DNA base flipping

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. 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 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. 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. 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. 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. 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. 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. DNA nucleotides are held together with hydrogen bonds, which are relatively weak and can be easily broken. Base flipping occurs on a millisecond timescale by breaking the hydrogen bonds between bases and unstacking the base from its neighbors. The base is rotated out of the double helix by 180 degrees., typically via the major groove, and into the active site of an enzyme. This opening leads to small conformational changes in the DNA backbone which are quickly stabilized by the increased enzyme-DNA interactions. 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. There are two mechanisms of DNA base flipping: active and passive. 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. Research has demonstrated both mechanisms: uracil-DNA glycosylase follows the passive mechanism and Tn10 transposase follows the active mechanism. 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. 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 and into the specificity pocket of a glycosylase which hydrolyzes the glycosidic bond and removes the base. 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. Base flipping is used to repair mutations such as 8-Oxoguanine (oxoG) and thymine dimers created by UV radiation.