Base Flipping

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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 completely 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 methyltransferase 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 systems 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

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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, glycosylases, DNA methyltransferases, and various enzymes such as integrases and recombinases, which rearrange DNA segments. For proteins such as transcription 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 phosphodiester 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 contained both the methyltransferase and its cofactor, S-adenosyl-L-methionine (AdoMet), but no DNA. From the structure, and the accumulated biochemical 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

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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 structures 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).

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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 combina- tion of modiﬁcations at the position of the target cytosine within the recognition sequence in the two DNA strands. Table 2 lists the oligonucleotides used in the M.HhaI ternary complexes and the nomenclatures used.

5-FLUORO-20-DEOXYCYTIDINE, 20-DEOXYCYTIDINE, AND 5-METHYL-20-DEOXYCY- TIDINE The first three structures listed in Table 2 contain identical self- complementary DNA sequences. The complex F13 contains 5-fluoro-20-deoxycytidine (5FC or F) and was crystallized in the presence of AdoMet, which resulted in the formation of a covalent linkage between the enzyme and DNA and generation of 5-methyl-5-fluoro-20-deoxydihydrocytidine and AdoHcy (9). The N13 structure contains unmodified deoxycytidine, whereas the M13 contains 5-methyl-20-deoxycytidine (5mC or M) (17). The structures are rather similar, with one of the target nucleotides flipped out of the DNA helix and fitting snugly into the active site of the enzyme (Figure 1, C-1, color section). The 5mC residue in the fully methylated oligonucleotide in M13, the reaction product, is flipped out of the DNA helix in the same manner as with the C in N13 and 5FC in F13. This is surprising but consistent with biochemical data, which suggest that the binding specificity for M.HhaI is asymmetric 50-GXGC- 30/30-CGCG-50 and determined by the nucleotides neighboring the target (X)

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Table 2 DNA sequences used in M.HhaI ternary structures

DNAa Xb Cofactor NameResolution (A)û Reference

50-TGATAGCXG TATC -30 F AdoMet F13 2.8 9 30- CTATCGXGATAGT-50 C AdoHcy N13 2.7 17 M AdoHcy M13 2.7 17

50-TGTCAGGCX A-3TGG 0C AdoHcy HM13 2.7 18 30-GCA TCGMGTACCT-50 S AdoMet S13 2.05 19 Z AdoHcy Z13 2.5 c d 50-TGTCAGGX CATGG -30 AP AdoHcy AP13 2.4 30-GCA TCGCGTACCT -50 d 50-TGTCAGCGCATGG -30 A AdoHcy GA13 2.8 d 30-GTCA CGX GTACC T-50 U AdoHcy GU13 2.7 aThe recognition sequence is in italics, and the ﬂipped nucleotide is in bold and underlined. bA adenine, C cytosine, U uracil, M 5-methyl-2 -deoxycytidine, F 5-ﬂuoro-2 -deoxycytidine, = = = = 0 = 0 S 40-thio-20-deoxycytidine, Z 5,6-dihydro-5-azacytidine, AP abasic sugar. =cJK Christman, R Sheikhnejad,= V Marquez, C Marasco, J Sufrin,= M O’Gara, X Cheng, unpublished data. dM O’Gara, JR Horton, RJ Roberts, X Cheng, unpublished data.

nucleotide (22). In other words, the methyltransferase does not depend on the flippable base for its binding specificity. The HM13 structure (the fourth in Table 1) contained a hemimethylated DNA substrate with a nonpalindromic sequence (18). The two strands of DNA are clearly nonequivalent, because only the unmethylated target C flips out of the DNA helix, whereas the 5mC on the complementary strand remains stacked in the DNA helix. This seems to be an important aspect of DNA methylation; that is, the ability of DNA methyltransferases to distinguish DNA substrates with methyl groups on one strand (hemimethylated DNA) from those that carry no methyl groups. However, many of the bacterial type II enzymes such as M.HhaI are equally active on unmethylated and hemimethylated DNA, with an increased affinity for asymmetrically methylated DNA. Consequently they function as both maintenance and de novo methyltransferases. Both the N4 (NH2) group and the methyl group of 5mC on the complementary strand interact with the protein (18); these interactions may enable the de novo methyltransferase to recognize both unmodified cytosine and methylated cytosine.

40-THIO-20-DEOXYCYTIDINE AND DIHYDRO-5-AZACYTIDINE That M.HhaI does not show much binding specificity for the flippable base may reflect a need to leave that base unencumbered by recognition contacts. This provides an oppor- tunity to probe the structural and chemical interactions involved in sequence- specific recognition and catalysis using nucleotide analogs incorporated into synthetic oligonucleotides in the position of the target nucleotide of M.HhaI. So

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far, two nucleotide analogs—a 40-thionucleoside and 5,6-dihydro-5-azacytosine —have been used in our studies. When 40-thio-20-deoxycytidine is incorporated as the target cytosine in the recognition sequence for M.HhaI, binding to the 40-thio–modified DNA is almost identical to that of the unmodified DNA under equilibrium conditions (19). In contrast, the methyl transfer was strongly inhibited in solution. Sur- prisingly, the flipped 40-thio-20-deoxycytidine in the S13 crystal structure was partially methylated (19). These results show that 40-thio-20-deoxycytidine does not disrupt DNA recognition, binding, or base flipping by M.HhaI. Instead they suggest that it interferes with a step in the methylation reaction after flipping but prior to methyl transfer. 5,6-Dihydro-5-azacytosine (DHAC) riboside was originally synthesized to obtain a hydrolytically stable replacement for the antileukemic drug 5-azacytosine riboside (23, 24). DHAC contains a cytosine-like ring lacking aromatic 3 character with an sp -hybridized carbon (CH2 group) at position 6 and an NH group at position 5, resembling the transition state of a dihydrocytosine intermediate in the reaction of cytosine-5 DNA methyltransferases (25). The Z13 structure, containing DHAC as the target (Table 2), showed that DHAC is also flipped out of the DNA helix similarly to C, 5mC, and 5FC, but with no covalent bond formed between the sulfur atom of nucleophile Cys81 and the pyrimidine C6 carbon (JK Christman, R Sheikhnejad, V Marquez, C Marasco, J Sufrin, M O’Gara & X Cheng, unpublished data). This result indicates that the DHAC-containing DNA is sufficient to produce strong inhibition of the DNA methyltransferase and that the DHAC moiety occupies the active site of M.HhaI as a transition-state mimic.

MISMATCHED BASES (G:A, G:U, AND G:AP) Following the discovery of base flipping by M.HhaI, the effects of replacing the target cytosine by mismatched bases, including adenine, guanine, thymine, and uracil, were investigated (22, 26). By electrophoretic mobility shift analysis, M.HhaI and M.HpaII were found to bind even more tightly to such mismatched substrates; the highest affinity was for a gap formed by removal of the target nucleotide and both phosphodiester linkages (22). Furthermore, the uracil can be enzymatically methylated and converted to thymine at low efficiencies (22, 26), and the binding of these methyltransferases at the G:U mismatch prevented its repair by uracil DNA glycosylase in vitro (26). So far, three well-refined ternary structures of M.HhaI complexed with AdoHcy and a nonpalindromic oligonucleotide containing a G:A, G:U, or G:AP (AP apurinic/apyrimidinic abasic) mismatch at the target base pair, respectively,= have been determined= (M O’Gara, JR Horton, RJ Roberts & X Cheng, unpublished data). The mismatched adenine, uracil, and abasic site

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are flipped out and located in the enzyme’s active site, respectively. It seems likely that this active-site pocket is nonspecific for binding but specific for methylation. In light of the nonspecific binding pocket, the DNA methyltransferase may be related more to repair enzymes such as 3-methyladenine DNA glycosylase II and endonuclease III, which have broad substrate specificity. On the other hand, the methylation reaction is specific in that catalysis occurs only when the flipped base is cytosine or uracil (at low efficiency), more closely resembling uracil DNA glycosylase. HaeIII DNA Methyltransferase HaeIII DNA methyltransferase (M.HaeIII), which also belongs to the mono- meric type II bacterial methyltransferases, acts at its substrate site in palindromic DNA and modifies the recognition sequence in two independent methyl transfer events—50-GGCC-30/30-CCGG-50—in double-stranded DNA. It methylates either of the underlined cytosines. In the structure of M.HaeIII bound to hemimethylated DNA, the substrate cytosine is flipped out from the DNA helix (Figure 2, C-1, color section), as observed for M.HhaI. M.HaeIII differs from M.HhaI in that the flipping out of the cytosine is accompanied by rearrangement of the remaining recognition bases in their pairing (11). Between M.HhaI and M.HaeIII, the protein-base contacts in the recognized sequence are expected to differ because of their different specificity. Indeed, the folding of the corresponding DNA-recognition domains is different (11). How- ever, these protein-base contacts share two important features (27). For both methyltransferase-DNA complexes, six phosphates on the methylated strand contact the protein: three on the 50 side and three on the 30 side of the target cytosine, regardless of the position of the target nucleotide within the recognition sequence (second of four for M.HhaI, 50-GCGC-30, and third of four for M.HaeIII, 50-GGCC-30). From the protein side, a conserved arginine is responsible for the recognition of the guanine 50 to the target cytosine. These shared recognition patterns may be common to other 5mC methyltransferases recognizing a guanine 50 to the target base (27, 28). Human Uracil DNA Glycosylase Human uracil DNA glycosylase (UDG) is responsible for the removal of uracil residues within either single- or double-stranded DNA. The crystal structures of the human (29, 30) and herpes simplex viral (31, 32) forms of the enzyme have been solved. They revealed an extraordinarily specific binding pocket that excludes normal DNA bases and uracil within RNA from specific binding. Clearly these interactions could not form if the uracil was positioned inside a B-DNA helix, so it seemed likely that base flipping could be involved. The involvement of base flipping has now been confirmed with the description

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of a co-crystal structure for human UDG complexed with a uracil-containing double-stranded DNA (Figure 3, C-2, color section) (14). This structure, which was solved using a double mutant of UDG (Leu272Arg and Asp145Asn) showed that the uracil, the deoxyribose, and the 50 phosphate are rotated 180 from their starting structure within DNA, with Arg272 inserted into the position previously occupied by uracil. Even though the catalytic efficiency of this double mutant was severely reduced, the N-Cl0 glycosidic bond had been broken (Figure 3, C-2, color section). The DNA conformation essentially maintained the B form, except for the flipped nucleotide. The conformation of the flipped-out nucleotide is very similar to that observed in the M.HhaI-DNA complex, and the protein-DNA interactions are concentrated along the sugar-phosphate backbone of the strand containing the flipped-out uracil (14). Thus, the four structurally characterized base-flipping proteins, M.HhaI, M.HaeIII, human UDG, and T4 endonuclease V (see below), have all shown that the DNA phosphate-protein contacts are made mainly on one particular strand (reviewed in 27). It was suggested that UDG uses a push-and-pull mechanism of base flipping to ultimately achieve specific binding and catalysis (14). The push (protein invasion) is contributed by Leu272: It displaces the target base through hydrophobic interactions with DNA. The pull (trapping the flipped nucleotide) is derived by satisfying the specific binding pocket with uracil. T4 Endonuclease V T4 endonuclease V is a DNA glycosylase/AP lyase that can initiate repair of cis-syn cyclobutane pyrimidine dimers in DNA by cleaving the glycosydic bond of the 50 pyrimidine and then cleaving the phosphodiester backbone (33). Un- like M.HhaI and UDG, T4 endonuclease V kinks the dimer-containing DNA, at an angle of approximately 60 (Figure 4, C-3, color section) (12). In contrast to UDG, endonuclease V does not flip out the damaged bases into a binding pocket. Instead, it moves the nucleotide opposite the 50 pyrimidine of the dimer into a binding pocket on the surface of the enzyme (Figure 4, C-3, color section). In the co-crystal structure, a flipped adenine lies in proximity of and sandwiched between two layers of protein atoms, both of which are arranged parallel to the base plane. Vassylyev et al (12) suggested that the adenine was stabilized by van der Waals interactions. Interestingly, the pocket into which the adenine flips does not provide specific contacts that allow unambiguous recognition of the base. This lack of specific recognition has been seen biochemically (34; unpublished data cited in 12) and shows that the glycosylase activity is unaffected by the nature of the base opposite the 50-lesion. However, the key feature associated with endonuclease V’s base flipping is that the hole in DNA, which is created by movement of the base, is filled by

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the enzyme inserting its active-site residues into that hole. Thus, through the change in the structure of the DNA, the enzyme is correctly positioned to carry out a nucleophilic attack on Cl0 of the 50 pyrimidine of the dimer.

PROBABLE BASE-FLIPPING SYSTEMS Several crystal structures have appeared for proteins that interact with DNA. In these structures, it seems improbable that proper interaction could occur if the DNA maintained its normal B conformation. However, in these cases modeling with DNA containing a flipped base gives a much more reasonable chance of interaction and suggests that base flipping is used in these systems. Several recent reviews have appeared that discuss flipping in the context of enzymes involved in the repair of DNA lesions (35–38). These systems are described individually below and summarized in Table 3. Although definitive proof of base flipping for those enzymes listed in Table 3 awaits successful co- crystallization studies, the locations of concave active sites within deep clefts in these enzymes strongly suggest that a simple base flipping in the DNA takes place rather than a complicated refolding of the protein. The Amino-Methyltransferases M.TaqI and M.PvuII One reason to flip out a DNA nucleotide is to subject it to chemical modi- fication within a catalytic pocket (13, 27), as noted above for the cytosine-5 DNA methyltransferases (Figures 1 and 2, C-1, color section). Another class of DNA methyltransferases, the amino-methyltransferases, methylate the exo- cyclic amino groups of adenine (N6) or cytosine (N4). None of these amino- methyltransferases have been structurally characterized in complex with DNA,

Table 3 Probable base-ﬂipping systems

Speciﬁc protein Catalytic reaction Reference

TaqI DNA methyltransferase Forms TCG-6mA in DNA 39 PvuII DNA methyltransferase Forms CTG-4mC-AG in DNA 40 Escherichia coli photolyase Converts pyrimidine dimers to TT 41 E. coli endonuclease III Removes pyrimidine radiolysis 42 products from DNA 3-Methyladenine DNA glycosylase Removes 3-methyladenine from DNA 43, 44 E. coli exonuclease III Cleaves 50 to Ap sites 45 T4 -glucosyltransferase Transfers glucose residues to T4 DNA 46 E. coli Ada O6-methylguanine Removes Me from O6-Me-guanosine 47 DNA methyltransferase E. coli mismatch-speciﬁc uracil Removes thymine from DNA a DNA glycosylase aTE Barrett, R Savva, LH Pearl, unpublished data.

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but M.TaqI (adenine-N6 specific) (Figure 5, C-4, color section) and M.PvuII (cytosine-N4 specific) (Figure 6, C-5, color section) have a catalytic-domain structure with a concave active-site pocket very similar to that of M.HhaI (39, 40, 48–50). When these structures are modeled with normal B-DNA [Figures 5 (top)and6(bottom); see color section], the AdoMet is far away from the target base, but by flipping this base out of the helix the two reactants are brought into close proximity [Figures 5 (bottom), and 6 (bottom); see color section]. Thus, both N6A-methyltransferases and N4C-methyltransferases almost certainly flip out their target nucleotides. In the case of M.EcoRI, which forms GA(6meA)TTC, biochemical evidence in favor of base flipping has been obtained through a fluorescence-based assay to detect conformational alterations in DNA induced by protein binding (51). This method, which uses 2-aminopurine to replace the adenine in the substrate DNA, may have general applications to probe potential base flipping in other systems. Thus it has recently been shown that when M.HhaI and M.TaqI bind to oligonucleotides containing 2-aminopurine as the target base within their recognition sequences, the fluorescence intensity is greatly enhanced, consistent with base flipping (B Holz, S Klimasauskas, S Serva&EWeinhold, unpublished data). Biochemical evidence in favor of base flipping has also been developed for another N6A-methyltransferase, M.EcoRV (52). Here it has been shown that enhanced binding takes place when the target adenine, the first A in the recognition sequence GATATC, is replaced by a modified base that weakens base pairing. These results parallel those found for the known base-flipping cytosine-5 methyltransferase, M.HhaI (22). In all of these studies it is believed that destabilization of the base pair leads to an increased rate of base flipping and the concomitant appearance of enhanced binding. E. coli DNA Photolyase In addition to DNA repair enzymes such as UDG that function within the base excision repair pathway, DNA photolyase—an enzyme that acts through a direct reversal pathway—also appears to incorporate nucleotide flipping in its mechanism of action (41). E. coli photolyase uses a blue light–harvesting chromophore 5,10 methenyl-tetrahydrofolylpolyglutamate (MTHF) to absorb a photon and transfer the excitation energy to a catalytic chromophore, flavin adenine dinucleotide (FAD) (53). The enzyme further transfers an electron from FADH to the cyclobutane pyrimidine dimer to catalyze its fission to yield the two original pyrimidines (53). Examination of the solvent-accessible surface of the photolyase revealed that the FAD cofactor is accessible to the dimer, only by way of a cavity in the enzyme (Figure 7, C-6, color section). The dimensions of the putative binding-site cavity are sufficient to bind the bases of the dimer but to exclude

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the intradimer phosphate. The cavity is lined with polar residues on one side and hydrophobic residues on the other. Park et al (41) point out that the asymmetric polarity of the cavity matches well with the asymmetric polarity of the pyrimidine dimer, in which the cyclobutane ring is hydrophobic and the opposite edges of the thymine bases have nitrogens and oxygens capable of forming hydrogen bonds. E. coli Endonuclease III The X-ray crystal structure of endonuclease III showed that this enzyme pos- sesses an elongated bilobal structure with a deep cleft separating two distinct domains linked by two loops (Figure 8, C-6, color section) (42, 54). The two domains are -helical in structure, with one containing a 4Fe-4S cluster and the other containing a helix-hairpin-helix motif. The cleft separating the two domains can accommodate B-form DNA; thus both domains have been suggested to be involved in DNA binding. Endonu- clease III substrate binding was investigated by soaking the enzyme crystals in thymine glycol, a known inhibitor of glycosylase activity (54). The thymine glycol binding site was located within a water-filled pocket in the cleft. Thus, the damaged base is suggested to be flipped extrahelical and inserted into this pocket for catalysis to occur, in a manner similar to that of M.HhaI and UDG (Figure 8, C-6, color section). 3-Methyladenine DNA Glycosylase (AlkA) AlkA is an N-alkylpurine DNA glycosylase that can efficiently remove 3-methyladenine, 7-methylguanine, 3-methylguanine, O2-methylcytosine, and O2-methylthymine from double-stranded DNAs. The crystal structure was published by Yamagata et al (43) and Labahn et al (44). These studies revealed that AlkA is composed of three approximately equal-sized domains. Domain 1 has the topology and shape of the TATA-binding protein (2, 3, 55), whereas domains 2 and 3 have the same topology and fold as endonuclease III (see above). Domain 1 appears to serve as a platform for the other two domains. The interface of domains 2 and 3 creates the base-binding and catalytic site, and the flexibility of domain 3 permits the binding of various substrates. Examination of the surface topography of AlkA revealed a cleft at the junc- tion of domains 2 and 3, which is hydrophobic in nature and is dominated by aromatic amino acid side chains (Figure 9, C-7, color section) (43). The hydrophobic cleft also contains several charged residues, including the catalytically essential Asp. However, to position the methylated base correctly within the active site, it was suggested (44) that AlkA uses the electron- rich aromatic rings to interact with the electron-deficient, positively charged alkylated base, which is flipped out of the DNA helix. Thus, broad substrate

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specificity can likely be achieved through these strong donor-acceptor interactions. E. coli Exonuclease III The only hydrolytic AP endonuclease that has its structure solved to date is E. coli exonuclease III (45). The overall structure was shown to be similar to that of DNase I (56), even though there is less than 20% sequence similarity. It consists of two six-stranded sheets, flanked by four -helices forming a four-layered -sandwich motif. A ternary complex of exonuclease III with 2 Mn + and dCMP has also been reported (45). This complex showed the dCMP bound at one end of the sandwich, a region of the enzyme that contains a high concentration of basic residues. It was suggested that DNA-containing AP sites may be significantly distorted such that the abasic sugar may protrude into the active site to allow the chemistry to occur (45). As discussed above, M.HhaI is also able to flip an abasic sugar. Alternatively, Mol et al (45) suggest that the nucleotide opposite the AP site may be flipped into a pocket on the surface of the protein. If this flipping does occur, then exonuclease III will be an example similar to T4 endonuclease V. T4 -Glucosyltransferase Bacteriophage T4 uses 5-hydroxymethylcytosine in place of cytosine when polymerizing its DNA. Following DNA synthesis, most (if not all) of these hydroxymethylcytosines in duplex DNA are further modified by the addition of glucose. This reaction is catalyzed by the -glucosyltransferase, which transfers the glucose from uridine diphosphoglucose to the hydroxymethyl groups in 5-hydroxymethylcytosine. The structure of the enzyme was solved in both the presence and absence of the glucose donor, uridine diphosphoglucose (46). The structure comprises two domains of similar topology (Figure 10, C-7, color section). The two domains are separated by a cleft that contains a possible site for duplex DNA binding. The catalytic site can be inferred from the position of the UDP-glucose, which is deeply bound in a pocket at the bottom of the cleft. When normal B-DNA is modeled into the structure, the glucose donor and the target base are widely separated. However, as the authors suggest, base flipping of the 5-hydroxymethylcytosine would nicely juxtapose the two interacting moieties (46). E. coli Ada O6-Methylguanine DNA Methyltransferase O6-methylguanine DNA methyltransferase, otherwise known as the ada repair enzyme, is a suicidal DNA-repair protein that removes the dangerous methyl group from the promutagenic lesion O6-methylguanine and transfers it onto a cysteine in the protein. The resulting self-methylation of the active-site cysteine

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renders the protein inactive. A structure has been reported for the protein without DNA, but as noted (47), when B-DNA is modeled into the structure, a target base would be more than 20 A˚ away from the active center! Thus a signifi- cant conformational change would be required to bring the O6-methylguanine into close proximity with the buried catalytic cysteine that is the acceptor for the methyl group (Figure 11, C-8, color section). Although Moore et al (47) proposed a model in which the protein undergoes a drastic conformational distortion to bind DNA, it seems more likely that flipping the O6-methylguanine out of the DNA helix would solve the problem of accessibility. E. coli Mismatch-Specific Uracil DNA Glycosylase Mismatch-specific uracil DNA glycosylase (MUG) removes uracil by a gly- colytic mechanism from mismatches that contain uracil or thymine opposite guanine, which would arise from deamination of cytosine or 5-methylcytosine. The enzyme was originally discovered and characterized on the basis of its amino acid similarity to human thymine glycosylase (57). Recently a crystal structure has been obtained for E. coli MUG binding to DNA (TE Barrett, R Savva & LH Pearl, unpublished data). The structure shows great similarity to uracil DNA glycosylase, especially around the active site. In the crystal, the uracil base has been hydrolyzed and an abasic site is left. Interestingly, an arginine residue is intercalated on the opposite strand, and this may be partly responsible for the double-stranded DNA specificity of this enzyme. T7 ATP-Dependent DNA Ligase and T7 DNA Polymerase Finally, some proteins may mimic base flipping without actually flipping any- thing. The T7 ATP-dependentDNA ligase has a structure that broadly resembles that of DNA methyltransferases (58). Subramanya et al (58) have proposed that the reaction intermediate, which has AMP covalently bonded to the target nick in the DNA, is formally analogous to the DNA methyltransferase intermediates: The AMP is flipped out, even though no bases are missing from the target DNA. A recent crystal structure for T7 DNA polymerase in complex with a primer template, a nucleoside triphosphate, and its processivity factor (thioredoxin) has been obtained (S Doublie, S Tabor, A Long, CC Richardson & T Ellenberger, unpublished data). In this complex the template strand is sharply kinked such that the base immediately 50 of the template base paired to the incoming nucleotide is flipped out of the active site. This conformation exposes the template base for interactions with the polymerase and its nucleotide substrate.

MECHANISM We have little hard data about the mechanism of base flipping. Two theories are debated. In the first, it is suggested that base flipping is an active process

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in which the base is pushed out of the helix by some appropriate residues on the protein. Once flipped, it is pulled into the active site of the enzyme, where it remains trapped during the reaction. A three-step pathway has been proposed for the DNA methyltransferases in which they first recognize the target site and increase the interstrand phosphate-phosphate distance nearby. They then initiate base flipping by protein invasion of the DNA and finally trap the flipped DNA structure (27). An alternative possibility is that during the normal breathing of DNA, the bases naturally spend some time in the completely flipped-out position, and it is this transient conformation in DNA that is recognized and caught by the protein. Although some biophysical measurements of DNA motion have been made and lifetimes assigned to the flipped-out state, all such measurements rely on assumptions about the states being modeled (59). For instance, there is no direct experimental evidence showing that the lifetime assigned to the flipped-out state is correct. Most recently, nuclear magnetic resonance (NMR) techniques have been ap- plied to study the interaction of M.HhaI with DNA containing 5-fluorocytosine in solution (S Klimasauskas, T Szyperski, S Serva & K Wuthrich, unpublished data). Surprisingly, the dynamics of base-pair opening appear unaffected by binding M.HhaI alone. Only when the cofactor, or a cofactor analog, is added can a clear signal be detected that is attributable to the flipped state of the target base. Furthermore, these NMR studies suggest that at least three protein-DNA complexes are involved: (a) an initial binding complex that forms between the DNA and the protein in which the DNA maintains its normal stacked B conformation; (b) a complex, or series of complexes, in which the target base is flipped from the helix but is not yet locked into its final flipped position observed crystallographically; and (c) a final, catalytically competent complex in which the base is locked into the active site. This last complex would correspond to the complex seen in the crystal structure (9). Based on the NMR evidence, the initial complex and the flipped-out complex are major equilibrium species in solution, and all three types of complexes appear to be in rapid equilibrium until the cofactor binds and shifts the equilibrium to favor the final complex. This initial flipping is suggested to be an enzyme-assisted process even in the absence of cofactor. Unfortunately, there is no crystallographic evidence about the details of initial recognition. This evidence is needed to help unravel what has become a complicated reaction pathway. In addition, T4 endonuclease V has also been studied biochemically; evidence has been obtained for an initial binding complex in which the adenine residue is not flipped (A McCullough, ML Dodson, OD Scharer & RS Lloyd, unpublished data). What is clear from the recent structure between M.HhaI and an abasic site (M O’Gara, JR Horton, RJ Roberts & X Cheng, unpublished data) is that the enzyme does not require a flipped-out cytosine residue as part of its initial

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recognition mechanism. Similarly, T4 endonuclease V can flip bases other than its normal substrate adenine (34; also cited in 12). Both observations suggest that what is flipped is not the base, but rather the deoxyribose and/or its flanking phosphates. Many of the other enzymes that use base flipping, such as uracil DNA glycosylase, operate much more efficiently as enzymes than does M.HhaI. In this case it seems unlikely that UDG simply waits for the natural flip of the uracil residue.

THE ORIGINS OF BASE FLIPPING It has been proposed that base flipping is an ancient process that may have arisen even before the use of DNA as genetic material (13). The argument is based on two lines of reasoning: Base flipping is used by many enzymes involved in DNA repair, and the DNA methyltransferase M.HhaI can bind tightly to mismatches in DNA. In both cases abnormal base pairing is recognized with dramatic consequences. A possible connection between DNA methylation and repair has been suggested by Smith (60), who proposed that the human methyltransferase is a mismatch and DNA damage-recognition protein. Perhaps the DNA methyltransferases have evolved from DNA mismatch binding proteins by combining, into a single molecule, protein domains able to recognize mismatches in DNA, to perform sequence-specific recognition, to bind AdoMet, and to carry out the methylation reaction. In this scenario, the key original module is a mismatch recognition system that could accomplish base flipping. Imagine an early stage in evolution when DNA first assumed its role as the genetic material. Undoubtedly, the DNA polymerase of that time was less faith- ful than are current DNA polymerases, and incorrect bases would be inserted with alarming frequency. In addition, spontaneous deamination of cytosine to uracil could also lead to loss of fidelity, as would other chemical modifications of the bases that might disrupt pairing. In short, a repair system would be essential if this early DNA were to be competent to serve as the genetic material. How might this be accomplished? A first step must be recognition that a mismatch was present, followed by removal of the incorrect nucleotide and its replacement, most likely accomplished by the DNA polymerase having a second chance to copy faithfully. Because it is inconceivable that a full-blown repair process would have arisen immediately in a single enzyme, the repair process would require a series of steps, each involving a separate catalytic molecule. Base flipping seems tailor- made for the first step. One could imagine a small molecule, such as an RNA or peptide, being able to recognize and bind a mismatch and, in the process, flipping the mismatched base out of the DNA helix. Once the base is out of the helix, it would then be simple for a second molecule such as an endonuclease to

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cleave the phosphodiester chain and essentially create a new primer for the DNA polymerase. Experimental evidence shows that at least one small molecule, a metallo-porphyrin, can cause base flipping during intercalation into a B-DNA helix (61). The real strength of this proposal is that it allows the separation of mismatch recognition from the further steps of repair. Such stepwise ac- cumulation of functions in a single molecule is undoubtedly how modern-day proteins evolved. If base flipping was indeed used for mismatch detection in the early DNA polymers, one might reasonably ask how mismatch correction was dealt with in the earlier RNA world. Presumably, exactly the same scenario could be painted during the early course of RNA replication. Although there is no evidence for base flipping by enzymes involved in RNA metabolism, there is also a dearth of crystal structures for proteins involved in such processes.

FUTURE PROSPECTS The theme common to all examples of base flipping is the requirement for enzymes to perform chemistry on the bases normally embedded in a B-DNA helix. As more proteins that perform chemistry on DNA are examined, further examples of base flipping are likely to be discovered. Of particular interest will be studies of the various mismatch repair systems, such as the methyl-directed and the short patch-repair systems in E. coli, which might also be expected to use base flipping. Could there be other examples of base flipping within the realm of nucleic acid metabolism that have so far been elusive? Perhaps! If base flipping truly was an early evolutionary discovery, then one might anticipate its occurrence in other processes where it might prove advantageous. One such process is the local unwinding in DNA that is needed during the initiation of transcription or replication. We do not know how this is achieved. Examination of any of the DNA structures that contain flipped bases immediately suggests that base flipping would provide an easy answer. With one base flipped out of helix and the concomitant loss of both base pairing and stacking interactions, it would be simple to disrupt an adjacent base pair and so begin the unzipping of the helix. This is not the only mechanism that could be envisioned to initiate strand separation, but it should be considered a possibility. Within the area of RNA metabolism, all enzymes that perform chemistry on RNA must also be considered candidates to employ base flipping. The enzymes that modify tRNA or rRNA within base-paired regions, either by methylation or by introducing more complex side chains, might use this mechanism. Another likely candidate is double-stranded RNA adenosine deaminase. This enzyme converts adenosine residues in RNA into inosine. It has been implicated in

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Figure 1 M.HhaI complexed to its substrate DNA (pdb code 1mht) (9). DNA bases are shown in blue, sugar-phosphate backbone in red, and protein in gold. AdoHcy is shown in white.

Figure 2 M.HaeIII with DNA bound (pdb code 1dct) (11). DNA bases are shown in blue, sugar-phosphate backbone in red, and protein in gold.

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Figure 3 Human uracil DNA glysosylase complexed to DNA with a flipped uracil (coordinates provided by C Mol and J Tainer) (14). The N-C1' glycosylic bond of the flipped nucleotide has been cleaved, and the free uracil is bound in the specificity pocket. DNA bases are shown in blue, sugar-phosphate backbone in red, and protein in gold.

ROBERTS & CHENG C-3

Figure 4 T4 endonuclease V complexed to DNA containing a thymine cyclobutane dimer (pdb code 1 vas) (12). The adenine opposite the 5'-thymine residue of the pyrimidine dimer is flipped. DNA bases are shown in blue, sugar-phosphate backbone in red, and protein in gold.

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Figure 5 M.TaqI-DNA docking model (coordinates provided by G Schluckebier and W Saenger) (48) with (top) B-DNA and (bottom) DNA containing a flipped adenine. DNA bases are shown in blue, sugar-phosphate backbone in red, and protein in gold. AdoMet is shown in white.

ROBERTS & CHENG C-5

Figure 6 M.PvuII-DNA docking model (40) with (top) B-DNA and (bottom) DNA with a flipped cytosine. DNA bases are shown in blue, sugar-phosphate backbone in red, and protein in gold. AdoMet is shown in white.

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Figure 7 Ribbon diagram of Escherichia coli DNA photolyase (pdb code 1dnp) (41). The α/β domain is shown in white, methenyl-tetrahydrofolylpolyglutamate (MTHF) in light gray, and the helical domain in gold. The flavin mononucleotide group of flavin adenine dinucleotide (FAD) is shown in green, and the AMP unit is in red.

Figure 8 Escherichia coli endonuclease III−DNA docking model with a flipped nucleotide (coordinates provided by M Thayer and J Tainer) (42). DNA bases are shown in blue, sugar-phosphate backbone in red, and protein in gold. The HhH motif is shown in green.

ROBERTS & CHENG C-7

Figure 9 Escherichia coli 3-methyladenine DNA glycosylase II−DNA docking model with a flipped nucleotide (coordinates provided by Y Yamagata) (43). DNA bases are shown in blue, sugar-phosphate backbone in red, and protein in gold. The HhH motif is shown in green, and the catalytically essential Asp is in white.

Figure 10 T4 β-glucosyltransferase (pdb code 2bgu)-DNA docking model with a flipped nucleotide (coordinates provided by P Freemont) (46). DNA bases are shown in blue, sugar-phosphate backbone in red, and protein in gold. Uridine diphosphoglucose is shown in white.

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Figure 11 Ribbon diagram of ada O6-methylguanine DNA methyltransferase (pdb code 1sfe) docked with a B-DNA. DNA bases are shown in blue, sugar-phosphate backbone in red, and protein in gold. The side chain of the catalytic cysteine is shown in white with a red border.

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RNA editing and is suggested to resemble the DNA methyltransferases at the sequence level (62). In summary, the phenomenon of base flipping is appearing in a number of systems following its initial discovery in the DNA methyltransferases, and many additional enzyme systems must be considered candidates to use this novel mechanism. Unfortunately, detailed mechanistic information is lacking, and it remains to be proven whether base flipping is an active process in which the protein pushes the base out of the helix or a passive one in which the protein binds to a transiently flipped base. Most authors, including ourselves, favor the active process, but further work is needed to settle the issue. Particularly intriguing is the recent finding that when M.HhaI binds to an abasic site, it flips the deoxyribose ring and its flanking phosphates into the same conformation that is adopted during base flipping on its normal substrate. Thus if the process is active, one might expect that the push will take place not on the base, but rather on the sugar-phosphate backbone. Perhaps we should have termed the phenomenon the less evocative “DNA backbone rotation.”

ACKNOWLEDGMENTS We thank Drs S Kumar, S Pradhan, L Robow, and X Zhang for critical reading of the manuscript; Dr. S Kumar and C Lin for help with the preparation of ﬁgures; Drs T Barrett, P Freemont, C Mol, L Pearl, W Saenger, G Schluckebier, J Tainer, M Thayer, and Y Yamagata for providing coordinates; and T Ellenberger, S Klimasauskas, and E Weinhold for providing preprints. We also wish to thank most warmly the members of our laboratories whose hard work was responsible for much of the methyltransferase story. Work in our laboratories is supported by grants from the National Institutes of Health (GM46127 to RJR; GM/OD52117 and GM49245 to XC).

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