Structural evidence of a passive base-flipping mechanism for AGT, an unusual GT-B glycosyltransferase

The Escherichia coli T4 bacteriophage uses two glycosyltransferases to glucosylate and thus protect its DNA: the retaining alpha-glucosyltransferase (AGT) and the inverting beta-glucosyltransferase (BGT). They glucosylate 5-hydroxymethyl cytosine (5-HMC) bases of duplex DNA using UDP-glucose as the sugar donor to form an alpha-glucosidic linkage and a beta-glucosidic linkage, respectively. Five structures of AGT have been determined: a binary complex with the UDP product and four ternary complexes with UDP or UDP-glucose and oligonucleotides containing an A:G, HMU:G (hydroxymethyl uracyl) or AP:G (apurinic/apyrimidinic) mismatch at the target base-pair. AGT adopts the GT-B fold, one of the two folds known for GTs. However, while the sugar donor binding mode is classical for a GT-B enzyme, the sugar acceptor binding mode is unexpected and breaks the established consensus: AGT is the first GT-B enzyme that predominantly binds both the sugar donor and acceptor to the C-terminal domain. Its active site pocket is highly similar to four retaining GT-B glycosyltransferases (trehalose-6-phosphate synthase, glycogen synthase, glycogen and maltodextrin phosphorylases) strongly suggesting a common evolutionary origin and catalytic mechanism for these enzymes. Structure-guided mutagenesis and kinetic analysis do not permit identification of a nucleophile residue responsible for a glycosyl-enzyme intermediate for the classical double displacement mechanism. Interestingly, the DNA structures reveal partially flipped-out bases. They provide evidence for a passive role of AGT in the base-flipping mechanism and for its specific recognition of the acceptor base.     

Xenopus egg lysates repair heat-generated DNA nicks with an average patch size of 36 nucleotides.

Base excision repair (BER) is an essential DNA repair pathway since it processes spontaneous (endogenous) DNA damage such as abasic sites, oxidized and alkylated bases, as well as mismatches arising from deamination of cytosine and 5-methylcytosine. Some of these lesions are repaired by the exchange of a single deoxynucleotide [Dianov, G. et al. (1992) Mol. Cell. Biol. 12, 1605-1612; Wiebauer, K. and Jiricny, J. (1990) Proc. Natl. Acad. Sci. USA, 87, 5842-5845] or a few deoxynucleotides [Matsumoto, Y. et al. (1994) Mol. Cell. Biol., 14 6187-6197]. Here we report that DNA single strand breaks induced by hyperthermic conditions are repaired with an average patch size of approximately 36 nt in Xenopus laevis egg lysates.     

Book review

Fauna of New Zealand (Ko te Aitanga Pepeke o Aotearoa) No. 60. Carabidae (Insecta: Coleoptera): synopsis of supraspecific taxa. By André Larochelle and Marie‐Claude Larivière. Published in 2007 by Landcare Research, Manaaki Whenua Press. Online price: NZ$54.00. ISBN: 9780478093940. 188 p.     

Dynamic modes of the flipped-out cytosine during HhaI methyltransferase-DNA interactions in solution.

Flipping of a nucleotide out of a B-DNA helix into the active site of an enzyme has been observed for the HhaI and HaeIII cytosine-5 methyltransferases (M.HhaI and M.HaeIII) and for numerous DNA repair enzymes. Here we studied the base flipping motions in the binary M. HhaI-DNA and the ternary M.HhaI-DNA-cofactor systems in solution. Two 5-fluorocytosines were introduced into the DNA in the places of the target cytosine and, as an internal control, a cytosine positioned two nucleotides upstream of the recognition sequence 5'-GCGC-3'. The 19F NMR spectra combined with gel mobility data show that interaction with the enzyme induces partition of the target base among three states, i.e. stacked in the B-DNA, an ensemble of flipped-out forms and the flipped-out form locked in the enzyme active site. Addition of the cofactor analogue S-adenosyl-L-homocysteine greatly enhances the trapping of the target cytosine in the catalytic site. Distinct dynamic modes of the target cytosine have thus been identified along the reaction pathway, which includes novel base-flipping intermediates that were not observed in previous X-ray structures. The new data indicate that flipping of the target base out of the DNA helix is not dependent on binding of the cytosine in the catalytic pocket of M.HhaI, and suggest an active role of the enzyme in the opening of the DNA duplex.     

High resolution crystal structures of T4 phage beta-glucosyltransferase: induced fit and effect of substrate and metal binding

beta-Glucosyltransferase (BGT) is a DNA-modifying enzyme encoded by bacteriophage T4 that transfers glucose from uridine diphosphoglucose to 5-hydroxymethyl cytosine bases of phage T4 DNA. We report six X-ray structures of the substrate-free and the UDP-bound enzyme. Four also contain metal ions which activate the enzyme, including Mg(2+) in forms 1 and 2 and Mn(2+) or Ca(2+). The substrate-free BGT structure differs by a domain movement from one previously determined in another space group. Further domain movements are seen in the complex with UDP and the four UDP-metal complexes. Mg(2+), Mn(2+) and Ca(2+) bind near the beta-phosphate of the nucleotide, but they occupy slightly different positions and have different ligands depending on the metal and the crystal form. Whilst the metal site observed in these complexes with the product UDP is not compatible with a role in activating glucose transfer, it approximates the position of the positive charge in the oxocarbonium ion thought to form on the glucose moiety of the substrate during catalysis.     

The structure of recA protein-DNA filaments. 2 recA protein monomers unwind 17 base pairs of DNA by 11.5 degrees/base pair in the presence of adenosine 5'-O-(3-thiotriphosphate)

recA protein binds to duplex DNA in the presence of Mg2+ and adenosine 5'-O-(3-thiotriphosphate) forming a stiff nucleoprotein filament with a distinct axial repeat which contains 17 +/- 1 base pairs and spans 8-9 nm along the fiber (Di Capua, E., Engel, A., Stasiak, A., and Koller, Th. (1982) J. Mol. Biol. 157, 87-103; Dunn, K., Chrysogelos, S., and Griffith, J. (1982) Cell 28, 757-765). Measurement of the protein:DNA ratio in these filaments utilizing double label analysis and isopycnic density banding shows that there are 2 recA monomers for every 17 base pairs. The DNA is also partially unwound in this filament. Utilizing the recA-induced relaxation of naturally supertwisted SV40 DNA, we show that the DNA is unwound by 11.5 +/- 1.5 degrees/base pair which corresponds to 180-200 degrees for each repeat unit along the filament length.     

Observation of water molecules within the bimolecular d(G₃CT₄G₃C)₂G-quadruplex

G-Rich oligonucleotides with cytosine residues in their sequences can form G-quadruplexes where G-quartets are flanked by G·C Watson-Crick base pairs. In an attempt to probe the role of cations in stabilization of a structural element with two G·C base pairs stacked on a G-quartet, we utilized solution state nuclear magnetic resonance to study the folding of the d(G(3)CT(4)G(3)C) oligonucleotide into a G-quadruplex upon addition of (15)NH(4)(+) ions. Its bimolecular structure exhibits antiparallel strands with edge-type loops. Two G-quartets in the core of the structure are flanked by a couple of Watson-Crick G·C base pairs in a sheared arrangement. The topology is equivalent to the solution state structure of the same oligonucleotide in the presence of Na(+) and K(+) ions [Kettani, A., et al. (1998) J. Mol. Biol.282, 619, and Bouaziz, S., et al. (1998) J. Mol. Biol.282, 637). A single ammonium ion binding site was identified between adjacent G-quartets, but three sites were expected. The remaining potential cation binding sites between G-quartets and G·C base pairs are occupied by water molecules. This is the first observation of long-lived water molecules within a G-quadruplex structure. The flanking G·C base pairs adopt a coplanar arrangement and apparently do not require cations to neutralize unfavorable electrostatic interactions among proximal carbonyl groups. A relatively fast movement of ammonium ions from the inner binding site to bulk with the rate constants of 21 s(-1) was attributed to the lack of hydrogen bonds between adjacent G·C base pairs and the flexibility of the T(4) loops.     

Interaction of the tight-binding I12-X86 lac repressor with non operator DNA: salt dependence of complex formation.

The interaction of the wild-type lac repressor and its tight binding double mutant I12-X86 with a non operator-210 base pair-DNA fragment has been investigated using the nitrocellulose filter binding assay. While the affinity of the double mutant for this non specific DNA is increased as compared to that of the wild-type repressor, the number of ions released from the vicinity of the DNA upon complex formation is less important for the mutant than for the wild-type. These results demonstrate that the adaptation in the recognition surface of the repressor recently proposed by Mossing et al (J. Mol. Biol., 1985, 186, 295-305) in the case of an Oc mutant may be a more general phenomenon.     

Structure of the chromosomal copy of yeast ARS1

We have used deoxyribonuclease I (DNase I) and methidium-propyl-EDTA.Fe(II) digestion to characterize the chromosomal structure of the single-copy autonomously replicating sequence ARS1. The major feature of this chromatin is a region of strong hypersensitivity to both cleavage agents. The hypersensitive region contains most of the DNA sequences which have been suggested by in vitro mutagenesis studies [Celniker, S., Sweder, K., Srienc, F., Bailey, J., & Campbell, J. (1984) Mol. Cell. Biol. 4, 2455-2466] to be important in ARS function. It lies at the downstream end of the TRP1 gene. A chromosomal DNase I footprinting analysis was carried out on the hypersensitive region. These data give direct evidence for several localized DNA/protein contacts within the hypersensitive region. The most prominent of these chromatin-dependent contacts is located on the functionally most important 11 base pairs of ARS DNA. On the TRP1 side of the hypersensitive region, there are positioned nucleosomes. On the other side of the hypersensitive region, there is a complex (and possibly heterogeneous) structure.     

Solvation effects on the sequence variability of DNA double helical conformations

The role of solvation on the sequence dependent conformational variabilities in DNA has been studied by calculating hydration free energies from solvent accessible surface areas for several base steps, as a function of various helical parameters, roll, twist and propeller twist. The results of roll calculations suggest opposite trends for AA and GG steps, with the former tending to have a compressed minor groove and the latter a compressed major groove. These trends are consistent with the experimental findings on sequence preferences and the nature of anisotropic bending of DNA observed in nucleosomes (Drew, H.R. and Travers, A.A., J. Mol. Biol. 186, 773-790 (1985); Satchwell, S.C., Drew, H.R. and Travers, A.A., J. Mol. Biol. 191, 659-675 (1986)) and CAP-DNA interactions (Gartenberg, M.R. and Crothers, D.M., Nature 333, 824-829, (1988)). Solvation energy profiles also indicate preferences for the base pairs in GG and AA steps to adopt low and high propeller twists, respectively. Such agreements may either reflect a coincidence of solvation effects with other energy terms or a dominance of solvent effects. The results are discussed in the context of the crystallographic observations of structural tendencies.     

Escherichia coli DNA helicase I catalyzes a site- and strand-specific nicking reaction at the F plasmid oriT.

A site- and strand-specific nick, introduced in the F plasmid origin of transfer, initiates conjugal DNA transfer during bacterial conjugation. Recently, molecular genetic studies have suggested that DNA helicase I, which is known to be encoded on the F plasmid, may be involved in this nicking reaction (Traxler, B. A., and Minkley, E. G., Jr. (1988) J. Mol. Biol. 204, 205-209). We have demonstrated this site- and strand-specific nicking event using purified helicase I in an in vitro reaction. The nicking reaction requires a superhelical DNA substrate containing the F plasmid origin of transfer, Mg2+ and helicase I. The reaction is protein concentration-dependent but, under the conditions used, only 50-70% of the input DNA substrate is converted to the nicked species. Genetic data (Everett, R., and Willetts, N. (1980) J. Mol. Biol. 136, 129-150) have also suggested the involvement of a second F-encoded protein, the TraY protein, in the oriT nicking reaction. Unexpectedly, the in vitro nicking reaction does not require the product of the F plasmid traY gene. The implications of this result are discussed. The phosphodiester bond interrupted by helicase I has been shown to correspond exactly to the site nicked in vivo suggesting that helicase I is the site- and strand-specific nicking enzyme that initiates conjugal DNA transfer. Thus, helicase I is a bifunctional protein which catalyzes site- and strand-strand specific nicking of the F plasmid in addition to the previously characterized duplex DNA unwinding (helicase) reaction.     

Chemical display of thymine residues flipped out by DNA methyltransferases.

The DNA cytosine-C5 methyltransferase M. Hha I flips its target base out of the DNA helix during interaction with the substrate sequence GCGC. Binary and ternary complexes between M. Hha I and hemimethylated DNA duplexes were used to examine the suitability of four chemical methods to detect flipped-out bases in protein-DNA complexes. These methods probe the structural peculiarities of pyrimidine bases in DNA. We find that in cases when the target cytosine is replaced with thymine (GTGC), KMnO4proved an efficient probe for positive display of flipped-out thymines. The generality of this procedure was further verified by examining a DNA adenine-N6 methyltransferase, M. Taq I, in which case an enhanced reactivity of thymine replacing the target adenine (TCGT) in the recognition sequence TCGA was also observed. Our results support the proposed base-flipping mechanism for adenine methyltransferases, and offer a convenient laboratory tool for detection of flipped-out thymines in protein-DNA complexes.     

Orientation of the Lac repressor DNA binding domain in complex with the left lac operator half site characterized by affinity cleaving.

Lac repressor (LacR) is a helix-turn-helix motif sequence-specific DNA binding protein. Based on proton NMR spectroscopic investigations, Kaptein and co-workers have proposed that the helix-turn-helix motif of LacR binds to DNA in an orientation opposite to that of the helix-turn-helix motifs of lambda repressor, lambda cro, 434 repressor, 434 cro, and CAP [Boelens, R., Scheek, R., van Boom, J. and Kaptein, R., J. Mol. Biol. 193, 1987, 213-216]. In the present work, we have determined the orientation of the helix-turn-helix motif of LacR in the LacR-DNA complex by the affinity cleaving method. The DNA cleaving moiety EDTA.Fe was attached to the N-terminus of a 56-residue synthetic protein corresponding to the DNA binding domain of LacR. We have formed the complex between the modified protein and the left DNA half site for LacR. The locations of the resulting DNA cleavage positions relative to the left DNA half site provide strong support for the proposal of Kaptein and co-workers.     

Association of Escherichia coli lac repressor with poly[d(A-T)] monitored with 8-anilino-1-napthalenesulfonate.

The association of lac repressor with poly[d(A-T)] was monitored with the fluorescent prob 8-anilino-1-naphthalenesulfonate (Ans). Excess poly[d(A-T)] decreased the emission intensity of the repressor--Ans complex by 30%. Fluorescence titrations indicated that 33 +/- 4 base pairs were required to bind all of the repressor. Sedimentation studies indicated, however, that all of the repressor sedimented as a protein--DNA complex with as few as 10 to 15 base pairs per tetramer, even in the presence of Ans. These data are interpreted with two models: one where repressors bind to both sides of the DNA (Butler, A. P., et al. (1977) Biochemistry 16, 4757: Zingsheim, H.P., et al. (1977) J. Mol. Biol. 115, 565), the other where a double layer of repressors bind to a single side of the DNA. Removal of the amino-terminal regions from the repressor decreased the fluorescence from bound Ans by 77%. The amino-terminal fragments alone did not enhance Ans fluorescence.     

The biochemical basis of 5-bromouracil-induced mutagenesis. Heteroduplex base mispairs involving bromouracil in G x C----A x T and A x T----G x C mutational pathways

We have investigated the mechanism of bromouracil-induced transition mutations in vitro using synthetic DNA templates and purified T4 DNA polymerase. Evidence is presented for the occurrence of bromouracil-guanine base pairs in product DNA in the G x C----A x T pathway where guanine is present in the DNA template and bromouracil is present as the deoxynucleoside triphosphate substrate 5-bromodeoxyuridine triphosphate. This finding supports a widely known but as yet untested model proposed by Freese (Freese, E. (1959) J. Mol. Biol. 1, 87-105) in which bromouracil-guanine base pairs are intermediates in 5-bromodeoxyuridine-induced transition mutation pathways. We find that the newly formed B x G base pairs are proofread with an efficiency of 75-85% by the 3' -exonuclease of T4 polymerase. The insertion of bromouracil occurring in direct competition with cytosine deoxyribonucleotides opposite template guanine sites is 1.1 +/- 0.14% (mean +/- S.E.), and the misincorporation ratio, inc(B)/inc(C), is reduced 6-fold by the action of the proofreading exonuclease to 0.16 +/- 0.02% (mean +/- S.E.). A previous study by Trautner et al. (Trautner, T. A., Swartz, M. N., and Kornberg, A. (1962) Proc. Natl. Acad. Sci. U. S. A. 48, 449-455) suggested that, while template bromouracil stimulates incorporation of dGMP in the A x T----G x C transition mutation pathway, it may not be occurring exclusively by the pathway proposed by Freese. We concur with these earlier results, and, in addition, we find the surprising result that the 3'-exonuclease activity of wild-type T4 polymerase removes little or no incorporated dGMP on bromouracil-containing templates.     

Chemical mapping of cytosines enzymatically flipped out of the DNA helix

Haloacetaldehydes can be employed for probing unpaired DNA structures involving cytosine and adenine residues. Using an enzyme that was structurally proven to flip its target cytosine out of the DNA helix, the HhaI DNA methyltransferase (M.HhaI), we demonstrate the suitability of the chloroacetaldehyde modification for mapping extrahelical (flipped-out) cytosine bases in protein-DNA complexes. The generality of this method was verified with two other DNA cytosine-5 methyltransferases, M.AluI and M.SssI, as well as with two restriction endonucleases, R.Ecl18kI and R.PspGI, which represent a novel class of base-flipping enzymes. Our results thus offer a simple and convenient laboratory tool for detection and mapping of flipped-out cytosines in protein-DNA complexes.     

A base-flipping mechanism for the T4 phage beta-glucosyltransferase and identification of a transition-state analog

T4 phage beta-glucosyltransferase (BGT) modifies T4 DNA. We crystallized BGT with UDP-glucose and a 13mer DNA fragment containing an abasic site. We obtained two crystal structures of a ternary complex BGT-UDP-DNA at 1.8A and 2.5A resolution, one with a Tris molecule and the other with a metal ion at the active site. Both structures reveal a large distortion in the bound DNA. BGT flips the deoxyribose moiety at the abasic site to an extra-helical position and induces a 40 degrees bend in the DNA with a marked widening of the major groove. The Tris molecule mimics the glucose moiety in its transition state. The base-flipping mechanism, which has so far been observed only for glycosylases, methyltransferases and endonucleases, is now reported for a glucosyltransferase. BGT is unique in binding and inserting a loop into the DNA duplex through the major groove only. Furthermore, BGT compresses the backbone DNA one base further than the target base on the 3'-side.