuta L-strand discontinuities can be either simple DNA-DNA junctions, sealable by either T4 or E. coli DNA ligases, or small gaps of 4-6 nucleotides (Birken- meyer et al., 1987). The L-strand nicks occur at a single site within the universal minicircle sequence in either conserved region as shown in Figs. 5b and c. The universal minicircle sequence overlaps the 5’ terminus on the 3’ side of the gap. In the gapped molecules up to 6 nucleotides can be removed from the 5’ terminus by alkaline hydrolysis, suggesting that L-strand synthesis is initiated by an RNA primer of at least 6 nucleotides. The gap could result from partial excision of a more extensive RNA primer or from chain termination prior to encountering the 5’ terminus of the “primer.” Surprisingly, the A residue located on the 5’ side of the nick site is not hydrolyzed from the terminus of gapped molecules. The resistance to alkaline hydrolysis may be due to that nucleotide being a modified ribonucleotide as implied for the H-strand primer. A less likely alter- native is that the primer excision mechanism results in the removal of the first deoxyri- bonucleotide. The nicked molecules may re- flect a later step in the processing of the primer prior to ligation. The localization of the L-strand discontinuities within the universal minicircle sequence suggests that this sequence may be involved in the initiation of the L-strand primer and/or in the transition from RNA to DNA synthesis.

In T. equiperdum, which has only a single conserved sequence region per minicircle,

some of the newly replicated minicircles contain a single gap of about 8- 10 nucleotides (Ntambi and Englund, 1985). The 5’ terminus on the 3’ side of the gap in molecules lacking terminal ribonucleotides is within the universal minicircle sequence and corresponds exactly with the nick sites found in C. fusciculata minicircles (Fig. 5d). Some molecules in the population have also been found to contain one or two terminal rA residues (Ntambi ef al., 1986). In some cases the penultimate A is resistant to removal by al- kali, indicating the presence of either a de- oxynucleotide or a modified ribonucleotide. The finding of gapped molecules reattached to the network and having terminal ribonucleotides indicates that the complete excision of RNA primers, gap filling, and ligation to yield covalently closed minicircles all can occur after reattachment. In fact, there ap pears to be some mechanism that assures that progeny minicircles are not covalently closed until after the network divides (Eng- lund, 1978).

MODELS OF MINICIRCLE REPLICATION

The studies presented here allow us to for- mulate an overall mechanism of minicircle replication. Although some important details still are missing, it appears that minicircles replicate by mechanisms similar to those of some bacterial plasmids.

The Cairns-type replication intermediates observed by Englund (1979) possibly represent early intermediates in the replication process. The various nicked and gapped molecules that have been isolated so far all represent later intermediates. While these latter molecules provide significant insights into the mechanisms by which they were pro- duced, they are not sufficient to define the precise replication mechanism. In particular, the timing of second-strand synthesis is uncertain. On the one hand, rare single-strand circles have been observed in the pool of free minicircles. If these molecules represent products of replication, then the process may be entirely asymmetric as in the case of sin-

186 DAN S. RAY

gle-stranded bacteriophages where one strand is completely replicated before initiation of synthesis of the complementary strand. A difficulty of this model of replication is that there would be no need for synthesis of the second strand to be discontinuous. The observation that synthesis of the C. fisciculata L-strand appears to be continuous while that of the H-strand appears to be highly discontinuous suggests that both strands undergo replication simultaneously, with the L-strand being the leading strand and the H-strand being the lagging strand. The observation that one of the branches of the Cairns-type structures appears single stranded has been suggested to resemble the “expanded D-loop” structures involved in the replication of animal mtDNA. The D- loop mechanism of replication of mtDNA involves the RNA-primed initiation of one strand of the mtDNA to form a triplex D- loop structure as an early intermediate (Clayton, 1982). Synthesis of this strand continues around the ring until the origin of the complementary strand is exposed in a single stranded form and initiation occurs. This latter initiation involves RNA priming by a mitochondrial DNA primase at a specific site on the single-stranded template (Wong and Clayton, 1985).

Such a model for initiation at the Crithidia A and B origins is shown in Fig. 6. In this case, the sites of initiation of the two strands

3 v 5 L 5’ A 3 H

4

3’ f, 5’ 5’ 3

+S=A--5 3

FIG. 6. D-loop model of initiation within the A and B origins of the C. fusciculufa minicircle. The open and closed triangles indicate the origins of the H- and L- strands shown in Fig. 4. RNA priming is indicated by sawtoothed lines. The L-strand is initiated first and is extended continuously while displacing the parental L- strand. After the L-strand progresses beyond the H- strand origin, the H-strand is initiated on a single- stranded template and is extended in the opposite direction.

FIG. 7. Model for replication of C. /iiciculuta minicircle DNA. Site-specific priming of nascent DNA strands within the A and B regions is indicated by sawtoothed lines as in Fig. 6. Random priming of Okazaki- like fragments of the H-strand are indicated by small open circles. A detailed description of the various steps in the model is provided in the text. From Birkenmeyer er al. (1987).

are separated by only about 100 bp within each origin. This D-loop model for minicircle replication is attractive because it could account for the observed site-specific discontinuities and the highly discontinuous nature of H-strand synthesis. In the case of minicircles such as those from C. fasciculata where duplicate origins are present on opposite sides of the minicircle, the model becomes analogous to the D-loop model proposed for unicircular dimeric mouse mtDNA (Clay- ton, 1982). A replication model based on this mechanism of initiation within each origin is shown in Fig. 7. This model illustrates replication from a single origin (B). Similar structures would result from initiation at the opposite origin (A). Both origins appear to be used with equal frequency since L-strand nicks and gaps are found within the A and B regions about equally often (Birkenmeyer and Ray, unpublished).

In this model initiation of the L-strand of C. jizciculata can occur at either of the two L-strand origins overlapping the universal minicircle sequence. However, unlike the H- strand, the apparent absence of half-minicircle-length L-strands suggests that once initiation occurs at one of the L-strand origins,


synthesis of a full-length strand occurs before initiation can take place at the second origin. Alternatively, some specific mechanism may exist that prevents initiation at the second origin. Elongation of the nascent L-strand displaces the parental L-strand and reveals the H-strand origin in a single stranded form after extending the L-strand by more than 100 nucleotides. H-strand synthesis may then be initiated by a mitochondrial DNA primase. However, upon elongation of the H-strand by about 100 nucleotides to the site of initiation of the L-strand, further synthesis of the H-strand may be blocked due to the duplex nature of the template at that point. As L-strand synthesis proceeds around the ring, the displaced parental L-strand can serve as template for multiple, possibly random secondary initiations of the H-strand. After L-strand synthesis proceeds beyond the second primary origin of the H-strand, a second specific priming appears to occur. The mechanism of initiation of the Okazaki-like fragments in between the primary origins may differ from those at the two primary origins of H-strand synthesis since discontinuities at the latter two sites are refractory to covalent closure whereas the other H-strand discontinuities are joined rapidly both in vivo (Kitchin et al., 1984, 1985) and in the organelle system (Birkenmeyer and Ray, 1986; Birkenmeyer et al., 1987). As the L-strand nears completion the two daughter molecules segregate, giving rise to progeny of two different kinds: molecules having a circular parental H-strand and a nearly unit-length nascent L-strand, and molecules having a circular parental L-strand and a highly discontinuous nascent H-strand. Joining of the Okazaki-like fragments of the H-strand con- verts the latter species to a form having half- length H-strands. The gap filling and joining reactions can take place after rejoining of the daughter minicircles to the network. The final steps in the maturation of both the H- and the L-strands involves removal of the primers at the primary origins, gap filling and ligation. It will be of interest to see what role the putative modified nucleotides in the

RNA primers might play in blocking these final steps until the network is completely replicated and divided.

REPLICATION ENZYMES

The replication model proposed here invokes certain specific enzymes in the minicircle replication mechanism. In addition to the obvious need for one or more DNA polymerases, as many as two or three priming enzymes may be required. These include enzymes for priming L-strand initiation on the duplex minicircle, priming at the H-strand primary origins on single-stranded tem- plates, and priming of the Okazaki-like fragments of the H-strand. If this possible diversity of priming enzymes seems excessive, one need only recall the multiple priming mechanisms encoded in the E. coli genome (Kom- berg, 1980). These range from the simple priming mechanism for phage M 13 that uti- lizes only the RNA polymerase holoenzyme and the SSB protein to the complex mechanism for phage 4X174 that requires a complex multiprotein “primosome.” In addition to a primase, this latter complex consists of some 20 polypeptides with a total mass near lo6 Da. Still other enzymes that will be required include activities for unwinding DNA strands, altering topology, processing RNA primers, filling gaps, and. ligating nicks.

Multiple DNA polymerases have been detected in whole-cell extracts of C. fusciculutu (Holmes ef al., 1984). Two of the enzymes appear to be similar to the usual (Y- and /3- type enzymes of mammalian cells (Korn- berg, 1980). However, there are significant differences and the identification of these activities has to be considered tentative. The range of properties of mtDNA polymerases is so wide that enzymatic criteria for identification of an mtDNA polymerase are even more uncertain (Bolden et al., 1977; Yama- guchi et al., 1980; Wernette and Kaguni, 1986). In trypanosomes this identification ultimately may depend on immunofluorescent localization of specific polymerases using antibodies prepared against homogeneous enzymes.

188 DAN S. RAY

Two DNA topoisomerases have been purified from C. fksciculata: an ATPdependent type II enzyme (Shlomai and Zadok, 1983) and an ATP-independent type I enzyme (Melendy and Ray, 1987). Although the cellular localization of the type II enzyme is unknown, the specificities of the enzyme suggest that it could play a role in the release of minicircles from the network and the rejoining of newly replicated minicircles to the network. Interestingly, network-associated covalently closed minicircles are released efficiently from the network by the type II enzyme whereas the release of network-associated minicircles nicked by a C. fasciculata nicking enzyme is inhibited (Shlomai and Linial, 1986). However, no reduction in the rate of minicircle release was observed for networks nicked randomly by DNAse I. It has been suggested that such specific nicks in the L-strand of newly replicated minicircles might be a signal for the discrimination between replicated and unreplicated minicircles to ensure that each minicircle replicates only once during the cell cycle.

The type I topoisomerase is a highly abun- dant enzyme in whole-cell extracts of C. J%- ciculata. Homogeneous enzyme has been used to raise antibodies and to localize the enzyme by double-antibody immunofluores- cence techniques. This particular topoisomerase appears not to be involved in minicircle replication since it is largely or entirely localized in the nucleus (Melendy and Ray, 1987).

PROSPECTS FOR THE FUTURE

The unusual network structure of kDNA has attracted considerable attention over the past 10 years. The enzymatic machinery re- sponsible for the precise cleavage of the fully duplicated network and for the subsequent signaling of the covalent closure of the progeny minicircles surely will be exciting and rewarding areas of investigation in the future. The possible roles of bent helical regions of the minicircles in the organization of minicircles in the condensed network or in

aspects of network duplication remain to be elucidated.

The question of whether minicircles encode protein products still is controversial. If so, these proteins need to be isolated and their functions determined. If not, what al- ternative role do the minicircles play in the biology of these organisms?

The finding that minicircles replicate free of the network has focused attention recently on the sequence organization and replication mechanisms of the minicircles. The isolation of replication intermediates, the identification of specific origins of replication, and the association of the universal minicircle sequence with the RNA-primed initiation of one strand represent important steps toward defining the minicircle replication mechanism. The development of an in vitro system for minicircle replication and the isolation of the numerous proteins required for minicircle replication will be essential for revealing the details of this mechanism. In view of the coordination of kDNA replication with the nuclear S phase, a possible reward of this substantial effort could be the opening of an avenue of approach to the question of the biochemical mechanisms involved in coordinating DNA replication with the cell cycle. While individual minicircles replicate at various times during S phase, some specific mechanism must coordinate minicircle rep lication with that of the maxicircles and with the nuclear S phase.

The full realization of the potential of the minicircle system for addressing general questions regarding eukaryotic DNA replication mechanisms and their control would be accelerated greatly by the development of genetic and recombinant DNA techniques to complement the biochemical approaches. There currently is no reliable method of DNA transformation of trypanosomes, nor are there suitable plasmid vectors. Intensive efforts in these areas could be highly rewarding.

The progress in understanding the structure and replication of kDNA minicircles has been enormous. Whereas the kinetoplast


network is a highly unusual structure, the minicircles that constitute most of the network share many features with bacterial plasmids. Hopefully, the study of these novel mitochondrial plasmids will lead to a better understanding of basic cellular control mechanisms.

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