The Role of Base Flipping in Damage Recognition and Catalysis by T4 Endonuclease V

Abstract

The process of moving a DNA base extrahelical (base flipping) has been shown in the co-crystal structure of a UV-induced pyrimidine dimer-specific glycosylase, T4 endonuclease V, with its substrate DNA. Compared with other enzymes known to use base flipping, endonuclease V is unique in that it moves the base opposite the target site extrahelical, rather than moving the target base itself. Utilizing substrate analogs and catalytically inactive mutants of T4 endonuclease V, this study investigates the discrete steps involved in damage recognition by this DNA repair enzyme. Specifically, fluorescence spectroscopy analysis shows that fluorescence changes attributable to base flipping are specific for only the base directly opposite either abasic site analogs or the 5 -thymine of a pyrimidine dimer, and no changes are detected if the 2-aminopurine is moved opposite the 3 -thymine of the pyrimidine dimer. Interestingly, base flipping is not detectable with every specific binding event suggesting that damage recognition can be achieved without base flipping. Thus, base flipping does not add to the stability of the specific enzyme-DNA complex but rather induces a conformational change to facilitate catalysis at the appropriate target site. When used in conjunction with structural information, these types of analyses can yield detailed mechanistic models and critical amino acid residues for extrahelical base movement as a mode of damage recognition.The initiating events in base excision repair are performed by a class of enzymes, DNA glycosylases. These damage-specific enzymes are responsible for recognizing and binding to damaged bases and catalyzing the cleavage of the N-C 1 Ј glycosydic bond linking the damaged base to the sugar phosphate backbone. Glycosylases thus provide the specificity to base excision repair. The precise mechanism by which these enzymes discriminate between nontarget and target DNA bases is beginning to be elucidated for a few glycosylases due to insights from high resolution x-ray crystallographic structures (reviewed in Ref. 1). In particular, uracil DNA glycosylases and a catalytically inactive mutant of T4 endonuclease V have been co-crystallized with their product or substrate DNAs, respectively, and shown to move a base extrahelical (nucleotide), placing the base in a pocket within the enzyme.The mechanism of base flipping is not limited to DNA repair enzymes. In fact, it appears to be a generalized mechanism for catalytic DNA binding proteins (reviewed in Refs. 2 and 3). The first evidence that an enzyme flips a base extrahelical was revealed in the crystal structure of a DNA cytosine 5-methyltransferase, HhaI, complexed with its substrate DNA, in which the target cytosine was flipped out of the DNA helix and into the active site pocket of the enzyme where the methylation reaction can occur (4). A similar methyltransferase, HaeIII, has also been shown to move a base extrahelical (5), suggesting that this may be a conserved mechanism for this class of enzymes. As mentioned above, the two co...

Incision of DNA–protein crosslinks by UvrABC nuclease suggests a potential repair pathway involving nucleotide excision repair

“…UvrA was a necessary component to the DPC incision reactions. Thus, these data imply that despite the severity of the structural DNA distortion in the DPC complex (42,44), an even more distorted, bubble-like structure forms as a result of the initial damage recognition by UvrA 2 B. These, as well as sequential local structural alterations of the UvrA 2 B-DNA complex, may lead to the disruption of contacts between the DNA and T4-pdg.…”

“…According to the cocrystal structure, although the enzyme did not undergo major structural alterations, the DNA was kinked at a Ϸ60°bend with the nucleotide pairing partner to the 5Ј pyrimidine of the dimer flipped to an extrahelical position on a surface cleft of the enzyme. The stability of the DNA bend has not been experimentally confirmed under the conditions used to form the covalent complex, but fluorescence data measuring the local environment and extrahelical extrusion of a 2-aminopurine opposite a damaged site have revealed the formation of a stably flipped complex (44). Based on assumptions made concerning these data, it is predicted that the starting DPC substrate for incision by UvrABC is a highly distorted, kinked complex containing an extrahelical nucleotide, in which the enzymes' overall molecular footprint is Ϸ10 nucleotides.…”

Reconciling Structure and Function in HhaI DNA Cytosine-C-5 Methyltransferase

“…DNA bending by enzymes and other proteins is well known. Stabilization of extrahelical nucleosides has been observed via x-ray crystallography in M.HhaI, the HaeIII DNA methyltransferase (4), uracil-DNA glycosylase (17), and T4 endonuclease V (18). The nucleoside flipping process has been observed via 2-aminopurine fluorescence in M.EcoRI (6, 37), M.TaqI, and M.HhaI (16), and uracil-DNA glycosylase (38).…”

“…In the ternary complex, however, it is the methionine moiety in the binding pocket. Among a variety of protein contacts, the purine ring stacks with the indole ring of Trp 41 and is perpendicular to the plane of the phenyl group of Phe 18 . The image was generated with GRASP (57).…”

“…Furthermore, the AdoMet binding pocket revealed in M.HhaI crystal structures is expected to be common to protein, DNA, RNA, and small molecule AdoMet-dependent methyltransferases (14,15). Enzyme-mediated nucleoside flipping, first observed in M.HhaI, occurs with other DNA methyltransferases (6,16) and DNA repair enzymes (17,18). M.HhaI thus embodies structural and functional characteristics common to DNA repair enzymes, AdoMet-dependent enzymes, and DNA methyltransferases.…”