DNA base flipping

{{Use American English|date = February 2019}} {{Short description|Biochemical process}} thumb|400px | right |alt=Cartoon of DNA with a base flipped out|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.<ref>{{cite journal |last1=Roberts |first1=Richard J. |last2=Cheng |first2=Xiaodong |title=BASE FLIPPING |journal=Annual Review of Biochemistry |date=June 1998 |volume=67 |issue=1 |pages=181–198 |doi=10.1146/annurev.biochem.67.1.181 |pmid=9759487}}</ref> 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<ref>{{cite journal |last1=Reiter |first1=Nicholas J. |last2=Blad |first2=Heike |last3=Abildgaard |first3=Frits |last4=Butcher |first4=Samuel E. |title=Dynamics in the U6 RNA Intramolecular Stem−Loop: A Base Flipping Conformational Change |journal=Biochemistry |date=1 November 2004 |volume=43 |issue=43 |pages=13739–13747 |doi=10.1021/bi048815y |pmid=15504036 |s2cid=25391616}}</ref> or in the DNA:RNA intermediates formed during RNA transcription.

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.<ref name=original>{{cite journal |last1=Klimasauskas |first1=Saulius |last2=Kumar |first2=Sanjay |last3=Roberts |first3=Richard J. |last4=Cheng |first4=Xiaodong |title=Hhal methyltransferase flips its target base out of the DNA helix |journal=Cell |date=January 1994 |volume=76 |issue=2 |pages=357–369 |doi=10.1016/0092-8674(94)90342-5 |pmid=8293469|s2cid=23161543}}</ref> 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.<ref name=original /> 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.<ref name=original />

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.<ref name=original /> 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.<ref name="Brown">{{cite web|last=Brown|first=Tom|title=Nucleic Acids Book|url=https://atdbio.com/nucleic-acids-book/Base-flipping|access-date=26 February 2014 |website=ATDBio }}</ref><ref name="Huang2003">{{cite journal |last1=Huang |first1=Niu |last2=Banavali |first2=Nilesh K. |last3=MacKerell |first3=Alexander D. |title=Protein-facilitated base flipping in DNA by cytosine-5-methyltransferase |journal=Proceedings of the National Academy of Sciences |date=7 January 2003 |volume=100 |issue=1 |pages=68–73 |doi=10.1073/pnas.0135427100 |pmid=12506195|pmc=140885|bibcode=2003PNAS..100...68H|doi-access=free}}</ref><ref name="Grubmüller">{{cite web |last=Grubmüller |first=Helmut |title=DNA Base Flipping |url=http://www.mpibpc.mpg.de/259673/10-DNA-Base-Flipping |archive-url=https://web.archive.org/web/20170204004422/http://www.mpibpc.mpg.de/259673/10-DNA-Base-Flipping |archive-date=4 February 2017 |access-date=26 February 2014 |website=The Max Planck Institute for Biophysical Chemistry}}</ref>

==Mechanism==

[[File:Model of the EhMeth-DNA complex.jpg|thumb| 250px | left|alt=Model of interactions between DNA with flipped base and a methyltransferase |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<ref name=bouvier>{{cite journal |last1=Bouvier |first1=Benjamin |last2=Grubmüller |first2=Helmut |title=A Molecular Dynamics Study of Slow Base Flipping in DNA using Conformational Flooding |journal=Biophysical Journal |date=August 2007 |volume=93 |issue=3 |pages=770–786 |doi=10.1529/biophysj.106.091751 |pmid=17496048|pmc=1913169|bibcode=2007BpJ....93..770B}}</ref> by breaking the hydrogen bonds between bases and unstacking the base from its neighbors.<ref name=passive>{{cite journal |last1=Larivière |first1=Laurent |last2=Moréra |first2=Solange |title=Structural Evidence of a Passive Base-flipping Mechanism for β-Glucosyltransferase |journal=Journal of Biological Chemistry |date=August 2004 |volume=279 |issue=33 |pages=34715–34720 |doi=10.1074/jbc.M404394200 |pmid=15178685|doi-access=free}}</ref> The base is rotated out of the double helix by 180 degrees,<ref>{{cite book|editor-last=Grosjean |editor-first=Henri|title=DNA and RNA modification enzymes: structure, mechanism, function and evolution|year=2009|publisher=Landes Bioscience|location=Austin, Tex.|isbn=978-1-58706-329-9|url=https://www.landesbioscience.com/curie/chapter/4163/|access-date=2014-03-10|archive-url=https://web.archive.org/web/20140407075340/https://www.landesbioscience.com/curie/chapter/4163/|archive-date=2014-04-07|series=Molecular biology intelligence unit |oclc=316421190}}</ref> typically via the major groove,<ref name="Huang2003" /> and into the active site of an enzyme. This opening leads to small conformational changes in the DNA backbone<ref name=giudice>{{cite journal |last1=Giudice |first1=Emmanuel |last2=Várnai |first2=Péter |last3=Lavery |first3=Richard |title=Base pair opening within B-DNA: free energy pathways for GC and AT pairs from umbrella sampling simulations |journal=Nucleic Acids Research |date=1 March 2003 |volume=31 |issue=5 |pages=1434–1443 |doi=10.1093/nar/gkg239 |pmid=12595551|pmc=149832}}</ref> which are quickly stabilized by the increased enzyme-DNA interactions.<ref name="Huang2003" /> 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.<ref name="Huang2003" />

There are two mechanisms of DNA base flipping: active and passive.<ref name="O'neil dissertation">{{Cite thesis |title=Base Flipping: Detection, Structures and Energetics |last=O'Neil |first=Lauren L. |date=2008 |publisher=University of Notre Dame |doi=10.7274/9306sx63d8n}}</ref> 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.<ref name="passive"/> Research has demonstrated both mechanisms: uracil-DNA glycosylase follows the passive mechanism<ref name=passive /> and Tn10 transposase follows the active mechanism.<ref name=active>{{cite journal |last1=Bischerour |first1=Julien |last2=Chalmers |first2=Ronald |title=Base Flipping in Tn10 Transposition: An Active Flip and Capture Mechanism |journal=PLOS ONE |date=10 July 2009 |volume=4 |issue=7 |article-number=e6201 |doi=10.1371/journal.pone.0006201 |pmid=19593448|pmc=2705183|bibcode=2009PLoSO...4.6201B|doi-access=free}}</ref>

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.<ref name="Brown"/><ref name="Huang2003" /><ref name="Grubmüller"/>

==Biological processes==

===DNA modification and repair=== thumb|200px | right|alt=Model of uracil DNA glycosylase and flipped uracil residue|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<ref name="Huang2003" /> and into the specificity pocket of a glycosylase which hydrolyzes the glycosidic bond and removes the base.<ref name=textbook>{{cite book |last1=Watson |first1=James D. |last2=Baker |first2=Tania A. |last3=Bell |first3=Stephen P. |last4=Gann |first4=Alexander |last5=Levine |first5=Michael |last6=Losick |first6=Richard |title=Molecular biology of the gene |date=2014 |publisher=Pearson |location=Boston |isbn=978-0-321-76243-6 |edition=Seventh}}</ref> 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.<ref>{{cite journal |last1=Krokan |first1=Hans E |last2=Drabløs |first2=Finn |last3=Slupphaug |first3=Geir |title=Uracil in DNA – occurrence, consequences and repair |journal=Oncogene |date=16 December 2002 |volume=21 |issue=58 |pages=8935–8948 |doi=10.1038/sj.onc.1205996 |pmid=12483510}}</ref> Base flipping is used to repair mutations such as 8-Oxoguanine (oxoG)<ref>{{cite journal |last1=Banerjee |first1=Anirban |last2=Yang |first2=Wei |last3=Karplus |first3=Martin |last4=Verdine |first4=Gregory L. |title=Structure of a repair enzyme interrogating undamaged DNA elucidates recognition of damaged DNA |journal=Nature |date=March 2005 |volume=434 |issue=7033 |pages=612–618 |doi=10.1038/nature03458 |pmid=15800616|bibcode=2005Natur.434..612B|s2cid=4426014}}</ref> and thymine dimers created by UV radiation.<ref name=textbook /><ref>{{cite journal |last1=Fuxreiter |first1=Monika |last2=Luo |first2=Ning |last3=Jedlovszky |first3=Pál |last4=Simon |first4=István |last5=Osman |first5=Roman |title=Role of Base Flipping in Specific Recognition of Damaged DNA by Repair Enzymes |journal=Journal of Molecular Biology |date=November 2002 |volume=323 |issue=5 |pages=823–834 |doi=10.1016/S0022-2836(02)00999-3 |pmid=12417196 |hdl=2437/124488 }}</ref>

===Replication, transcription and recombination=== DNA replication and RNA transcription both make use of base flipping.<ref name="Huang2003" /> DNA polymerase is an enzyme that carries out replication. It can be thought of as a hand that grips the DNA single strand template.<ref name=textbook /> 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.<ref name="dna poly">{{cite journal |last1=Patel |first1=Premal H. |last2=Suzuki |first2=Motoshi |last3=Adman |first3=Elinor |last4=Shinkai |first4=Akeo |last5=Loeb |first5=Lawrence A. |title=Prokaryotic DNA polymerase I: evolution, structure, and "base flipping" mechanism for nucleotide selection |journal=Journal of Molecular Biology |date=May 2001 |volume=308 |issue=5 |pages=823–837 |doi=10.1006/jmbi.2001.4619 |pmid=11352575}}</ref> 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.<ref name=textbook /><ref>{{cite journal |last1=Lim |first1=Heon Man |last2=Lee |first2=Hee Jung |last3=Roy |first3=Siddhartha |last4=Adhya |first4=Sankar |title=A "master" in base unpairing during isomerization of a promoter upon RNA polymerase binding |journal=Proceedings of the National Academy of Sciences |date=18 December 2001 |volume=98 |issue=26 |pages=14849–14852 |doi=10.1073/pnas.261517398 |pmid=11734629 |pmc=64947|bibcode=2001PNAS...9814849L|doi-access=free}}</ref>

Base flipping occurs during latter stages of recombination.<ref>{{cite journal |last1=Voloshin |first1=Oleg N. |last2=Camerini-Otero |first2=R.Daniel |title=Synaptic Complex Revisited |journal=Molecular Cell |date=September 2004 |volume=15 |issue=6 |pages=846–847 |doi=10.1016/j.molcel.2004.09.010 |pmid=15383274|doi-access=free}}</ref> RecA is a protein that promotes strand invasion<ref name=textbook /> 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.<ref>{{cite journal |last1=Folta-Stogniew |first1=Ewa |last2=O'Malley |first2=Shawn |last3=Gupta |first3=Ravindra |last4=Anderson |first4=Karen S. |last5=Radding |first5=Charles M. |title=Exchange of DNA Base Pairs that Coincides with Recognition of Homology Promoted by ''E. coli'' RecA Protein |journal=Molecular Cell |date=September 2004 |volume=15 |issue=6 |pages=965–975 |doi=10.1016/j.molcel.2004.08.017 |pmid=15383285 |doi-access=free}}</ref> Other studies indicate that it is also involved in V(D)J Recombination.<ref>{{cite journal |last1=Bischerour |first1=Julien |last2=Lu |first2=Catherine |last3=Roth |first3=David B. |last4=Chalmers |first4=Ronald |title=Base Flipping in V(D)J Recombination: Insights into the Mechanism of Hairpin Formation, the 12/23 Rule, and the Coordination of Double-Strand Breaks |journal=Molecular and Cellular Biology |date=1 November 2009 |volume=29 |issue=21 |pages=5889–5899 |doi=10.1128/MCB.00187-09 |pmid=19720743 |pmc=2772739}}</ref>

===DNA methylation=== thumb|left|170px |alt=DNA methylation illustration|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.<ref name="Klose2006">{{cite journal |last1=Klose |first1=Robert J. |last2=Bird |first2=Adrian P. |title=Genomic DNA methylation: the mark and its mediators |journal=Trends in Biochemical Sciences |date=February 2006 |volume=31 |issue=2 |pages=89–97 |doi=10.1016/j.tibs.2005.12.008 |issn=0968-0004 |pmid=16403636}}</ref> 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.<ref>{{cite journal |last1=Nakao |first1=Mitsuyoshi |title=Epigenetics: interaction of DNA methylation and chromatin |journal=Gene |date=October 2001 |volume=278 |issue=1–2 |pages=25–31 |doi=10.1016/s0378-1119(01)00721-1 |pmid=11707319 }}</ref><ref>{{cite journal |last1=Plass |first1=Christoph |last2=Soloway |first2=Paul D |title=DNA methylation, imprinting and cancer |journal=European Journal of Human Genetics |date=1 January 2002 |volume=10 |issue=1 |pages=6–16 |doi=10.1038/sj.ejhg.5200768 |pmid=11896451 |doi-access=free }}</ref><ref>{{cite journal |last1=Esteller |first1=Manel |last2=Herman |first2=James G. |author2-link=James G. Herman |title=Cancer as an epigenetic disease: DNA methylation and chromatin alterations in human tumours |journal=The Journal of Pathology |date=January 2002 |volume=196 |issue=1 |pages=1–7 |doi=10.1002/path.1024 |pmid=11748635 |s2cid=35380651 }}</ref> 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.<ref name="Huang2003" />

===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.<ref>{{Cite web |url=http://arbl.cvmbs.colostate.edu/hbooks/genetics/biotech/enzymes/renzymes.html |title=Biology and Activity of Restriction Endonucleases |access-date=2014-04-03 |archive-url=https://web.archive.org/web/20140418083718/http://arbl.cvmbs.colostate.edu/hbooks/genetics/biotech/enzymes/renzymes.html |archive-date=2014-04-18 }}</ref> 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.<ref>{{cite journal | last1 = Horton | first1 = John R. | last2 = Zhang | first2 = Xing | last3 = Maunus | first3 = Robert | last4 = Yang | first4 = Zhe | last5 = Wilson | first5 = Geoffrey | last6 = Roberts | first6 = Richard | last7 = Cheng | first7 = Xiaodong | year = 2006 | title = DNA Nicking by HinP1I Endonuclease: Bending, Base Flipping and Minor Groove Expansion | journal = Nucleic Acids Research | volume = 34 | issue = 3| pages = 939–948 | doi=10.1093/nar/gkj484 | pmid=16473850 | pmc=1363774}}</ref> 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=== thumb|right|200px |alt= X-ray crystallography workflow|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.{{Citation needed|date=January 2025}} Klimasaukas and colleagues used this technique to observe the first base flipping phenomenon, in which their experimental procedure involved several steps:<ref name=original /> # Purification # Crystallization # Data Collection # 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.<ref>{{cite journal |last1=Kumar |first1=Sanjay |last2=Cheng |first2=Xiaodong |last3=Pflugrath |first3=James W. |last4=Roberts |first4=Richard J. |title=Purification, crystallization, and preliminary x-ray diffraction analysis of an M.HhaI-AdoMet complex |journal=Biochemistry |date=15 September 1992 |volume=31 |issue=36 |pages=8648–8653 |doi=10.1021/bi00151a035 |pmid=1390649}}</ref> 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.<ref name="ReferenceA">{{cite journal |last1=Cheng |first1=Xiaodong |last2=Kumar |first2=Sanjay |last3=Posfai |first3=Janos |last4=Pflugrath |first4=James W. |last5=Roberts |first5=Richard J. |title=Crystal structure of the Hhal DNA methyltransferase complexed with S-adenosyl-l-methionine |journal=Cell |date=July 1993 |volume=74 |issue=2 |pages=299–307 |doi=10.1016/0092-8674(93)90421-l |pmid=8343957 |s2cid=54238106}}</ref> 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<ref name="ReferenceA"/> where the search models X-PLOR, MERLOT, and TRNSUM were used to solve the rotation and translation functions.<ref>{{cite book |last1=Brünger |first1=Axel T. |title=X-PLOR, Version 3.1: a system for X-ray crystallography and NMR |date=1992 |publisher=Yale University Press |location=New Haven |isbn=978-0-300-05402-6}}</ref><ref>{{cite journal |last1=Fitzgerald |first1=P. M. D. |title=MERLOT, an integrated package of computer programs for the determination of crystal structures by molecular replacement |journal=Journal of Applied Crystallography |date=1 June 1988 |volume=21 |issue=3 |pages=273–278 |doi=10.1107/s0021889887012299 |bibcode=1988JApCr..21..273F }}</ref> 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.{{Citation needed|date=January 2025}} In addition, NMR can provide a variety of information including structure, reaction states, chemical environment of the molecules, and dynamics.<ref>{{cite book |last1=Guéron |first1=Maurice |last2=Leroy |first2=Jean-Louis |title=Studies of base pair kinetics by NMR measurement of proton exchange |series=Methods in Enzymology |date=1995 |volume=261 |pages=383–413 |doi=10.1016/s0076-6879(95)61018-9 |pmid=8569504 |isbn=978-0-12-182162-3 }}</ref><ref>{{cite journal | last1 = Leijon | first1 = M. | last2 = Graslund | first2 = A. | year = 1992 | title = Effects of sequence and length on imino proton-exchange and basepair opening kinetics in DNA oligonucleotide duplexes | journal = Nucleic Acids Res | volume = 20 | issue = 20| pages = 5339–5343 | doi=10.1093/nar/20.20.5339| pmid = 1331987 | pmc = 334339 }}</ref> 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 <sup>19</sup>F chemical shift analysis could be performed. Once the <sup>19</sup>F 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.<ref name=grosjean>{{cite book |last1=Klimašauskas |first1=Saulius |last2=Liutkevičiūtė |first2=Zita |editor1-last=Grosjean |editor1-first=Henri |title=DNA and RNA modification enzymes: structure, mechanism, function and evolution |date=2009 |publisher=Landes Bioscience |location=Austin, Texas |isbn=978-1-58706-329-9 |chapter=Experimental Approaches to Study DNA Base Flipping}}</ref>

===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.<ref name=grosjean /> 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.<ref name=flurores>{{cite journal|last=Holz|first=B|title=2-Aminopurine as a fluorescent probe for DNA base flipping by methyltransferases|journal=Nucleic Acids Research|date=15 February 1998|volume=26|issue=4|pages=1076–1083|doi=10.1093/nar/26.4.1076|pmid=9461471|pmc=147370}}</ref> 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)<ref name=6map>{{cite journal |last1=Yang |first1=Kongsheng |last2=Matsika |first2=Spiridoula |last3=Stanley |first3=Robert J. |title=6MAP, a Fluorescent Adenine Analogue, Is a Probe of Base Flipping by DNA Photolyase |journal=The Journal of Physical Chemistry B |date=1 September 2007 |volume=111 |issue=35 |pages=10615–10625 |doi=10.1021/jp071035p |pmid=17696385}}</ref> and Pyrrolo‑C (3-[β-D-2-ribofuranosyl]-6-methylpyrrolo[2,3-d]pyrimidin-2(3H)-one).<ref>{{cite journal |last1=Yang |first1=Kongsheng |last2=Stanley |first2=Robert J. |title=The Extent of DNA Deformation in DNA Photolyase– Substrate Complexes: A Solution State Fluorescence Study |journal=Photochemistry and Photobiology |date=May 2008 |volume=84 |issue=3 |pages=741–749 |doi=10.1111/j.1751-1097.2007.00251.x |pmid=18086248|s2cid=44506405}}</ref><ref>{{cite journal |last1=Berry |first1=David A. |last2=Jung |first2=Kee-Yong |last3=Wise |first3=Dean S. |last4=Sercel |first4=Anthony D. |last5=Pearson |first5=William H. |last6=Mackie |first6=Hugh |last7=Randolph |first7=John B. |last8=Somers |first8=Robert L. |title=Pyrrolo-dC and pyrrolo-C: fluorescent analogs of cytidine and 2′-deoxycytidine for the study of oligonucleotides |journal=Tetrahedron Letters |date=March 2004 |volume=45 |issue=11 |pages=2457–2461 |doi=10.1016/j.tetlet.2004.01.108}}</ref> 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.<ref name=time-resolved>{{cite journal |last1=Neely |first1=Robert K. |last2=Tamulaitis |first2=Gintautas |last3=Chen |first3=Kai |last4=Kubala |first4=Marta |last5=Siksnys |first5=Virginijus |last6=Jones |first6=Anita C. |title=Time-resolved fluorescence studies of nucleotide flipping by restriction enzymes |journal=Nucleic Acids Research |date=November 2009 |volume=37 |issue=20 |pages=6859–6870 |doi=10.1093/nar/gkp688 |pmid=19740769 |pmc=2777440}}</ref>

===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.<ref name=potass-perman-probe>{{cite journal |last1=Serva |first1=Saulius |last2=Weinhold |first2=Elmar |last3=Roberts |first3=Richard J. |last4=Klimasauskas |first4=Saulius |title=Chemical display of thymine residues flipped out by DNA methyltransferases |journal=Nucleic Acids Research |date=1 August 1998 |volume=26 |issue=15 |pages=3473–3479 |doi=10.1093/nar/26.15.3473 |pmid=9671807|pmc=147733}}</ref> Chloroacetaldehyde is used to detect cytosine residues flipped out by the HhaI DNA cytosine-5 methyltransferase (M. HhaI).<ref name="cca probe">{{cite journal |last1=Daujotyte |first1=D. |last2=Liutkeviciute |first2=Z. |last3=Tamulaitis |first3=G. |last4=Klimasauskas |first4=S. |title=Chemical mapping of cytosines enzymatically flipped out of the DNA helix |journal=Nucleic Acids Research |date=15 April 2008 |volume=36 |issue=10 |pages=e57 |doi=10.1093/nar/gkn200 |pmid=18450817 |pmc=2425465}}</ref>

thumb|800px|center|alt=DNA showing a hybridization probe|A hybridization probe is added to a DNA molecule.

==References== {{reflist|2}}

Category:Molecular biology Base flipping