14.13 Base flipping is used by methylases and glycosylases
Figure 14.29 A glycosylase removes a base from DNA by cleaving the bond to the deoxyribose.
Figure 14.30 A glycosylase hydrolyzes the bond between base and deoxyribose (using H20), but a lyase takes the reaction further by pening the sugar ring (using NH2).
As an alternative to the conventional removal of part of a polynucleotide chain by nuclease activity, glycosylases and lyases can remove bases from the chain. Figure 14.29 shows that a glycosylase cleaves the bond between the damaged or mismatched base and the deoxyribose. Figure 14.30 shows that some glycosylases are also lyases that can take the reaction a stage further by using an amino (NH2) group to attack the deoxyribose ring. Although the results of the glycosylase and lyase reaction appear different, the basic mechanisms of their attack on the DNA are similar (for review see McCullough, Dodson, and Lloyd, 1999).
Figure 14.31 A methylase "flips" the target cytosine out of the double helix in order to modify it. Photograph kindly provided by Rich Roberts.
The interaction of these enzymes with DNA is remarkable. It follows the model first demonstrated for methyltransferases Xenzymes that add a methyl group to cytosine in DNA. The methylase flips the target cytosine completely out of the helix (Klimasauskas et al., 1994). Figure 14.31 shows that it enters a cavity in the enzyme where it is modified. Then it is returned to its normal position in the helix. All this occurs without input of an external energy source.
One of the most common reactions in which a base is directly removed from DNA is catalyzed by uracil-DNA glycosylase. Uracil occurs in DNA most typically because of a (spontaneous) deamination of cytosine. It is recognized by the glycosylase and removed. The reaction is similar to that of the methylase: the uracil is flipped out of the helix and into the active site in the glycosylase (Savva et al., 1995; Mol et al., 1995).
The common feature of the methylase and the uracil-DNA glycosylase is the flipping of the target base into the enzyme structure. A variation on this theme is used by T4 endonuclease V, now renamed T4-pdg (pyrimidine dimer glycosylase) to reflect its mode of action. It flips out the adenine base that is complementary to the thymine on the 5’ side of the pyrimidine dimer (Vassylyev et al., 1995). So in this case, the target for the catalytic action of the enzyme remains in the DNA duplex, and the enzyme uses flipping as an indirect mechanism to get access to its target.
This section updated 5-16-2000
Reviews
McCullough, A. K. , Dodson, M. L. , and Lloyd, R. S. (1999). Initiation of base excision repair: glycosylase mechanisms and structures.. Ann. Rev. Biochem 68, 255-285.
Research
Klimasauskas, S., Kumar, S., Roberts, R. J. and Cheng, X. (1994). HhaI methyltransferase flips its target base out of the DNA helix.. Cell 76, 357-369.
Mol, D. D. et al. (1995). Crystal structure and mutational analysis of human uracil-DNA glycosylase: structural basis for specificity and catalysis.. Cell 80, 869-878.
Savva, R. et al. (1995). The structural basis of specific base-excision repair by uracil-DNA glycosylase.. Nature 373, 487-493.
Vassylyev, D. G. et al. (1995). Atomic model of a pyrimidine dimer excision repair enzyme complexed with a DNA substrate: structural basis for damaged DNA recognition.. Cell 83, 773-782.