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Base Flipping P1: DPI/shd/rvt P2: NBL/rpk May 16, 1998 14:32 Annual Reviews AR057-08 Annu. Rev. Biochem. 1998. 67:181–98 Copyright c 1998 by Annual Reviews. All rights reserved BASE FLIPPING Richard J. Roberts New England Biolabs, Beverly, Massachusetts 01915; e-mail: [email protected] Xiaodong Cheng Department of Biochemistry, Emory University School of Medicine, Atlanta, Georgia 30322; e-mail: [email protected] KEY WORDS: DNA repair enzymes, DNA methyltransferases, DNA-modifying enzymes, evolution ABSTRACT Base flipping is the phenomenon whereby a base in normal B-DNA is swung com- pletely out of the helix into an extrahelical position. It was discovered in 1994 when the first co-crystal structure was reported for a cytosine-5 DNA methyltrans- ferase binding to DNA. Since then it has been shown to occur in many systems where enzymes need access to a DNA base to perform chemistry on it. Many DNA glycosylases that remove abnormal bases from DNA use this mechanism. This review describes systems known to use base flipping as well as many sys- tems where it is likely to occur but has not yet been rigorously demonstrated. The mechanism and evolution of base flipping are also discussed. CONTENTS INTRODUCTION ...........................................................182 KNOWN BASE-FLIPPING SYSTEMS ..........................................183 HhaI DNA Methyltransferase ...............................................184 HaeIII DNA Methyltransferase ..............................................187 Human Uracil DNA Glycosylase .............................................187 T4 Endonuclease V .......................................................188 PROBABLE BASE-FLIPPING SYSTEMS........................................189 The Amino-Methyltransferases M.TaqI and M.PvuII .............................189 E. coli DNA Photolyase ...................................................190 E. coli Endonuclease III ...................................................191 3-Methyladenine DNA Glycosylase (AlkA) .....................................191 E. coli Exonuclease III ....................................................192 T4β-Glucosyltransferase ...................................................192 E. coli Ada O6-Methylguanine DNA Methyltransferase ..........................192 E. coli Mismatch-Specific Uracil DNA Glycosylase ..............................193 181 0066-4154/98/0701-0181$08.00 P1: DPI/shd/rvt P2: NBL/rpk July 25, 1998 10:34 Annual Reviews AR057-08 182 ROBERTS & CHENG T7 ATP-Dependent DNA Ligase and T7 DNA Polymerase .........................193 MECHANISM ..............................................................194 THE ORIGINS OF BASE FLIPPING ............................................195 FUTURE PROSPECTS .......................................................196 INTRODUCTION The binding of proteins to DNA is crucial to life. Whereas some proteins bind to control transcription and replication, others both bind to DNA and catalyze chemical reactions on the DNA. The latter proteins include nucleases, gly- cosylases, DNA methyltransferases, and various enzymes such as integrases and recombinases, which rearrange DNA segments. For proteins such as tran- scription factors and repressors, whose function rests with DNA binding, some common structural features have been identified, such as zinc fingers and helix- turn-helix motifs (1). The binding to DNA by these proteins frequently involves small deformations in the usual B helix, although in some cases extreme dis- tortions can be found, such as the saddle structure on which the TATA-binding protein sits (2, 3) or the bends induced by integration host factor (4). Some nucleases, such as R.EcoRI (5) and R.EcoRV (6), which cleave the phospho- diester backbone, also induce conformational changes in the DNA helix, but for the most part these changes are not dramatic. This is probably because the target phosphodiester bonds lie on the outside of the helix and thus are readily accessible to the enzyme. For proteins that need to interact with the bases rather than the phosphodiester backbone and then perform chemistry on those bases, accessibility is less clear. How might such proteins gain access to the interior of the helix? Although it seems possible that the bases might become accessible by distortion of the helix through bending and kinking, it is by no means simple to envision. Obviously, a structure is needed for such a protein actually binding to DNA. In 1993 a structure was obtained for the cytosine-5 DNA methyltransferase, M.HhaI, an enzyme that needed access to its target cytosine base to perform the chemistry of methylation within the aromatic ring (7, 8). The crystal con- tained both the methyltransferase and its cofactor, S-adenosyl-L-methionine (AdoMet), but no DNA. From the structure, and the accumulated biochemi- cal knowledge about the enzyme, some clear predictions could be made about where the DNA would be located. However, the structure gave little hint of the rather dramatic conformational change in DNA that would take place upon its binding. That change was revealed in 1994, when a ternary structure was reported for a complex between M.HhaI, its DNA substrate, and the reaction product, S-adenosyl-L-homocysteine (9). Surprisingly, the enzyme did not dis- tort the DNA in some crude fashion by bending or kinking, but rather the target P1: DPI/shd/rvt P2: NBL/rpk July 25, 1998 10:34 Annual Reviews AR057-08 BASE FLIPPING 183 cytosine had swung completely out of the helix and into the active-site pocket of the enzyme (Figure 1, C-1, color section at end of volume). The base had undergone a conformational shift of 180, and the first example of base flipping had been discovered. In retrospect, this shift makes sense, because it probably provides the simplest method for a protein to access the interior of a DNA helix. Because of the overall similarity at the primary sequence level of all cytosine-5 DNA methyltransferases (10), it seemed reasonable that base flipping was not just an isolated quirk unique to M.HhaI but would be found in all members of this set of methyltransferases. It was thus reassuring when a structure for a second methyltransferase, M.HaeIII, appeared (11). It showed essentially the same phenomenon, albeit with some differences that could be attributed to differences in the recognition sequences. The big question, though, was whether base flipping would show up in other situations. In our original pa- per we proposed that other classes of DNA methyltransferases and some DNA glycosylases might also use base flipping to gain access to the bases within the helix (9). Both of these predictions proved accurate, although in the case of T4 endonuclease V (12), the base flipping is not quite as we had envisioned (see below). Many other examples have now appeared, either as co-crystal struc- tures of DNA-protein complexes or from structures of the native enzymes with or without cofactors. In the latter cases, it has often proved possible to infer that base flipping may be involved because modeling of normal B-DNA into these structures fails to bring the target base close to the active site—a situation that is remedied if the base is flipped. It has been argued that base flipping is an ancient process that may even have preceded the use of DNA as the genetic material (13). If that is the case, then one might expect to find other examples of base flipping among RNA enzymes and in other proteins that interact with DNA. One might even imagine that base flipping could nucleate the opening of a helix, as would be required during the initiation of replication or transcription. In this review we summarize the progress that has been made in studies of base flipping since its discovery in 1994 and discuss, somewhat speculatively, its mechanism and evolution. We present the range of processes where base flipping has been proven to occur, further cases where it is postulated to occur, and a few examples where it might be worth looking for its involvement. KNOWN BASE-FLIPPING SYSTEMS Base flipping has been directly observed in four systems (Table 1): in the co- crystal structures for the cytosine-5 DNA methyltransferases M.HhaI (9) and M.HaeIII (11), in a catalytically compromised form of T4 endonuclease V (12), and in human uracil DNA glycosylase (14). P1: DPI/shd/rvt P2: NBL/rpk July 25, 1998 10:34 Annual Reviews AR057-08 184 ROBERTS & CHENG Table 1 Known base-flipping systems Specific protein Catalytic reaction Reference HhaI DNA methyltransferase Forms G-5mC-GC in DNA 9 HaeIII DNA methyltransferase Forms GG-5mC-C in DNA 11 T4 endonuclease V Removes pyrimidine dimers from DNA 12 Human uracil DNA glycosylase Removes uracil from DNA 14 HhaI DNA Methyltransferase HhaI DNA methyltransferase (M.HhaI), a 327–amino acid residue protein, methylates the internal cytosine of its recognition sequence 50-GCGC-30/30- CGCG-50 (15, 16). Despite its palindromic recognition sequence, the protein functions as a monomer, methylating only one strand at a time. The methyl donor AdoMet is required for enzymatic activity and is converted to S-adenosyl- L-homocysteine (AdoHcy) during methylation. The structure of M.HhaI has been characterized extensively by X-ray crystal- lography in complexes with various forms of its DNA substrate (9, 17–19). The protein has two lobes: a conserved AdoMet-dependent catalytic domain and a DNA-recognition domain (7, 8, 20, 21). Nine ternary structures of M.HhaI- DNA-cofactor have been determined, with either AdoMet or AdoHcy. Each structure contains a 13-mer oligonucleotide duplex with a different
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