Non-helical models of DNA structure

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Three double-stranded DNA structures occur naturally. A-DNA (dehydrated structure), B-DNA (most common structure), Z-DNA (strain-releif structure during transcription).

In the history of molecular biology, non-helical or "side-by-side" models of DNA were proposed in the 1970s as a challenge to the now accepted double-helical model of DNA structure. The non-helical models attempted to solve problems relating to the topology of circular DNA chromosomes during replication. These theories were briefly considered seriously as a minority viewpoint,[1] but they were later largely rejected due to experimental evidence from X-ray crystallography and solution NMR (of both DNA alone, and bound to DNA-binding proteins), as well as the discovery of the topoisomerases responsible for circular chromosome separation. Although localised or transient non-duplex helical structures exist,[2] non-helical models are not currently accepted by the mainstream scientific community.[3]

Terminology[edit]

Plectonemic circular strands
Paranemic circular strands
Top: A rope model of a circularized plectonemic (helical) duplex, showing that when one cannot separate the strands without breaking at least one of them, a tangled knot results. Bottom: A rope model of a circularized paranemic (non-helical) duplex, the strands of which can be easily separated.

The strands of a helical DNA duplex, if covalently closed into a circular structure, are topologically linked, and thus cannot be separated without breaking one or both strands. This sort of structure is referred to as "plectonemic". In circular DNA replication, these twists must be removed for the daughter strands to separate. In contrast, the strands of a non-helical DNA duplex would be topologically non-linked, and can be separated without strand breakage. This sort of structure is referred to as "paranemic".[4][5]

Note that the term "non-helical" refers to net helicity - there can be no helical twist at all, as in the figure at the top of this article, or there could be an equal number of right-handed and left-handed twists. The abbreviation "TN", to be used to refer to any DNA structure whose strands are topologically non-linked, has been proposed.[4]

History[edit]

Background[edit]

The double-helix model of DNA structure was first published in 1953[6] (further details in 1954[7]) based on X-ray diffraction images of DNA as well as base-pairing chemical and biochemical information.[8][9] The initial double helix model discovered, now termed B-form DNA is by far the most common conformation in cells.[10] Two additional rarer helical conformations that also naturally occur were identified in the 1970s: A-form DNA, and Z-form DNA.[11]

In 1963, autoradiographs of the E. coli chromosome demonstrated that it was a single circular molecule that is replicated at a pair of replication forks at which both new DNA strands are being synthesized.[12] It was recognized at the time that the helical twists between the daughter strands presented a topological problem, as they would have to be removed for the strands to separate during cell replication. It was initially proposed that the twists were unwound by means of a hypothetical "swivel" around which the chromosome would be free to rotate as necessary for the un-winding and re-winding operations.[13] However, such a structure would have to have about 400,000 helical twists in B-form DNA, each of which would have to be removed in as little as 20 minutes, if the strands are to separate during cell replication. This leads to the apparent need of the chromosome - or at least some part of it - to be spinning at speeds up to 6900–9000 rpm throughout the life of the bacterial cell.[14] It was this apparent contradiction that early non-helical models attempted to address until the discovery of topoisomerases in 1970 resolved the problem.[4][15]

Proposals of non-helical structure[edit]

Non-helical models were initially speculated in the 1970s to provide a theoretical solution to the perceived angular momentum problem in DNA replication.[16][17] The topological concerns that originally gave rise to non-helical structure proposals had been laid to rest by the lale 1970s,[18] and non-helical models, suffered from a lack of experimental support.[18]

However, as further evidence developed in support of the helical structure (see below), non-helical models were dropped from mainstream science.[4] Although alternative non-helical models were subsequently proposed during the 1980s[19] and into the 2000s,[20] these have been rejected by the scientific community as of insufficient evidence and of solving a solved problem.[3] Nevertheless, isolated theories persist in fringe journals.[21]

Confirmation of helical structure[edit]

In early discussions of the in vivo structure of DNA, it was proposed that the observed helical structure was an artifact of the X-ray crystallography process. However the structure of DNA was subsequently confirmed in solution via gel electrophoretic methods[22] and later via solution NMR,[23] indicating that the crystallography process did not distort it. The structure of DNA in complex with nucleosomes, helicases, and numerous other DNA binding proteins also supports its biological relevance.[24]

The discovery of topoisomerases, enzymes that can change the linking number of circular nucleic acids and thus "unwind" the replicating bacterial chromosome, was largely seen by the mainstream scientific community to satisfy the topological objection to the B-form DNA helical structure.[25] The later X-ray crystallography data for the nucleosome, which showed a helical DNA structure wrapped around the protein nucleosome core particle, was considered to provide further affirmation for the existence of the helical structure in vivo. Non-double-helical models are not currently accepted by the mainstream scientific community.[3] Enzymes capable of unwinding and rewinding DNA helical twists, namely topoisomerases and gyrases, are essential for life, and necessary for DNA replication in various in vitro synthesis systems.

Proposed non-helical models[edit]

Theoretical proposals[edit]

Linear tetraplex model proposal (1969)[26]
Side-By-Side model structure proposal (1976)[16]

In 1969, a linear tetraplex structure was proposed with straight, extended backbones and intercalating bases.[26]

In 1976, a subsequent non-helical DNA structure with alternating sections of right-handed and left-handed helix was independently proposed by two different groups.[16][27] Termed the "side-by-side" (SBS) structure, it was a hypothetical attempt solve the angular momentum problem in DNA replication, which was thought by its proponents at the time to be otherwise unsolvable.[16][28] The structure was initially presented with the helicity changin direction every half turn. However one set of proponents later clarified they had not intended alternating half turns, but rather long stretches of each helical direction.[29]

Experimental studies of circular DNA structure[edit]

One key observation in support of a double-helical structure is that the individual single strands of small circular viral and plasmid DNA are inseparable under the usual types of denaturation conditions, supporting a plectonemic, i.e., twisted structure whose strands are topologically locked together. While eukaryotic and bacterial chromosomes are too large to isolate intact (breaking into fragments when extracted from cells), smaller viral chromosomes and bacterial plasmids are remain intact when extracted. Strands of duplex circular chromosomes do not separate when subjected to conditions which denature linear DNA.[30] When the circular chromosomes of viruses are boiled, nothing happens to the native structure. The failure of the strands of circular chromosomes to separate under conditions where the strands of linear DNA would readily do so is evidence that helical strands are plectonemically wound together (i.e., topologically linked).

Attempts to generate SBS DNA experimentally centered around comparing a circular chromosome to the form created by isolating the separate circular single strands and re-annealing them with no net twist. The prepared DNA, termed "Form V" (extending the nomenclature of Forms I–IV used in sedimentation studies of DNA), had electrophoretic mobility different from that of the native chromosome, indicating a different overall structure.[31] However this prepared chromosome did not establish that "Form V" has the SBS structure. While the strands of native circular DNA are not separable under common denaturing conditions, in 1996 Tai Te Wu reported having found conditions under which fully intact single strands of two different circular duplex plasmids could be separated.[32] Wu believed that, because these plasmids have D-loops with DNA–RNA duplexes from RNA transcription only on their sense side, and since DNA–RNA interactions are stronger than DNA–DNA interactions,[33] the sense and anti-sense strands would thus have different electrophoretic mobilities. He employed a very low voltage, so that the electrophoresis required 48 hours to go to completion. DNA sequencing then indicated that each of the two bands were enriched in one of the two strands of the chromosome.

In 2009, Youcheng Xu noted that brief re-annealing times (i.e. 20–30 minutes) resulted in anomalous structures which did not co-migrate with routinely prepared DNA topoisomers, while prolonged re-annealing times (in his case, 72 hours at 4º) resulted in structures which did co-migrate with the topoisomers, suggesting normal base-pairing. They interpreted their data as supporting the possibility that the two strands inside the native DNA double helix are winding ambidextrously rather than plectonemically, with left-handed and right-handed regions coexisting in a zero linking number topoisomer.[34][35] The finding that the two circular strands of supercoiled or relaxed plasmid can be gently dissociated in low salt aqueous solution or pure water provides additional evidence supporting the ambidextrous double helix model.

Recently, Dr. Pawan Kumar from the All India Institute of Medical Sciences, New Delhi has demonstrated that two strands of pUC19 plasmid can be separated into individual circular DNA strands with the addition of sodium hydroxide (NaOH).[36] The separated strands of plasmid DNA reannealed to form the double-stranded plasmid under suitable conditions. Moreover, plasmid DNA formed with the reannealing of individual strands was found to be similar to native plasmid in its properties. These findings showed that two strands of a plasmid DNA can be reversibly separated into individual strands and contradicted the Watson and Crick model of DNA structure.

References[edit]

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