Other Forms of DNAs:
Depending upon the sequences, the DNA can take different micro-heterogenerity and structural forms that shows how the DNA structurally shows dynamic features. It is not a structural form that was predicted by Watson and Crick model, it is not a dumb rigid and not modulable rigid and dumb form of DNA.
Figure: Dynamic DNA (you can visualize as you like it).
FIGure: Shows alternate forms of DNA
Depending upon sequences DNA can assume specific structural forms. One such structural distortion is cruciform. The requirement for such structural deviation is a specific sequence, the length of the sequence, the temperature and kind of cat-ions.
Inverted repeats: CGATCTGG-CCAGATCG
Mirror repeats: GGTTGGCC-CCGGTTGG
Direct repeats: GGTTGGCC-GGTTGGCC
When such sequences have a length of 10 or more base pairs with a center of symmetry, dsDNA can assume cruciform like a hairpin or a single strand DNA can assume stem loop structure. Such regions perhaps may be present in regulatory segments for recognition by specific protein factors.
Triple Helical DNA:
Three strands of DNA, which are complementary to each other, have propensity for triple helix formation. Homo or hetero polypurine or polypyrimidine tracts can assume triple strand conformation. Such triple stranded structures have been demonstrated and many diseases have been attributed to them. Triplex strands can affect transcription, replication and gene expression and they even prevent specific protein binding. Triplex DNA structures can be either intermolecular or intramolecular forms.
Intermolecular forms: They are easily formed with polypurine- polypyrimidine tracts. In this the third strand can pair in two different orientations, which depends on its strand nature. However the base pairing requires protonation (low pH) of cytosine and the base pairing is by Hoogsteen bonding. Such type of triple strand formation is possible during homologous mode of recombination, where one of the nicked strand from the homologous segment intercalates into a helical duplex that is paired and produce triple strand structures, and such structures are required for the said function.
Intramolecular triplex forms: These structures form easily, if the DNA segment contains mirror repeat symmetry, in addition to PuPy sequences. IMT DNA may contain repeats of G, GA, GGAA, GGGA, or AAAG x n. Third strand pairing requires protonation of C for pairing with G (requires lower pH). It can also exist in four different isoforms. Super coiling of DNA favors IMT. The third strand can be produced during replication by a process called slippage process, where the polymerase in certain regions after replicating a segment, traverses back and replicates the same strand second time and it can be repeated several times (very rarely it happens).
Note: This Figure shows Triple stranded DNA
Ex. (GAA) n (TTC) n forms intramolecular triplex, exhibits four different isoforms. The third strand pairs with double strand, threads into major groove of the dsDNA.
Fig: This represents another form of Base pairing in triple stranded slipped DNA repeats
Base pairing In Triple stranded DNA:
T = A = T: A&T = W/C bonding and T&A = Hoogsteen bonding.
A = A = T: A = T= W/C bonding. A&A = Hoogsteen
C = G = C: G&C = W/C bonding, C&G = Hoogsteen.
G = G = C: G&C = W/C bonding, G&G = Hoogsteen.
In slipped DNA one of the strands contain sequence repeats; the repeats can be of n x times. Slipped DNA arises during replication by DNA polymerases, where it can create greater length of DNA in one strand and deletions in the other strand.
Slipped strands have been identified and isolated. Nine different loci in humans have been identified and they are found to be very unstable. The repeats found are CTGnCAGn, CTGnCAGn. CTGnCAGn slipped DNAs have been found to move slower in gels. Surprisingly ssDNAs have been found to be stable and they are found as small loops. If such loops are longer they can still exist stably with intra-strand hairpin structures.
Single stranded oligo’s of CCGs or CGGs can form intermolecular duplexes or intramolecular duplexes. Such stable slipped segments can expand and become heritable. They can affect gene expression, protein binding, transcriptional initiation and possibly replication.
Curved and Bent DNA:
DNA by its nature is a very flexible helix and the binding of proteins, which organizes genomic DNA into a very compact structure, makes them nonflexible, thus it is not subjected ware and tear. But certain fragments of DNA, when run on a gel they migrate so slowly as if they are longer DNA. Kinetoplast DNA of Crithedia, when a 414 bp long DNA run on a gel, it behaves as if it is 828 long bp segment. This behavior is attributed to rigid regions alternating with normal segments and such DNA exhibits curvature. If DNA is flexible it can snake through the gel pores, but if the DNA is curved and rigid, it cannot do so, so the slow movement.
Segments of such DNA when analyzed showed they contained repeats of 4 to 5 A s, proceeded by a C and followed by a T. The runs A s are phased by 10 bps.
Fig: Represents curved form of DNA
Ex. CCC (5A) TCTC (6A) TAGGC (6A) TGCC (5A) TCCCAAC.
Wherever a 10 bases phasing with 5 or more runs of ‘A’ s leads to curvature, because runs of ‘A’s assume rigid structures. Between such rigid structures, if flexible regions are found automatically the DNA becomes curved for the flexible regions forms kink or bend between the rod like structures. The ‘A’ tract regions show base pairs having high propeller twist, which is due to strong base pair stacking interactions. This stability is due to stacks of ‘A’s share hydrogen bonding with ‘T’ s stacks found below. And ‘A’ helical repeats have only 10 bp per turn. The ‘A’ tract regions also have a narrow minor groove. These features are unique to DNA with rigid ‘A’ tracts. Sequences such as GA3T3C, G2A3T3C2, G3A3T3C3 and G2A3T3C2 also show curvatures. Such segments exhibit non B-DNA structural forms. The B-DNA shows bend or kinks between B-DNA and Z-DNA, such transitional regions show sequences such as CAAATCGC or CAAAAAATGC. Foot printing of ‘A’-tract DNA, using hydroxyl radicals, indicates that ‘A’ tract DNA is not cut as the normal B-DNA. Kinetoplast DNA has a sequence of GAATTC [CA5-6T] GT [CA5-6T] AGG [CA5-6T] GC [CAAAAT] CCCAAAC; it moves anomalously in the gel. Binding of proteins or cations or any other chemical adducts can cause distortions, and torsions, which are transmitted along the length of the DNA.
Fig: Represents sequence based Bent form of ssDNA
Importance of Curved DNA:
During transcriptional initiation or activation the upstream regions of DNA have been found to be bent and curved, so the regulatory proteins bound at far regions, by curving or bending of the DNA, bring distant regions near to each other. Such curved DNA is also used during initiation of replication. Location of curvature segments may be present in the upstream, downstream or with in the promoter regions of the gene. Such DNA s can be used in site-specific integration or recombination, and very many times used during DNA repair. Curving of DNA is one of the most efficient methods of compacting the genomic DNA, which is often of very large size.
Fig: Shows bent or curved forms of DNA; one with the bound protein and another without it.
Sequences like CTGnCAGn have greater propensity for nucleosome assembly than any other DNA sequences and CGGnCCGn sequences are the least favored for nucleosome formation; this is because the DNA with such sequences have greater flexibility, i.e. they easily be made curved than any other known sequences, so they greatly favored for nucleosomal assembly, however methylated CGGnCCGn at C and with short repeats they can easily assemble into nucleosome, but if the length is longer than n=13, they don’t assemble into 'nu' structures. Both the above-mentioned repeats are flexible and each having different biological properties in terms of chromatin organization.
Contrary to the curved DNAs, certain sequences like CTGnCAGn and CGGnCCGn exhibit 20% faster movement than the normal B DNA in a gel. This unusual mobility depends upon the length of repeats, temperature and percentage of the acrylamide gel. Faster mobility is attributed to the change in “h” value i.e. Rotation per residue of the said sequences, and depending upon the length of such repeats it may induce greater avidity for writhe (super coiling), hence faster movement, and such a DNA is called flexible DNA.
For a quite period of time no body thought, even in dreams that a DNA can also exist as a four-stranded structure. Theoretically it is possible to construct a quadruplex DNA structure by using Watson-Crick and Hoogsteen base paring. Repeats of CGG in a single strand can produce quadruplex forms. In the presence of potassium, Na^+ or lithium^+ ions, CGG repeats readily form quadruplex structures, which can be monitored by running the samples on a gel. Quadruplex DNA moves faster than other forms of DNA. Chemical modification studies clearly suggest (protection of 7’position of Guanine) that Guanines are involved in four stranded structures with Hoogsteen hydrogen bonding. Segments of 2 x GCGC tetrads flanked by two repeats of GGGG (G-quartets) produce quadruplex forms.
Fig: Shows chromosomal ends with Telomeric DNA sequences.
Fig: Just diagrammatic representation of Human chromosomes with Telomeric ends and centromeric positions. Also note the positions of secondary constriction in chromosomes 13,14,15,21 and 22
Fig: Just shows the possible quadruples form of structures if there is G=G=G=G hoogsteen Hydrogen bonding (???). Does it happen in live situations.
Fig: Represents parallel and antiparallel strands.
Telomeric DNA is a par excellent example for quadruplex DNA, which is found in the extreme tips of Eukaryotic chromosomes; Telomeric DNA shows short sequences made of Gs and Ts. They are very characteristics of individual species. Such species-specific sequences are repeated hundred to thousand times in Telomeric DNA. Some members of Eukaryotic telomeric DNA have the following sequence repeats,
Tetrahymena macronucleus 3”CCCCAA5’
Oxytricha (ciliate) 3’CCCAAAA5’
Trypanosome (minichromosome) 3’CCCTA 5’
Dictyostelium 3’CCCTA 5’
Arabidopsis thaliana 3’CCCTAAA 5’
Homo sapien 3’CCCTAA 5’
Such sequences are repeated several 100 times or thousand times; they in turn can organize into the following structural forms
Fig: Just a diagrammatic representation of parallel strand that can form a quadruples DNA structural form.
Fig: Shows the 3-D form of Quadruplex form of DNA
Eukaryotic chromosomal DNA is double stranded, linear and runs from one end of the chromosome to the other end. It is expected that if the ends are open structures, they are susceptible for exonuclease digestion, but it is not the case. The ends are protected by quadruplex structural organization and furthermore they are associated with specific telomeric DNA binding proteins, which provides additional stability, protection and constancy.
In Tetrahymena, a sequence of 3’CCCCAACCCCAA 5’ in telomeric region doesn’t remain open but they form closed loop. Surprisingly repeated segments of telomeric DNA undergo reduction with repeated cell cycles, hence the lost DNA has to be replenished which is done by a unique enzyme called Telomerase. The enzyme is a complex of an RNA of 100 or 110 ntds long and the enzyme is similar to Reverse Transcriptase. Some genes and gene products of the Telomerase complex have been identified. The length of RNA in Tetrahymena is159 ntds and in Euplotes it is 192 ntds long. The RNA has 15 to 22 base repeats of 3’CCCCAA5’. Using the RNA repeats the enzyme pairs with the 5’GGGGTT3’ strand, and the free end is used as the primer and reverse transcriptase extends the top strand, then dissociate and reassociate and extends, this process can be repeated several times till sufficient length is reached. How the length of telomeric DNA is controlled is not known. The GGGGTT strand that is extended on CCCCAA can loop back and by G and G base pairing by non-Watson-Crick but Hoogsteen base pairing, it can generate a quartet with loops. Proteins specific to Telomeric DNA sequences further stabilize such structures.
3’- - - - - - --------------CCCCCAACCCCAACCCCAA - - - - - -5’
3’- - - -CCCCAACCCCAACCCCAA- - - - - - - -5’
Fig: Represents the Telomeric ends forming a structural feature with their associated proteins that protects their ends.
The size and the stability of telomere are implicated in aging process. In older or aged cells the length of Telomeric DNA is short and that of young and proliferating cells the Telomeric DNA is longer. The controversy is whether reduction of telomeric DNA causes aging of cells or aging of cells causes reduction in the length of the Telomeric DNA? An aged cell has less amount of Tel DNA. If such cells are stimulated to divide and redivide or transformed into a tumor cell, then its Tel DNA shows greater number of repeats.
Fig : Model showing the curved form of DNA
Fig: Shows how an extended single stranded DNA protrudes and curves to base pairs to generate DNA loop all extended.