DNA base flipping

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The DNA double helix with a Cytosine base flipped out 180deg Dna-base-flipping.svg
The DNA double helix with a Cytosine base flipped out 180°

DNA base flipping, or nucleotide flipping, is a mechanism in which a single nucleotide base, or nucleobase, is rotated outside the nucleic acid double helix. [1] This occurs when a nucleic acid-processing enzyme needs access to the base to perform work on it, such as its excision for replacement with another base during DNA repair. It was first observed in 1994 using X-ray crystallography in a methyltransferase enzyme catalyzing methylation of a cytosine base in DNA. Since then, it has been shown to be used by different enzymes in many biological processes such as DNA methylation, various DNA repair mechanisms, and DNA replication. It can also occur in RNA double helices [2] or in the DNA:RNA intermediates formed during RNA transcription.

Contents

DNA base flipping occurs by breaking the hydrogen bonds between the bases and unstacking the base from its neighbors. This could occur through an active process, where an enzyme binds to the DNA and then facilitates rotation of the base, or a passive process, where the base rotates out spontaneously, and this state is recognized and bound by an enzyme. It can be detected using X-ray crystallography, NMR spectroscopy, fluorescence spectroscopy, or hybridization probes.

Discovery

Base flipping was first observed in 1994 when researchers Klimasauskas, Kumar, Roberts, and Cheng used X-ray crystallography to view an intermediate step in the chemical reaction of a methyltransferase bound to DNA. [3] The methyltransferase they used was the C5-cytosine methyltransferase from Haemophilus haemolyticus (M. HhaI). This enzyme recognizes a specific sequence of the DNA (5'-GCGC-3') and methylates the first cytosine base of the sequence at its C5 location. [3] Upon crystallization of the M. HhaI-DNA complex, they saw the target cytosine base was rotated completely out of the double helix and was positioned in the active site of the M. HhaI. It was held in place by numerous interactions between the M. HhaI and DNA. [3]

The authors theorized that base flipping was a mechanism used by many other enzymes, such as helicases, recombination enzymes, RNA polymerases, DNA polymerases, and Type II topoisomerases. [3] Much research has been done in the years subsequent to this discovery and it has been found that base flipping is a mechanism used in many of the biological processes the authors suggest. [4] [5] [6]

Mechanism

Model of Entamoeba histolytica DNMT2, Demonstrates a base flipped out of the double helix and into the active site of a methyltransferase Model of the EhMeth-DNA complex.jpg
Model of Entamoeba histolytica DNMT2, Demonstrates a base flipped out of the double helix and into the active site of a methyltransferase

DNA nucleotides are held together with hydrogen bonds, which are relatively weak and can be easily broken. Base flipping occurs on a millisecond timescale [7] by breaking the hydrogen bonds between bases and unstacking the base from its neighbors. [8] The base is rotated out of the double helix by 180 degrees., [9] typically via the major groove, [10] and into the active site of an enzyme. This opening leads to small conformational changes in the DNA backbone [11] which are quickly stabilized by the increased enzyme-DNA interactions. [12] Studies looking at the free-energy profiles of base flipping have shown that the free-energy barrier to flipping can be lowered by 17 kcal/mol for M.HhaI in the closed conformation. [10]

There are two mechanisms of DNA base flipping: active and passive. [13] In the active mechanism, an enzyme binds to the DNA and then actively rotates the base, while in the passive mechanism a damaged base rotates out spontaneously first, then is recognized and bound by the enzyme. [8] Research has demonstrated both mechanisms: uracil-DNA glycosylase follows the passive mechanism [8] and Tn10 transposase follows the active mechanism. [14]

Furthermore, studies have shown that DNA base flipping is used by many different enzymes in a variety biological processes such as DNA methylation, various DNA repair mechanisms, RNA transcription and DNA replication. [4] [5] [6]

Biological processes

DNA modification and repair

A uracil residue flipped out of the DNA double helix and into the specificity pocket of Uracil DNA glycosylase Uracil base glycosidase.jpg
A uracil residue flipped out of the DNA double helix and into the specificity pocket of Uracil DNA glycosylase

DNA can have mutations that cause a base in the DNA strand to be damaged. To ensure genetic integrity of the DNA, enzymes need to repair any damage. There are many types of DNA repair. Base excision repair utilizes base flipping to flip the damaged base out of the double helix [5] and into the specificity pocket of a glycosylase which hydrolyzes the glycosidic bond and removes the base. [15] DNA glycosylases interact with DNA, flipping bases to determine a mismatch. An example of base excision repair occurs when a cytosine base is deaminated and becomes a uracil base. This causes a U:G mispair which is detected by Uracil DNA glycosylase. The uracil base is flipped out into the glycosylase active pocket where it is removed from the DNA strand. [16] Base flipping is used to repair mutations such as 8-Oxoguanine (oxoG) [17] and thymine dimers created by UV radiation. [15] [18]

Replication, transcription and recombination

DNA replication and RNA transcription both make use of base flipping. [5] DNA polymerase is an enzyme that carries out replication. It can be thought of as a hand that grips the DNA single strand template. [15] As the template passes across the palm region of the polymerase, the template bases are flipped out of the helix and away from the dNTP binding site. [19] During transcription, RNA polymerase catalyzes RNA synthesis. During the initiation phase, two bases in the -10 element flip out from the helix and into two pockets in RNA polymerase. These new interactions stabilize the -10 element and promote the DNA strands to separate or melt. [15] [20]

Base flipping occurs during latter stages of recombination. [21] RecA is a protein that promotes strand invasion [15] during homologous recombination. Base flipping has been proposed as the mechanism by which RecA can enable a single strand to recognize homology in duplex DNA. [22] Other studies indicate that it is also involved in V(D)J Recombination. [23]

DNA methylation

DNA molecule that is methylated on both strands on the center cytosine DNA methylation.jpg
DNA molecule that is methylated on both strands on the center cytosine

DNA methylation is the process in which a methyl group is added to either a cytosine or adenine. [24] This process causes the activation or inactivation of gene expression, thereby resulting in gene regulation in eukaryotic cells. DNA methylation process is also known to be involved in certain types of cancer formation. [25] [26] [27] In order for this chemical modification to occur, it is necessary that the target base flips out of the DNA double helix to allow the methyltransferases to catalyze the reaction. [5]

Target recognition by restriction endonucleases

Restriction endonucleases, also known as restriction enzymes are enzymes that cleave the sugar-phosphate backbone of the DNA at specific nucleotides sequences that are usually four to six nucleotides long. [28] Studies performed by Horton and colleagues have shown that the mechanism by which these enzymes cleave the DNA involves base flipping as well as bending the DNA and the expansion of the minor groove. [29] In 2006, Horton and colleagues, x-ray crystallography evidence was presented showing that the restriction endonuclease HinP1I utilizes base flipping in order to recognize its target sequence. This enzyme is known to cleave the DNA at the palindromic tetranucleotide sequence G↓CGC.

Experimental approaches for detection

X-ray crystallography

Workflow for solving the structure of a molecule by X-ray crystallography X ray diffraction.png
Workflow for solving the structure of a molecule by X-ray crystallography

X-ray crystallography is a technique that measures the angles and intensities of crystalline atoms in order to determine the atomic and molecular structure of the crystal of interest. Crystallographers are then able to produce and three-dimensional picture where the positions of the atoms, chemical bonds as well as other important characteristics can be determined. [30] Klimasaukas and colleagues used this technique to observe the first base flipping phenomenon, in which their experimental procedure involved several steps: [3]

  1. Purification
  2. Crystallization
  3. Data Collection
  4. Structure determination and refinement

During purification, Haemophilus haemolyticus methyltransferase was overexpressed and purified using a high salt back-extraction step to selectively solubilize M.HhaI, followed by fast protein liquid chromatography (FPLC) as done previously by Kumar and colleagues. [31] Authors utilized a Mono-Q anion exchange column to remove the small quantity of proteinaceous materials and unwanted DNA prior to the crystallization step. Once M.HhaI was successfully purified, the sample was then grown using a method that mixes the solution containing the complex at a temperature of 16 °C and the hanging-drop vapor diffusion technique to obtain the crystals. Authors were then able to collect the x-ray data according to a technique used by Cheng and colleagues in 1993. [32] This technique involved the measurement of the diffraction intensities on a FAST detector, where the exposure times for 0.1° rotation were 5 or 10 seconds. For the structure determination and refinement, Klimasaukas and colleagues used the molecular replacement of the refined apo structure described by Cheng and colleagues in 1993 [32] where the search models X-PLOR, MERLOT, and TRNSUM were used to solve the rotation and translation functions. [33] [34] This part of the study involves the use of a variety of software and computer algorithms to solve the structures and characteristics of the crystal of interest.

NMR spectroscopy

NMR spectroscopy is a technique that has been used over the years to study important dynamic aspects of base flipping. This technique allows researchers to determine the physical and chemical properties of atoms and other molecules by utilizing the magnetic properties of atomic nuclei. [35] In addition, NMR can provide a variety of information including structure, reaction states, chemical environment of the molecules, and dynamics. [36] [37] During the DNA base flipping discovery experiment, researchers utilized NMR spectroscopy to investigate the enzyme-induced base flipping of HhaI methyltransferase. In order to accomplish this experiment, two 5-fluorocytosine residues were incorporated into the target and the reference position with the DNA substrate so the 19F chemical shift analysis could be performed. Once the 19F chemical shift analysis was evaluated, it was then concluded that the DNA complexes existed with multiple forms of the target 5-fluorocytosine along the base flipping pathway. [38]

Fluorescence spectroscopy

Fluorescence spectroscopy is a technique that is used to assay a sample using a fluorescent probe. DNA nucleotides themselves are not good candidates for this technique because they do not readily re-emit light upon light excitation. [39] A fluorescent marker is needed to detect base flipping. 2-Aminopurine is a base that is structurally similar to adenine, but is very fluorescent when flipped out from the DNA duplex. [40] It is commonly used to detect base flipping and has an excitation at 305‑320 nm and emission at 370 nm so that it well separated from the excitations of proteins and DNA. Other fluorescent probes used to study DNA base flipping are 6MAP (4‑amino‑6‑methyl‑7(8H)‑pteridone) [41] and Pyrrolo‑C (3-[β-D-2-ribofuranosyl]-6-methylpyrrolo[2,3-d]pyrimidin-2(3H)-one). [42] [43] Time-resolved fluorescence spectroscopy is also employed to provide a more detailed picture of the extent of base flipping as well as the conformational dynamics occurring during base flipping. [44]

Hybridization probing

Hybridization probes can be used to detect base flipping. This technique uses a molecule that has a complementary sequence to the sequence you would like to detect such that it binds to a single-strand of the DNA or RNA. Several hybridization probes have been used to detect base flipping. Potassium permanganate is used to detect thymine residues that have been flipped out by cytosine-C5 and adenine-N6 methyltransferases. [45] Chloroacetaldehyde is used to detect cytosine residues flipped out by the HhaI DNA cytosine-5 methyltransferase (M. HhaI). [46]

A hybridization probe is added to a DNA molecule LightCycler Probes.jpg
A hybridization probe is added to a DNA molecule

See also

Related Research Articles

<span class="mw-page-title-main">DNA</span> Molecule that carries genetic information

Deoxyribonucleic acid is a polymer composed of two polynucleotide chains that coil around each other to form a double helix. The polymer carries genetic instructions for the development, functioning, growth and reproduction of all known organisms and many viruses. DNA and ribonucleic acid (RNA) are nucleic acids. Alongside proteins, lipids and complex carbohydrates (polysaccharides), nucleic acids are one of the four major types of macromolecules that are essential for all known forms of life.

Deamination is the removal of an amino group from a molecule. Enzymes that catalyse this reaction are called deaminases.

<span class="mw-page-title-main">5-Methylcytosine</span> Chemical compound which is a modified DNA base

5-Methylcytosine is a methylated form of the DNA base cytosine (C) that regulates gene transcription and takes several other biological roles. When cytosine is methylated, the DNA maintains the same sequence, but the expression of methylated genes can be altered. 5-Methylcytosine is incorporated in the nucleoside 5-methylcytidine.

<span class="mw-page-title-main">Transcription (biology)</span> Process of copying a segment of DNA into RNA

Transcription is the process of copying a segment of DNA into RNA. The segments of DNA transcribed into RNA molecules that can encode proteins are said to produce messenger RNA (mRNA). Other segments of DNA are copied into RNA molecules called non-coding RNAs (ncRNAs). mRNA comprises only 1–3% of total RNA samples. Less than 2% of the human genome can be transcribed into mRNA, while at least 80% of mammalian genomic DNA can be actively transcribed, with the majority of this 80% considered to be ncRNA.

<span class="mw-page-title-main">DNA polymerase</span> Form of DNA replication

A DNA polymerase is a member of a family of enzymes that catalyze the synthesis of DNA molecules from nucleoside triphosphates, the molecular precursors of DNA. These enzymes are essential for DNA replication and usually work in groups to create two identical DNA duplexes from a single original DNA duplex. During this process, DNA polymerase "reads" the existing DNA strands to create two new strands that match the existing ones. These enzymes catalyze the chemical reaction

<span class="mw-page-title-main">DNA methyltransferase</span> Class of enzymes

In biochemistry, the DNA methyltransferase family of enzymes catalyze the transfer of a methyl group to DNA. DNA methylation serves a wide variety of biological functions. All the known DNA methyltransferases use S-adenosyl methionine (SAM) as the methyl donor.

In molecular biology and genetics, transcriptional regulation is the means by which a cell regulates the conversion of DNA to RNA (transcription), thereby orchestrating gene activity. A single gene can be regulated in a range of ways, from altering the number of copies of RNA that are transcribed, to the temporal control of when the gene is transcribed. This control allows the cell or organism to respond to a variety of intra- and extracellular signals and thus mount a response. Some examples of this include producing the mRNA that encode enzymes to adapt to a change in a food source, producing the gene products involved in cell cycle specific activities, and producing the gene products responsible for cellular differentiation in multicellular eukaryotes, as studied in evolutionary developmental biology.

<span class="mw-page-title-main">Helicase</span> Class of enzymes to unpack an organisms genes

Helicases are a class of enzymes thought to be vital to all organisms. Their main function is to unpack an organism's genetic material. Helicases are motor proteins that move directionally along a nucleic acid phosphodiester backbone, separating two hybridized nucleic acid strands, using energy from ATP hydrolysis. There are many helicases, representing the great variety of processes in which strand separation must be catalyzed. Approximately 1% of eukaryotic genes code for helicases.

<span class="mw-page-title-main">Ribonucleotide</span> Nucleotide containing ribose as its pentose component

In biochemistry, a ribonucleotide is a nucleotide containing ribose as its pentose component. It is considered a molecular precursor of nucleic acids. Nucleotides are the basic building blocks of DNA and RNA. Ribonucleotides themselves are basic monomeric building blocks for RNA. Deoxyribonucleotides, formed by reducing ribonucleotides with the enzyme ribonucleotide reductase (RNR), are essential building blocks for DNA. There are several differences between DNA deoxyribonucleotides and RNA ribonucleotides. Successive nucleotides are linked together via phosphodiester bonds.

<span class="mw-page-title-main">DNA repair</span> Cellular mechanism

DNA repair is a collection of processes by which a cell identifies and corrects damage to the DNA molecules that encodes its genome. In human cells, both normal metabolic activities and environmental factors such as radiation can cause DNA damage, resulting in tens of thousands of individual molecular lesions per cell per day. Many of these lesions cause structural damage to the DNA molecule and can alter or eliminate the cell's ability to transcribe the gene that the affected DNA encodes. Other lesions induce potentially harmful mutations in the cell's genome, which affect the survival of its daughter cells after it undergoes mitosis. As a consequence, the DNA repair process is constantly active as it responds to damage in the DNA structure. When normal repair processes fail, and when cellular apoptosis does not occur, irreparable DNA damage may occur, including double-strand breaks and DNA crosslinkages. This can eventually lead to malignant tumors, or cancer as per the two hit hypothesis.

DNA glycosylases are a family of enzymes involved in base excision repair, classified under EC number EC 3.2.2. Base excision repair is the mechanism by which damaged bases in DNA are removed and replaced. DNA glycosylases catalyze the first step of this process. They remove the damaged nitrogenous base while leaving the sugar-phosphate backbone intact, creating an apurinic/apyrimidinic site, commonly referred to as an AP site. This is accomplished by flipping the damaged base out of the double helix followed by cleavage of the N-glycosidic bond.

<span class="mw-page-title-main">Base excision repair</span> DNA repair process

Base excision repair (BER) is a cellular mechanism, studied in the fields of biochemistry and genetics, that repairs damaged DNA throughout the cell cycle. It is responsible primarily for removing small, non-helix-distorting base lesions from the genome. The related nucleotide excision repair pathway repairs bulky helix-distorting lesions. BER is important for removing damaged bases that could otherwise cause mutations by mispairing or lead to breaks in DNA during replication. BER is initiated by DNA glycosylases, which recognize and remove specific damaged or inappropriate bases, forming AP sites. These are then cleaved by an AP endonuclease. The resulting single-strand break can then be processed by either short-patch or long-patch BER.

<span class="mw-page-title-main">Methyltransferase</span> Group of methylating enzymes

Methyltransferases are a large group of enzymes that all methylate their substrates but can be split into several subclasses based on their structural features. The most common class of methyltransferases is class I, all of which contain a Rossmann fold for binding S-Adenosyl methionine (SAM). Class II methyltransferases contain a SET domain, which are exemplified by SET domain histone methyltransferases, and class III methyltransferases, which are membrane associated. Methyltransferases can also be grouped as different types utilizing different substrates in methyl transfer reactions. These types include protein methyltransferases, DNA/RNA methyltransferases, natural product methyltransferases, and non-SAM dependent methyltransferases. SAM is the classical methyl donor for methyltransferases, however, examples of other methyl donors are seen in nature. The general mechanism for methyl transfer is a SN2-like nucleophilic attack where the methionine sulfur serves as the leaving group and the methyl group attached to it acts as the electrophile that transfers the methyl group to the enzyme substrate. SAM is converted to S-Adenosyl homocysteine (SAH) during this process. The breaking of the SAM-methyl bond and the formation of the substrate-methyl bond happen nearly simultaneously. These enzymatic reactions are found in many pathways and are implicated in genetic diseases, cancer, and metabolic diseases. Another type of methyl transfer is the radical S-Adenosyl methionine (SAM) which is the methylation of unactivated carbon atoms in primary metabolites, proteins, lipids, and RNA.

<span class="mw-page-title-main">Palindromic sequence</span> DNA or RNA sequence that matches its complement when read backwards

A palindromic sequence is a nucleic acid sequence in a double-stranded DNA or RNA molecule whereby reading in a certain direction on one strand is identical to the sequence in the same direction on the complementary strand. This definition of palindrome thus depends on complementary strands being palindromic of each other.

<span class="mw-page-title-main">DNA adenine methylase</span> Prokaryotic enzyme

DNA adenine methylase, (Dam) (also site-specific DNA-methyltransferase (adenine-specific), EC 2.1.1.72, modification methylase, restriction-modification system) is an enzyme that adds a methyl group to the adenine of the sequence 5'-GATC-3' in newly synthesized DNA. Immediately after DNA synthesis, the daughter strand remains unmethylated for a short time. It is an orphan methyltransferase that is not part of a restriction-modification system and regulates gene expression. This enzyme catalyses the following chemical reaction

<span class="mw-page-title-main">Pyrimidine dimer</span> Type of damage to DNA

Pyrimidine dimers are molecular lesions formed from thymine or cytosine bases in DNA via photochemical reactions, commonly associated with direct DNA damage. Ultraviolet light induces the formation of covalent linkages between consecutive bases along the nucleotide chain in the vicinity of their carbon–carbon double bonds. The photo-coupled dimers are fluorescent. The dimerization reaction can also occur among pyrimidine bases in dsRNA —uracil or cytosine. Two common UV products are cyclobutane pyrimidine dimers (CPDs) and 6–4 photoproducts. These premutagenic lesions alter the structure of the DNA helix and cause non-canonical base pairing. Specifically, adjacent thymines or cytosines in DNA will form a cyclobutane ring when joined together and cause a distortion in the DNA. This distortion prevents replication or transcription machinery beyond the site of the dimerization. Up to 50–100 such reactions per second might occur in a skin cell during exposure to sunlight, but are usually corrected within seconds by photolyase reactivation or nucleotide excision repair. In humans, the most common form of DNA repair is nucleotide excision repair (NER). In contrast, organisms such as bacteria can counterintuitively harvest energy from the sun to fix DNA damage from pyrimidine dimers via photolyase activity. If these lesions are not fixed, polymerase machinery may misread or add in the incorrect nucleotide to the strand. If the damage to the DNA is overwhelming, mutations can arise within the genome of an organism and may lead to the production of cancer cells. Uncorrected lesions can inhibit polymerases, cause misreading during transcription or replication, or lead to arrest of replication. It causes sunburn and it triggers the production of melanin. Pyrimidine dimers are the primary cause of melanomas in humans.

<span class="mw-page-title-main">Nucleic acid secondary structure</span>

Nucleic acid secondary structure is the basepairing interactions within a single nucleic acid polymer or between two polymers. It can be represented as a list of bases which are paired in a nucleic acid molecule. The secondary structures of biological DNAs and RNAs tend to be different: biological DNA mostly exists as fully base paired double helices, while biological RNA is single stranded and often forms complex and intricate base-pairing interactions due to its increased ability to form hydrogen bonds stemming from the extra hydroxyl group in the ribose sugar.

<span class="mw-page-title-main">Complementarity (molecular biology)</span> Lock-and-key pairing between two structures

In molecular biology, complementarity describes a relationship between two structures each following the lock-and-key principle. In nature complementarity is the base principle of DNA replication and transcription as it is a property shared between two DNA or RNA sequences, such that when they are aligned antiparallel to each other, the nucleotide bases at each position in the sequences will be complementary, much like looking in the mirror and seeing the reverse of things. This complementary base pairing allows cells to copy information from one generation to another and even find and repair damage to the information stored in the sequences.

Nucleic acid NMR is the use of nuclear magnetic resonance spectroscopy to obtain information about the structure and dynamics of nucleic acid molecules, such as DNA or RNA. It is useful for molecules of up to 100 nucleotides, and as of 2003, nearly half of all known RNA structures had been determined by NMR spectroscopy.

DNA-deoxyinosine glycosylase is an enzyme with systematic name DNA-deoxyinosine deoxyribohydrolase. This enzyme is involved in DNA damage repair and targets hypoxanthine bases.

References

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  29. ↑ Horton, John R.; Zhang, Xing; Maunus, Robert; Yang, Zhe; Wilson, Geoffrey; Roberts, Richard; Cheng, Xiaodong (2006). "DNA Nicking by HinP1I Endonuclease: Bending, Base Flipping and Minor Groove Expansion". Nucleic Acids Research. 34 (3): 939–948. doi:10.1093/nar/gkj484. PMC   1363774 . PMID   16473850.
  30. ↑ X-ray crystallography
  31. ↑ Kumar, S; Cheng, X; Pflugrath, JW; Roberts, RJ (1992). "Purification, Crystallization, and Preliminary X-ray Diffraction Analysis of an M.HhaI-AdoMet Complex". Biochemistry. 31 (36): 8648–8653. doi:10.1021/bi00151a035. PMID   1390649.
  32. 1 2 Cheng, X; Kumar, S; Posfai, J; Pflugrath, JW; Roberts, RJ (1993). "Crystal Structure of the HhaI DNA Methyltransferase Complexed with S-adenosyl-L-methionine". Cell. 74 (2): 299–307. doi:10.1016/0092-8674(93)90421-l. PMID   8343957. S2CID   54238106.
  33. ↑ Brunger A.T. (1992)"X-PLOR, Version 3.1 : A system for x-ray crystallography and NMR"(New Haven, Connecticut: Yale University Press)
  34. ↑ Fitzgerald, P.M.D. (1988). "MERLOT, an integral package of computer programs for the determination of crystal structure by molecular replacement". J. Appl. Crystallogr. 21 (3): 273–288. doi:10.1107/s0021889887012299.
  35. ↑ NMR spectroscopy
  36. ↑ Gueron, M., and J. L. Leroy. 1995. Studies of basepair kinetics by NMR measurement of proton exchange. In Nuclear Magnetic Resonance And Nucleic Acids. Academic Press, San Diego, CA.
  37. ↑ Leijon, M.; Graslund, A. (1992). "Effects of sequence and length on imino proton-exchange and basepair opening kinetics in DNA oligonucleotide duplexes". Nucleic Acids Res. 20 (20): 5339–5343. doi:10.1093/nar/20.20.5339. PMC   334339 . PMID   1331987.
  38. ↑ Klimasaukas, Salius, and Zita Liutkeviciute. "Experimental Approaches to Study DNA Base Flipping." DNA and RNA Modification Enzymes: Structure, Mechanism, Function and Evolution. Landes Bioscience, 2009. 37-50. Web. 16 Mar. 2014. <https://www.landesbioscience.com/pdf/04GrosjeanKlimasauskas.pdf Archived 2014-04-07 at the Wayback Machine >.
  39. ↑ Grosjean, [edited by] Henri (2009). DNA and RNA modification enzymes : structure, mechanism, function and evolution (PDF). Austin, Tex.: Landes Bioscience. p. 43. ISBN   978-1-58706-329-9.{{cite book}}: |first= has generic name (help)
  40. ↑ Holz, B (15 February 1998). "2-Aminopurine as a fluorescent probe for DNA base flipping by methyltransferases". Nucleic Acids Research. 26 (4): 1076–1083. doi:10.1093/nar/26.4.1076. PMC   147370 . PMID   9461471.
  41. ↑ Yang, K; Matsika, S; Stanley, RJ (Sep 6, 2007). "6MAP, a fluorescent adenine analogue, is a probe of base flipping by DNA photolyase". The Journal of Physical Chemistry B. 111 (35): 10615–25. doi:10.1021/jp071035p. PMID   17696385. S2CID   4998287.
  42. ↑ Yang, K; Stanley, RJ (May–Jun 2008). "The extent of DNA deformation in DNA photolyase-substrate complexes: a solution state fluorescence study". Photochemistry and Photobiology. 84 (3): 741–9. doi:10.1111/j.1751-1097.2007.00251.x. PMID   18086248. S2CID   44506405.
  43. ↑ Berry, David A.; Jung, Kee-Yong; Wise, Dean S.; Sercel, Anthony D.; Pearson, William H.; Mackie, Hugh; Randolph, John B.; Somers, Robert L. (March 2004). "Pyrrolo-dC and pyrrolo-C: fluorescent analogs of cytidine and 2′-deoxycytidine for the study of oligonucleotides". Tetrahedron Letters. 45 (11): 2457–2461. doi:10.1016/j.tetlet.2004.01.108.
  44. ↑ Neely, R. K.; Tamulaitis, G.; Chen, K.; Kubala, M.; Siksnys, V.; Jones, A. C. (8 September 2009). "Time-resolved fluorescence studies of nucleotide flipping by restriction enzymes". Nucleic Acids Research. 37 (20): 6859–6870. doi:10.1093/nar/gkp688. PMC   2777440 . PMID   19740769.
  45. ↑ Serva, S (1 August 1998). "Chemical display of thymine residues flipped out by DNA methyltransferases". Nucleic Acids Research. 26 (15): 3473–3479. doi:10.1093/nar/26.15.3473. PMC   147733 . PMID   9671807.
  46. ↑ Daujotyte, D.; Liutkeviciute, Z.; Tamulaitis, G.; Klimasauskas, S. (15 April 2008). "Chemical mapping of cytosines enzymatically flipped out of the DNA helix". Nucleic Acids Research. 36 (10): e57. doi:10.1093/nar/gkn200. PMC   2425465 . PMID   18450817.
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