Electron microscopy can be used to measure DNA supertwisting

Sperrazza, Joan M.; Register, James C.; Griffith, Jack D.

doi:10.1016/0378-1119(84)90190-2

Cited by 27 publications

(10 citation statements)

References 9 publications

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“…5B yielded a value of 15 Ϯ 3 per 1-kb minicircle. In a previous study of molecules of superhelical density in the range of Ϫ0.07, the number of crossovers detected by EM roughly equals the number of supertwists (36). This relationship is not established for highly twisted molecules like fraction S minicircles, and therefore, we can conclude only that supertwisting is extreme.…”

Section: Discussionmentioning

confidence: 86%

Role of p38 in Replication of Trypanosoma brucei Kinetoplast DNA

Liu

Molina

Kalume

et al. 2006

Molecular and Cellular Biology

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Trypanosomes have an unusual mitochondrial genome, called kinetoplast DNA, that is a giant network containing thousands of interlocked minicircles. During kinetoplast DNA synthesis, minicircles are released from the network for replication as -structures, and then the free minicircle progeny reattach to the network. We report that a mitochondrial protein, which we term p38, functions in kinetoplast DNA replication. RNA interference (RNAi) of p38 resulted in loss of kinetoplast DNA and accumulation of a novel free minicircle species named fraction S. Fraction S minicircles are so underwound that on isolation they become highly negatively supertwisted and develop a region of Z-DNA. p38 binds to minicircle sequences within the replication origin. We conclude that cells with RNAi-induced loss of p38 cannot initiate minicircle replication, although they can extensively unwind free minicircles.Kinetoplast DNA (kDNA) is the unusual mitochondrial genome of Trypanosoma brucei and related parasites (13,15,34). kDNA contains several thousand minicircles and a few dozen maxicircles catenated into a giant network. Each cell has one network, condensed into a disk-shaped structure, residing in the single mitochondrion. Maxicircles encode rRNAs and mRNAs for mitochondrial proteins, such as subunits of respiratory complexes, and minicircles encode guide RNAs that are templates for editing of maxicircle transcripts (15, 37).The network structure of kDNA requires a complex and unusual replication mechanism. One important feature of this mechanism is that many of the replication proteins are localized in discrete positions within or near the kDNA disk (13). Another key feature is that replication occurs during a distinct phase of the cell cycle, nearly concurrent with the nuclear S phase (44). The current working model of minicircle replication is outlined in the following paragraph.When replication begins, individual covalently closed minicircles are released from the network, by a topoisomerase, into a region of the mitochondrial matrix between the kDNA disk and the mitochondrial membrane near the flagellar basal body (3). Within this region, called the kinetoflagellar zone, the minicircles encounter proteins such as the origin recognition protein (universal minicircle sequence binding protein [UMSBP]) (1, 39), primase (12), and two DNA polymerases (11). Interaction of these and other proteins with the minicircle promotes unidirectional replication as -structures (26). The progeny minicircles are thought to segregate in the kinetoflagellar zone and then migrate to two antipodal sites, protein assemblies flanking the kDNA disk and positioned 180°apart (7). Within the antipodal sites, the next steps of replication occur. These include removal of RNA primers by structurespecific endonuclease-1 (6), filling in most (but not all) of the gaps between Okazaki fragments by DNA polymerase ␤ (38), and sealing the resulting nicks by DNA ligase k␤ (2). Then a topoisomerase II reattaches the progeny minicircles, still containing at least one ...

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Section: Discussionmentioning

confidence: 86%

Role of p38 in Replication of Trypanosoma brucei Kinetoplast DNA

Liu

Molina

Kalume

et al. 2006

Molecular and Cellular Biology

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“…We assume the number of supercoils in the liquid crystalline state to be about the same as that observed in electrophoresis. Support for this assumption is drawn from electron microscopy which has shown that the number of supertwists seen corresponds to the values obtained from gels (Sperrazza et al, 1984). We thus calculate 1 = 630 + 30A (i.e.…”

Section: Structural Parameters Of Supercoils In Liquid Crystalsmentioning

confidence: 92%

Supercoiled DNA is interwound in liquid crystalline solutions.

Torbet

DiCapua

1989

The EMBO Journal

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Two structures have been proposed for supercoiled DNA: it is idealized either as a toroidal ring or as a rod of two interwound duplex chains. The latter model is the most widely depicted but the evidence remains controversial. We have worked with monomers and dimers of two plasmids, pUC8 and pKS414, of similar size and natural superhelical density. pKS414 contains a bend promoting sequence whereas pUC8 does not. In concentrated solutions these plasmids form a partially ordered liquid crystalline phase which is found, using neutron diffraction, to consist of a hexagonally packed assembly of parallel rod-like particles. This shape strongly suggests an interwound conformation for which some structural parameters are deduced. The mass/unit length obtained by combining the area of the hexagonal lattice and the concentration is -3.6 times that of linear DNA. This implies a shallow superhelical pitch angle -36°which, when combined with the known number of supercoil turns, yields the pitch -360 A and radius -80 A for the supercoil. Oriented X-ray fibre diffraction patterns at 92% relative humidity indicate a B type duplex structure. Nicked circular plasmids also form liquid crystals but their behaviour, as a function of concentration, differs from that of the superhelical plasmids.

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“…Previous work on the shape of supercoiled doublestranded (ds) DNA in solution has led to controversial interpretations which have supported both a toroidal (Brady et al, 1976, Brady et al, 1987 and an interwound model of DNA supercoiling (Langowski, 1987;Torbet and DiCapua, 1989). Most electron microscopy data favor the interwound model (Rhoades and Thomas, 1968;Sperrazza et al, 1984;Boles et al, 1990). However, these routine electron microscopy preparation procedures can be misleading; it cannot be ruled out that supercoiled DNA molecules undergo a transition from a toroidal to an interwound form as a result of the adsorption and drying on the supporting film.…”

Section: Introductionmentioning

confidence: 99%

Direct visualization of supercoiled DNA molecules in solution.

et al. 1990

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The shape of supercoiled DNA molecules in solution is directly visualized by cryo‐electron microscopy of vitrified samples. We observe that: (i) supercoiled DNA molecules in solution adopt an interwound rather than a toroidal form, (ii) the diameter of the interwound superhelix changes from about 12 nm to 4 nm upon addition of magnesium salt to the solution and (iii) the partition of the linking deficit between twist and writhe can be quantitatively determined for individual molecules.

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Electron microscopy can be used to measure DNA supertwisting

Cited by 27 publications

References 9 publications

Role of p38 in Replication of Trypanosoma brucei Kinetoplast DNA

Role of p38 in Replication of Trypanosoma brucei Kinetoplast DNA

Supercoiled DNA is interwound in liquid crystalline solutions.

Direct visualization of supercoiled DNA molecules in solution.

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