RNA primer removal and gap filling on a model minicircle replication intermediate

Hines, Jane C.; Engel, Michele L.; Zhao, Hui

doi:10.1016/s0166-6851(01)00272-9

Cited by 15 publications

(19 citation statements)

References 21 publications

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“…Here they encounter SSE1 (10), an enzyme with RNase H activity that can remove RNA primers (11), and DNA polymerase ␤ (12), which presumably fills in most of the gaps between Okazaki fragments. Once this processing takes place, the newly replicated minicircles are reattached to the periphery of the kDNA network by a topoisomerase II (13) that also localizes to the antipodal sites (14).…”

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Trypanosoma brucei Has Two Distinct Mitochondrial DNA Polymerase β Enzymes

Saxowsky¹,

Choudhary²,

Klingbeil³

et al. 2003

Journal of Biological Chemistry

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In higher eukaryotes, DNA polymerase (pol) ␤ resides in the nucleus and participates primarily in DNA repair. The DNA polymerase ␤ from the trypanosomatid Crithidia fasciculata, however, was the first mitochondrial enzyme of this type described. Upon searching the nearly completed genome data base of the related parasite Trypanosoma brucei, we discovered genes for two pol ␤-like proteins. One is ϳ70% identical to the C. fasciculata pol ␤ and is likely the homolog of this enzyme. The other, although ϳ30% identical within the polymerase region, has unusual structural features including a short C-terminal tail and a long N-terminal extension rich in prolines, alanines, and lysines. Both proteins, when expressed recombinantly, are active as DNA polymerases and deoxyribose phosphate lyases, but their polymerase activity optima differ with respect to pH and KCl and MgCl 2 concentrations. Remarkably, green fluorescent protein fusion proteins and immunofluorescence demonstrate that both are mitochondrial, but their locations with respect to the mitochondrial DNA (kinetoplast DNA network) in this organism are strikingly different.

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confidence: 99%

Trypanosoma brucei Has Two Distinct Mitochondrial DNA Polymerase β Enzymes

Saxowsky¹,

Choudhary²,

Klingbeil³

et al. 2003

Journal of Biological Chemistry

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“…Topoisomerase II immunofluorescence is slightly different, showing strong fluorescence at the antipodal sites during S phase and diminishing, but not disappearing completely, during cytokinesis. By using purified recombinant proteins and a model substrate, SSE1 and polymerase ␤ were shown to be capable of RNA primer removal and gap filling to yield a ligatable substrate (27).…”

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confidence: 99%

Mitochondrial DNA ligase in Crithidia fasciculata

Sinha

Hines

Downey

2004

Proc. Natl. Acad. Sci. U.S.A.

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Kinetoplast DNA (kDNA), the form of mitochondrial DNA in trypanosomatids, consists of thousands of interlocked circular DNAs organized into a compact disk structure. A type II DNA topoisomerase, a DNA polymerase ␤, and a structure-specific endonuclease have been localized to antipodal sites flanking the kDNA disk along with nascent DNA minicircles. We have cloned a gene (LIG k) encoding a mitochondrial DNA ligase in the trypanosomatid Crithidia fasciculata, and we show that an epitope-tagged form of the ligase colocalizes with the other replication proteins at the antipodal sites and also at the two faces of the kDNA disk. DNA LIG k becomes adenylated in reactions with ATP, and the adenylate moiety is removed by incubation with pyrophosphate or nicked DNA. The ligase interacts physically with the ␤ polymerase and is proposed to be involved in the repair of gaps in the newly synthesized minicircles. In yeast and mammals, a single gene encodes both nuclear and mitochondrial forms of DNA ligase. The LIG K protein sequence has low similarity to mitochondrial DNA ligases in other eukaryotes and is distinct from the C. fasciculata nuclear DNA ligase (LIG I).T he mitochondrial DNA of trypanosomatid protozoa (kinetoplast DNA, or kDNA) consists of two forms of topologically interlocked circular DNAs, minicircles, and maxicircles (for recent reviews of kDNA structure and replication, see refs. 1-3). The kDNA of the trypanosomatid Crithidia fasciculata contains 5,000 minicircles of 2.5 kb each and 20-30 maxicircles of 37 kb each. The maxicircles encode genes for mitochondrial proteins and ribosomal RNAs. Minicircles encode small guide RNAs that serve as templates for RNA editing of maxicircle transcripts (4). In vivo, the kDNA exists as a condensed disk structure 1 m in diameter and Ϸ0.4 m thick. Each minicircle in the kDNA disk is linked to three other minicircles on average and is relaxed, unlike most other circular DNAs in nature, which are negatively supercoiled (5). Electron microscopic observation of sections through the kDNA disk shows fibers aligned parallel to the axis of the disk. This observation together with the thickness of the disk corresponding to approximately one half of the minicircle circumference (6) suggests that individual minicircles may have a topology like that of a rubber band stretched taut from opposite sides of the circle (7). Maxicircles appear to be present throughout the kDNA and exist as a catenated network within the overall network (8). The kDNA isolated from nonreplicating C. fasciculata is a planar elliptical network of interlocked circles with dimensions of Ϸ10 by 15 m.The kDNA disk is always located at the base of the flagellum, with the flagellum perpendicular to the face of the disk. Recent studies (9) have shown that in trypanosomes the kDNA disk is connected physically to the flagellar basal bodies and is segregated by them at cell division. The attachment was found to involve three components that penetrate the inner and outer mitochondrial membranes to link the kDNA physically ...

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“…The subject of this paper is the SSE1, a mitochondrial enzyme discovered in C. fasciculata by Engel and Ray (15)(16)(17). As mentioned above, this enzyme localizes to the antipodal sites.…”

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confidence: 89%

“…Within these sites, subsequent steps of replication occur, catalyzed by enzymes specifically localized therein. These reactions are thought to include primer removal by a structure-specific endonuclease-I (SSE1) (15)(16)(17) and repair of some (but not all) of the minicircle gaps by a DNA polymerase ␤ (18,19) and a DNA ligase (20,21). Finally, the progeny minicircles, still containing at least one nick or gap, are attached to the edge of the kDNA network by an antipodal topoisomerase II (22,23).…”

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Effects of RNA Interference of Trypanosoma brucei Structure-specific Endonuclease-I on Kinetoplast DNA Replication

Liu

Motyka

Englund

2005

Journal of Biological Chemistry

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Kinetoplast DNA, the mitochondrial DNA of trypanosomatid protozoa, is a network containing several thousand topologically interlocked DNA minicircles. Kinetoplast DNA synthesis involves release of minicircles from the network, replication of the free minicircles, and reattachment of the progeny back onto the network. One enzyme involved in this process is structure-specific endonuclease-I. This enzyme, originally purified from Crithidia fasciculata, has been proposed to remove minicircle replication primers (Engel, M. L., and Ray, D. S. (1998) Nucleic Acids Res. 26, 4773-4778). We have studied the structure-specific endonuclease-I homolog from Trypanosoma brucei, showing it to be localized in the antipodal sites flanking the kinetoplast DNA disk, as previously shown in C. fasciculata. RNA interference of structure-specific endonuclease-I caused persistence of a single ribonucleotide at the 5 end of both the leading strand and at least the first Okazaki fragment in network minicircles, demonstrating that this enzyme in fact functions in primer removal. Probably because of the persistence of primers, RNA interference also impeded the reattachment of newly replicated free minicircles to the network and caused a delay in kinetoplast DNA segregation. These effects ultimately led to shrinkage and loss of the kinetoplast DNA network and cessation of growth of the cell.Trypanosoma brucei is a protozoan parasite that causes sleeping sickness in humans and similar diseases in livestock. Trypanosomes, being one of the earliest branching eukaryotes, have unusual biological properties. Their mitochondrial DNA, known as kinetoplast DNA (kDNA), 2 is one of their most amazing features (see Refs. 1-3 for reviews on kDNA). kDNA is a giant network consisting of topologically interlocked DNA rings. Within the cell, the network is condensed into a disk-shaped structure located within a distended portion of the mitochondrial matrix adjacent to the flagellar basal body. There is a trans-membrane filament system linking the kDNA network to the basal body that facilitates segregation of daughter networks following replication (4, 5).The T. brucei kDNA network contains several thousand minicircles that are heterogeneous in sequence (each 1 kb) and a few dozen maxicircles (each 23 kb). Maxicircles, similar to mitochondrial DNAs of higher organisms, encode rRNAs and subunits of respiratory complexes. Minicircles encode small guide RNAs that control the editing of maxicircle transcripts. Editing is a remarkable process involving the addition or deletion of uridylate residues at specific sites in the transcript to form functional mRNA (reviewed in Refs. 6 and 7).kDNA replication has been studied in T. brucei and also in the related parasite Crithidia fasciculata (reviewed in Refs. 1, 3, and 8). In both species, kDNA synthesis occurs in approximate synchrony with nuclear DNA replication, but division of the kinetoplast occurs prior to that of the nucleus (9, 10). Prior to kDNA replication, during the G 1 phase of the cell cycle, all minicircl...

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RNA primer removal and gap filling on a model minicircle replication intermediate

Cited by 15 publications

References 21 publications

Trypanosoma brucei Has Two Distinct Mitochondrial DNA Polymerase β Enzymes

Trypanosoma brucei Has Two Distinct Mitochondrial DNA Polymerase β Enzymes

Mitochondrial DNA ligase in Crithidia fasciculata

Effects of RNA Interference of Trypanosoma brucei Structure-specific Endonuclease-I on Kinetoplast DNA Replication

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