A trypanosomal CCHC-type zinc finger protein which binds the conserved universal sequence of kinetoplast DNA minicircles: isolation and analysis of the complete cDNA from Crithidia fasciculata.

Abeliovich, Hagai; Tzfati, Yehuda; Shlomai, Joseph

doi:10.1128/mcb.13.12.7766

Cited by 36 publications

(28 citation statements)

References 51 publications

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“…The apparent protomer mass, as measured by SDS-polyacrylamide gel electrophoresis, is similar to the one calculated on the basis of the UMSBP encoding cDNA (18). Gel filtration data yielded a Stokes radius of 28 Å, as calculated by the method of Siegel and Monty (34) (Fig.…”

Section: Purification Of Crithidia Fasciculata Umsbp-mentioning

confidence: 51%

“…1). The partial sequencing of the shorter polypeptide chain by Edman degradation has revealed the absence of 11 amino acid residues at the protein N terminus compared with the sequence of the cDNA ORF (18). The identity of the longer polypeptide was verified by peptide mapping of the two polypeptide chains (not shown).…”

Section: Purification Of Crithidia Fasciculata Umsbp-mentioning

confidence: 99%

“…We have previously reported on the recognition of UMS by a unique sequence-specific single-stranded DNA binding protein from C. fasciculata (17) and on the isolation and analysis of the UMSBP-encoding cDNA (18). The amino acid sequence of the polypeptide, predicted from the cDNA, is 116 residues long and contains five Cys-X 2 -Cys-X 4 -His-X 4 -Cys (CCHC)-type zinc finger motifs.…”

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Universal Minicircle Sequence Binding Protein, a CCHC-type Zinc Finger Protein That Binds the Universal Minicircle Sequence of Trypanosomatids

Tzfati

Abeliovich

Avrahami

et al. 1995

Journal of Biological Chemistry

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Replication of kinetoplast DNA minicircles of trypanosomatids initiates at a conserved 12-nucleotide sequence, termed the universal minicircle sequence (UMS, 5-GGGGTTGGTGTA-3). A single-stranded nucleic acid binding protein that binds specifically to this origin-associated sequence was purified to apparent homogeneity from Crithidia fasciculata cell extracts. This UMS-binding protein (UMSBP) is a dimer of 27.4 kDa with a 13.7-kDa protomer. UMSBP binds single-stranded DNA as well as single-stranded RNA but not doublestranded or four-stranded DNA structures. Stoichiometry analysis indicates the binding of UMSBP as a protein dimer to the UMS site. The five CCHC-type zinc finger motifs of UMSBP, predicted from its cDNA sequence, are similar to the CCHC motifs found in retroviral Gag polyproteins. The remarkable conservation of this motif in a family of proteins found in eukaryotic organisms from yeast and protozoa to mammals is discussed.Kinetoplast DNA (kDNA) 1 is a unique extrachromosomal DNA network found in the single mitochondrion of parasitic flagellated protozoa of the family Trypanosomatidae. In Crithidia fasciculata, kDNA consists of about 5,000 DNA minicircles (2.5 kilobase pairs each) and about 50 DNA maxicircles (37 kilobase pairs each) interlocked topologically to form a huge DNA network (for review, see Refs. 1-3). Minicircles are heterogeneous in their nucleotide sequence but contain two short sequences, 70 -100 base pairs apart, that are conserved in all the species studied so far: the dodecamer sequence known as universal minicircle sequence (UMS), 5Ј-GGGGTTGGTGTA-3Ј, and the hexamer sequence 5Ј-ACGCCC-3Ј.On the basis of in vivo observations, Englund and co-workers (4 -8) have described the replication of kDNA minicircles as a process in which individual minicircles are detached from the central zone of the disc-shaped network, replicated, and reattached to the periphery of the disc. The network increases in size until it doubles and then divides and segregates into two daughter networks. Extensive studies of minicircle replication intermediates (9 -16) have suggested that replication begins at the UMS site with synthesis of an RNA primer and proceeds by continuous elongation of the leading light strand (L-strand). A single gap of 6 -10 nucleotides remains in the newly synthesized light strand at the UMS site (13) and is repaired only after replication of the minicircles and their reattachment to the network have been completed (8). Discontinuous synthesis of the lagging heavy strand (H-strand) starts when its origin, containing the conserved hexamer sequence, is exposed by the advancing replication fork. Highly gapped and nicked nascent H-strands are generated.We have previously reported on the recognition of UMS by a unique sequence-specific single-stranded DNA binding protein from C. fasciculata (17) and on the isolation and analysis of the UMSBP-encoding cDNA (18). The amino acid sequence of the polypeptide, predicted from the cDNA, is 116 residues long and contains five Cys-X 2 -Cys-X 4 -His-...

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Section: Purification Of Crithidia Fasciculata Umsbp-mentioning

confidence: 51%

Section: Purification Of Crithidia Fasciculata Umsbp-mentioning

confidence: 99%

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confidence: 99%

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Universal Minicircle Sequence Binding Protein, a CCHC-type Zinc Finger Protein That Binds the Universal Minicircle Sequence of Trypanosomatids

Tzfati

Abeliovich

Avrahami

et al. 1995

Journal of Biological Chemistry

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“…Some kDNA binding proteins are localized to the whole kinetoplast network, whereas others such as a DNA primase and a minicircle binding protein localize to specific zones (Abeliovich et al, 1993;Hines and Ray, 1998;Johnson and Englund, 1998;Abu-Elneel et al, 1999, 2001). The unilateral filaments may provide the basis for a structural anisometry plus a focused matrix necessary for ordered enzymatic functions required for replication of the network.…”

Section: Molecular Biology Of the Cell 1776mentioning

confidence: 99%

A High-OrderTrans-Membrane Structural Linkage Is Responsible for Mitochondrial Genome Positioning and Segregation by Flagellar Basal Bodies in Trypanosomes

Ogbadoyi

Robinson

Gull

2003

MBoC

234

279

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In trypanosomes, the large mitochondrial genome within the kinetoplast is physically connected to the flagellar basal bodies and is segregated by them during cell growth. The structural linkage enabling these phenomena is unknown. We have developed novel extraction/fixation protocols to characterize the links involved in kinetoplast-flagellum attachment and segregation. We show that three specific components comprise a structure that we have termed the tripartite attachment complex (TAC). The TAC involves a set of filaments linking the basal bodies to a zone of differentiated outer and inner mitochondrial membranes and a further set of intramitochondrial filaments linking the inner face of the differentiated membrane zone to the kinetoplast. The TAC and flagellum-kinetoplast DNA connections are sustained throughout the cell cycle and are replicated and remodeled during the periodic kinetoplast DNA S phase. This understanding of the high-order trans-membrane linkage provides an explanation for the spatial position of the trypanosome mitochondrial genome and its mechanism of segregation. Moreover, the architecture of the TAC suggests that it may also function in providing a structural and vectorial role during replication of this catenated mass of mitochondrial DNA. We suggest that this complex may represent an extreme form of a more generally occurring mitochondrion/cytoskeleton interaction. INTRODUCTIONThe African trypanosome Trypanosoma brucei belongs to a large order of pathogenic and free-living flagellated protozoa designated the kinetoplastida. The majority of these organisms possess a single mitochondrion containing a structural mass of proteins and catenated circular DNA molecules, the kinetoplast. The kinetoplast is essential for survival and cyclical transmission of the parasite between mammalian host and tsetse fly. Early light microscope descriptions of trypanosomes recognized the precise location of the kinetoplast in proximity to the base of the flagellum (Robertson, 1912(Robertson, , 1913. The positions of the flagellum and kinetoplast have become the central features in classifying the different life cycle stages of kinetoplastids (Hoare and Wallace, 1966;Vickerman, 1976). Electron microscopy revealed the kinetoplast to be a highly complex disk-shaped structure located within a distended portion of the mitochondrial matrix adjacent to the flagellum basal bodies (Vickerman, 1973). The T. brucei kinetoplast consists of multiple copies of topologically interlocked circular DNA molecules termed maxicircles and minicircles, along with specific kinetoplast proteins. The maxicircle composition of the T. brucei network is 25-50 copies and represents 10% of the network mass (each maxicircle is 20 kb and all have identical DNA sequence). Maxicircles encode the mitochondrial proteins and some guide RNAs (gRNAs), whereas the minicircles are present in several thousand copies (1 kb in length), are heterogeneous in sequence, and encode gRNAs. The gRNAs are essential for the RNA editing process whereby the maxic...

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“…The protein, which contains five tandemly arranged CCHC-type zinc-finger motifs, has been purified from cell extracts, and its encoding gene and genomic locus were cloned and analyzed (4)(5)(6)(7). Genes encoding orthologous proteins have been identified in other trypanosomatid species [ref.…”

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confidence: 99%

Mitochondrial origin-binding protein UMSBP mediates DNA replication and segregation in trypanosomes

Milman

Motyka

Englund

et al. 2007

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

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Kinetoplast DNA (kDNA) is the remarkable mitochondrial genome of trypanosomatids. Its major components are several thousands of topologically linked DNA minicircles, whose replication origins are bound by the universal minicircle sequence-binding protein (UMSBP). The cellular function of UMSBP has been studied in Trypanosoma brucei by using RNAi analysis. Silencing of the trypanosomal UMSBP genes resulted in remarkable effects on the trypanosome cell cycle. It significantly inhibited the initiation of minicircle replication, blocked nuclear DNA division, and impaired the segregation of the kDNA network and the flagellar basal body, resulting in growth arrest. These observations, revealing the function of UMSBP in kDNA replication initiation and segregation as well as in mitochondrial and nuclear division, imply a potential role for UMSBP in linking kDNA replication and segregation to the nuclear S-phase control during the trypanosome cell cycle.kDNA replication initiation ͉ kDNA segregation ͉ kinetoplast DNA ͉ trypanosomes cell cycle control ͉ universal minicircle sequence-binding protein K inetoplast DNA (kDNA) is a unique extrachromosomal DNA, found in the single mitochondrion of trypanosomatids. It consists, in the different species, of a few dozen maxicircles (20-40 kb each) and a few thousand minicircles (0.5-10 kb each), that are interlocked topologically into a DNA network (1, 2). Minicircles in most trypanosomatid species contain two short sequences that are associated with replication initiation: a dodecamer, designated the universal minicircle sequence (UMS), and a hexamer. These sequences have been mapped to the replication origins of the minicircle's light (L) and heavy (H) strands, respectively. kDNA replication occurs during S phase of the cell cycle, approximately in parallel with nuclear DNA replication (3). Minicircles are released from the network into the kinetoflagellar zone, located in the mitochondrial matrix, between the kDNA network and the flagellar basal body. Each minicircle replicates as an individual replicon, forming gapped and nicked progeny molecules. Minicircle replication intermediates then migrate onto two antipodal sites, flanking the kDNA disk, in which primer-removal, repair of the gaps between Okazaki fragments and reattachment of the progeny minicircles to the network occurs. The final gap-filling and sealing of the topologically linked minicircles take place before the network division (recently reviewed in refs. 1 and 2).We have previously reported the presence in Crithidia fasciculata of a UMS-binding protein (UMSBP). The protein, which contains five tandemly arranged CCHC-type zinc-finger motifs, has been purified from cell extracts, and its encoding gene and genomic locus were cloned and analyzed (4-7). Genes encoding orthologous proteins have been identified in other trypanosomatid species [ref. 8 and supporting information (SI) Table 1]. UMSBP binds specifically the two conserved sequences, located at the minicircle replication origins: the UMS dodecamer and an H-st...

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A trypanosomal CCHC-type zinc finger protein which binds the conserved universal sequence of kinetoplast DNA minicircles: isolation and analysis of the complete cDNA from Crithidia fasciculata.

Cited by 36 publications

References 51 publications

Universal Minicircle Sequence Binding Protein, a CCHC-type Zinc Finger Protein That Binds the Universal Minicircle Sequence of Trypanosomatids

Universal Minicircle Sequence Binding Protein, a CCHC-type Zinc Finger Protein That Binds the Universal Minicircle Sequence of Trypanosomatids

A High-OrderTrans-Membrane Structural Linkage Is Responsible for Mitochondrial Genome Positioning and Segregation by Flagellar Basal Bodies in Trypanosomes

Mitochondrial origin-binding protein UMSBP mediates DNA replication and segregation in trypanosomes

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