2-Aminopurine as a fluorescent probe for DNA base flipping by methyltransferases.

DNA base flipping, which was first observed for the C5-cytosine DNA methyltransferase M. Hha I, results in a complete removal of the stacking interactions between the target base and its neighbouring bases. We have investigated whether duplex oligodeoxynucleotides containing the fluorescent base analogue 2-aminopurine can be used to sense DNA base flipping. Using M. Hha I as a paradigm for a base flipping enzyme, we find that the fluorescence intensity of duplex oligodeoxynucleotides containing 2-aminopurine at the target site is dramatically enhanced (54-fold) in the presence of M. Hha I. Duplex oligodeoxynucleotides containing 2-aminopurine adjacent to the target cytosine show little fluorescence increase upon addition of M. Hha I. These results clearly demonstrate that duplex oligodeoxynucleotides containing 2-aminopurine at the target site can serve as fluorescence probes for base flipping. Another enzyme hypothesized to use a base flipping mechanism is the N6-adenine DNA methyltransferase M. Taq I. Addition of M. Taq I to duplex oligodeoxynucleotides bearing 2-aminopurine at the target position, also results in a strongly enhanced fluorescence (13-fold), whereas addition to duplex oligodeoxynucleotides containing 2-aminopurine at the 3'- or 5'-neighbouring position leads only to small fluorescence increases. These results give the first experimental evidence that the adenine-specific DNA methyltransferase M. Taq I also flips its target base.     

Nucleotide flipping by restriction enzymes analyzed by 2-aminopurine steady-state fluorescence

Many DNA modification and repair enzymes require access to DNA bases and therefore flip nucleotides. Restriction endonucleases (REases) hydrolyze the phosphodiester backbone within or in the vicinity of the target recognition site and do not require base extrusion for the sequence readout and catalysis. Therefore, the observation of extrahelical nucleotides in a co-crystal of REase Ecl18kI with the cognate sequence, CCNGG, was unexpected. It turned out that Ecl18kI reads directly only the CCGG sequence and skips the unspecified N nucleotides, flipping them out from the helix. Sequence and structure conservation predict nucleotide flipping also for the complexes of PspGI and EcoRII with their target DNAs (/CCWGG), but data in solution are limited and indirect. Here, we demonstrate that Ecl18kI, the C-terminal domain of EcoRII (EcoRII-C) and PspGI enhance the fluorescence of 2-aminopurines (2-AP) placed at the centers of their recognition sequences. The fluorescence increase is largest for PspGI, intermediate for EcoRII-C and smallest for Ecl18kI, probably reflecting the differences in the hydrophobicity of the binding pockets within the protein. Omitting divalent metal cations and mutation of the binding pocket tryptophan to alanine strongly increase the 2-AP signal in the Ecl18kI–DNA complex. Together, our data provide the first direct evidence that Ecl18kI, EcoRII-C and PspGI flip nucleotides in solution.     

Time-resolved fluorescence studies of nucleotide flipping by restriction enzymes

Restriction enzymes Ecl18kI, PspGI and EcoRII-C, specific for interrupted 5-bp target sequences, flip the central base pair of these sequences into their protein pockets to facilitate sequence recognition and adjust the DNA cleavage pattern. We have used time-resolved fluorescence spectroscopy of 2-aminopurine-labelled DNA in complex with each of these enzymes in solution to explore the nucleotide flipping mechanism and to obtain a detailed picture of the molecular environment of the extrahelical bases. We also report the first study of the 7-bp cutter, PfoI, whose recognition sequence (T/CCNGGA) overlaps with that of the Ecl18kI-type enzymes, and for which the crystal structure is unknown. The time-resolved fluorescence experiments reveal that PfoI also uses base flipping as part of its DNA recognition mechanism and that the extrahelical bases are captured by PfoI in binding pockets whose structures are quite different to those of the structurally characterized enzymes Ecl18kI, PspGI and EcoRII-C. The fluorescence decay parameters of all the enzyme-DNA complexes are interpreted to provide insight into the mechanisms used by these four restriction enzymes to flip and recognize bases and the relationship between nucleotide flipping and DNA cleavage.     

Ligand-induced changes in 2-aminopurine fluorescence as a probe for small molecule binding to HIV-1 TAR RNA

Replication of human immunodeficiency virus type 1 (HIV-1) is regulated in part through an interaction between the virally encoded trans-activator protein Tat and the trans-activator responsive region (TAR) of the viral RNA genome. Because TAR is highly conserved and its interaction with Tat is required for efficient viral replication, it has received much attention as an antiviral drug target. Here, we report a 2-aminopurine (2-AP) fluorescence-based assay for evaluating potential TAR inhibitors. Through selective incorporation of 2-AP within the bulge (C23 or U24) of a truncated form of the TAR sequence (delta TAR-ap23 and delta TAR-ap24), binding of argininamide, a 24-residue arginine-rich peptide derived from Tat, and Neomycin has been characterized using steady-state fluorescence. Binding of argininamide to the 2-AP deltaTAR constructs results in a four- to 11-fold increase in fluorescence intensity, thus providing a sensitive reporter of that interaction (KD approximately 1 mM). Similarly, binding of the Tat peptide results in an initial 14-fold increase in fluorescence (KD approximately 25 nM), but is then followed by a slight decrease that is attributed to an additional, lower-affinity association(s). Using the deltaTAR-ap23 and TAR-ap24 constructs, two classes of Neomycin binding sites are detected; the first molecule of antibiotic binds as a noncompetitive inhibitor of Tat/argininamide (KD approximately 200 nM), whereas the second, more weakly bound molecule(s) becomes associated in a presumably nonspecific manner (KD approximately 4 microM). Taken together, the results demonstrate that the 2-AP fluorescence-detected binding assays provide accurate and general methods for quantitatively assessing TAR interactions.     

The role of Arg165 towards base flipping, base stabilization and catalysis in M.HhaI

Arg165 forms part of a previously identified base flipping motif in the bacterial DNA cytosine methyltransferase, M.HhaI. Replacement of Arg165 with Ala has no detectable effect on either DNA or AdoMet affinity, yet causes the base flipping and restacking transitions to be decreased approximately 16 and 190-fold respectively, thus confirming the importance of this motif. However, these kinetic changes cannot account for the mutant's observed 10(5)-fold decreased catalytic rate. The mutant enzyme/cognate DNA cocrystal structure (2.79 A resolution) shows the target cytosine to be positioned approximately 30 degrees into the major groove, which is consistent with a major groove pathway for nucleotide flipping. The pyrimidine-sugar chi angle is rotated to approximately +171 degrees, from a range of -95 degrees to -120 degrees in B DNA, and -77 degrees in the WT M.HhaI complex. Thus, Arg165 is important for maintaining the cytosine positioned for nucleophilic attack by Cys81. The cytosine sugar pucker is in the C2'-endo-C3'-exo (South conformation), in contrast to the previously reported C3'-endo (North conformation) described for the original 2.70 A resolution cocrystal structure of the WT M.HhaI/DNA complex. We determined a high resolution structure of the WT M.HhaI/DNA complex (1.96 A) to better determine the sugar pucker. This new structure is similar to the original, lower resolution WT M.HhaI complex, but shows that the sugar pucker is O4'-endo (East conformation), intermediate between the South and North conformers. In summary, Arg165 plays significant roles in base flipping, cytosine positioning, and catalysis. Furthermore, the previously proposed M.HhaI-mediated changes in sugar pucker may not be an important contributor to the base flipping mechanism. These results provide insights into the base flipping and catalytic mechanisms for bacterial and eukaryotic DNA methyltransferases.     

M.TaqI facilitates the base flipping via an unusual DNA backbone conformation

MD simulations have been carried out to understand the dynamical behavior of the DNA substrate of the Thermus aquaticus DNA methyltransferase (M.TaqI) in the methylation process at N6 of adenine. As starting structures, an x-ray structure of M.TaqI in complex with DNA and cofactor analogue (PDB code: 1G 38) and free decamer d(GTTCGATGTC)(2) were taken. The x-ray structure shows two consecutive BII substates that are not observed in the free decamer. These consecutive BII substates are also observed during our simulation. Additionally, their facing backbones adopt the same conformations. These double facing BII substates are stable during the last 9 ns of the trajectories and result in a stretched DNA structure. On the other hand, protein-DNA contacts on 5' and 3' phosphodiester groups of the partner thymine of flipped adenine have changed. The sugar and phosphate parts of thymine have moved further into the empty space left by the flipping base without the influence of protein. Furthermore, readily high populated BII substates at the GpA step of palindromic tetrad TCGA rather than CpG step are observed in the free decamer. On the contrary, the BI substate at the GpA step is observed on the flipped adenine strand. A restrained MD simulation, reproducing the BI/BII pattern in the complex, demonstrated the influence of the unusual backbone conformation on the dynamical behavior of the target base. This finding along with the increased nearby interstrand phosphate distance is supportive to the N6-methylation mechanism.     

Crystal structures of restrictocin-inhibitor complexes with implications for RNA recognition and base flipping.

The cytotoxin sarcin disrupts elongation factor binding and protein synthesis by specifically cleaving one phosphodiester bond in ribosomes. To elucidate the molecular basis of toxin action, we determined three cocrystal structures of the sarcin homolog restrictocin bound to different analogs that mimic the target sarcin/ricin loop (SRL) structure of the rat 28S rRNA. In these structures, restrictocin contacts the bulged-G motif and an unfolded form of the tetraloop of the SRL RNA. In one structure, toxin loops guide selection of the target site by contacting the base critical for recognition (G4319) and the surrounding S-shaped backbone. In another structure, base flipping of the tetraloop enables cleavage by placing the target nucleotide in the active site with the nucleophile nearly inline for attack on the scissile bond. These structures provide the first views of how a site-specific protein endonuclease recognizes and cleaves a folded RNA substrate.     

Influence of base stacking and hydrogen bonding on the fluorescence of 2-aminopurine and pyrrolocytosine in nucleic acids

Fluorescent nucleobase analogues are used extensively to probe the structure and dynamics of nucleic acids. The fluorescence of the adenine analogue 2-aminopurine and the cytosine analogue pyrrolocytosine is significantly quenched when the bases are located in regions of double-stranded nucleic acids. To allow more detailed structural information to be obtained from fluorescence studies using these bases, we have studied the excited-state properties of the bases at the CIS and TDB3LYP level in hydrogen-bonded and base-stacked complexes. The results reveal that the first excited state (the fluorescent state) of a hydrogen-bonded complex containing 2-aminopurine and thymine is just the first excited state of 2-aminopurine alone. However, the same cannot be said for structures in which 2-aminopurine is base stacked with other nucleobases. Stacking causes the molecular orbitals involved in the fluorescence transition to spread over more than one base. The predicted rate for the fluorescence transition is reduced, thus reducing the fluorescence quantum yield. The decrease in radiative rate varies with the stacking arrangement (e.g., A- or B-form DNA) and with the identity of the nucleobase with which 2-aminopurine is stacked. Stacking 2-aminopurine between two guanine moieties is shown to significantly decrease the energy gap between the first and second excited states. We do not find reliable evidence for a low-energy charge-transfer state in any of the systems that were studied. In the case of pyrrolocytosine, base stacking was found to reduce the oscillator strength for the fluorescence transition, but very little spreading of molecular orbitals across more than one base was observed.     

Dimerization of short RNA fragments of avian retroviruses as revealed using 2-aminopurine fluorescence

Dimerization of retroviral RNA is known in detail for several groups of viruses, especially the human immunodeficiency virus (HIV). The dimerization region seems to involve short RNA sequences directly responsible for the formation of dimers of two types, kissing loop-loop (KD) and linear (LD). The 5′-terminal sequences, where dimers are mostly formed, substantially differ between avian retroviruses and HIV, while the mechanisms of their RNA dimerization are the same. RNA dimerization was studied using the adenine analog 2-aminopurine (2-AP), which was incorporated into the loop sequence of short fragments of the avian leucosis virus (ALV) RNA. A temperature dependence of 2-AP fluorescence was used to study the KD formation under various conditions. Magnesium ions and the aminoglycoside antibiotic paromomycin were tested for the effect on KD stability. The highest KD stability was observed at >1 mM Mg²⁺ and >2.5 μM paromomycin.     

The coupling of tight DNA binding and base flipping: identification of a conserved structural motif in base flipping enzymes

Val(121) is positioned immediately above the extrahelical cytosine in HhaI DNA C(5)-cytosine methyltransferase, and replacement with alanine dramatically interferes with base flipping and catalysis. DNA binding and k(cat) are decreased 10(5)-fold for the Val(121) --> Ala mutant that has a normal circular dichroism spectrum and AdoMet affinity. The magnitude of this loss of function is comparable with removal of the essential catalytic Cys(81). Surprisingly, DNA binding is completely recovered (increase of 10(5)-fold) with a DNA substrate lacking the target cytosine base (abasic). Thus, interfering with the base flipping transition results in a dramatic loss of binding energy. Our data support an induced fit mechanism in which tight DNA binding is coupled to both base flipping and protein loop rearrangement. The importance of the proximal protein segment (His(127)-Thr(132)) in maintaining this critical interaction between Val(121) and the flipped cytosine was probed with single site alanine substitutions. None of these mutants are significantly altered in secondary structure, AdoMet or DNA affinity, k(methylation), k(inactivation), or k(cat). Although Val(121) plays a critical role in both extrahelical base stabilization and catalysis, its position and mobility are not influenced by individual residues in the adjacent peptide region. Structural comparisons with other DNA methyltransferases and DNA repair enzymes that stabilize extrahelical nucleotides reveal a motif that includes a positively charged or polar side chain and a hydrophobic residue positioned adjacent to the target DNA base and either the 5'- or 3'-phosphate.     

Dynamics of fluorescence marker concentration as a probe of mobility.

We have developed an effective experimental system for the characterization of molecular and structural mobility. It incorporates a modified fluorescence microscope geometry and a variety of analytical techniques to measure effective diffusion coefficients ranging over almost six orders of magnitude, from less than 10(-11) cm2/s to greater than 10(-6) cm2/s. Two principal techniques, fluorescence correlation spectroscopy (FCS) and fluorescence photobleaching recovery (FPR), are employed. In the FPR technique, translational transport rates are measured by monitoring the evolution of a spatial inhomogeneity of fluorescence that is produced photochemically in a microscopic volume by a short burst of intense laser radiation. In contrast, FCS uses laser-induced fluorescence to probe the spontaneous concentration fluctuations in microscopic sample volumes. The kinetics are analyzed by computing time-correlation functions of the stochastic fluctuations of the measured fluorescence intensity. The optical system and digital photocount correlator designed around a dedicated minicomputer are described and discussed. The general power of these techniques is demonstrated with examples from studies conducted on bulk solutions, lipid bilayer membranes, and mammalian cell plasma membranes.     

Use of 2-aminopurine and tryptophan fluorescence as probes in kinetic analyses of DNA polymerase beta

Although the use of 2-aminopurine (2-AP) as a probe in stopped-flow analyses of DNA polymerase beta (Pol beta) had provided important mechanistic insight, the conditions used were limited by the location of 2-AP and the use of a combination of tryptophan (Trp) and 2-AP fluorescence. This study examined different DNA substrates to identify several factors that can affect the observed signal in stopped-flow experiments. Both Trp and 2-AP emissions were separately excited and monitored. It was found that both probes show a fast phase and a slow phase of fluorescence changes, but the direction and the amplitude vary greatly between the two probes and between different DNA substrates. Detailed analyses suggested that the location of 2-AP in the template has a significant impact on the fluorescence properties of 2-AP and that a location opposite the incoming dNTP, which has been used in all such studies in the past, is not optimal. In particular, the results show that placing 2-AP one base after the templating base greatly enhances the signal intensity, which suggests a significant change in base stacking interactions at this position during nucleotide incorporation. These results allowed us to derive an improved set of conditions which were then used to reevaluate results from previous reports. It also allows greater freedom in the type of base pairs studied, since 2-AP is not the templating base in the nascent base pair. Kinetic constants were determined for dNTP and catalytic Mg(2+). The results obtained from stopped-flow experiments were compared to results from chemical quench. Stopped flow of incorrect dNTP incorporation and the reverse reaction are also reported, which provide useful information to the mechanism of Pol beta.     

Dimerization of short RNA fragments from avian leukosis virus as revealed with 2-aminopurine fluorescence

The dimerization of genomic retroviral RNA is well studied for several groups of viruses, the dimerization of human immunodeficiency (HIV) RNA being investigated in more detail. Regions of dimerization apparently involve the short sequences RNA which are directly responsible for the formation of two type dimers: kissing loop-loop (KD) and linear (LD). The 5'-end sequences from RNA avian viruses, where the dimers are basically formed, considerably differ from those of HIV. However, as it was described earlier, the mechanism of dimerization of RNA from human immunodeficiency and from avian leukosis viruses are identical. The fluorescence of adenine analogue 2-aminopurine (2-AP) incorporated into loop sequence of short fragments RNA ALV was used for analysis of dimers formation. Using the temperature dependence of fluorescence intensity 2-AP we have determined RNA melting temperature under various conditions for KD RNA ALV formed by two strands. Effects of magnesium and aminoglycoside antibiotic paromomycin on stabilization of kissing loop-loop dimer RNA have been studied. Under the experimental conditions KD RNA ALV was found to have the stability at the magnesium concentration higher than 1 mM and at paromomycin concentration higher than 2.5 mkM.     

Unusual 2-aminopurine fluorescence from a complex of DNA and the EcoKI methyltransferase

The methyltransferase, M.EcoKI, recognizes the DNA sequence 5'-AACNNNNNNGTGC-3' and methylates adenine at the underlined positions. DNA methylation has been shown by crystallography to occur via a base flipping mechanism and is believed to be a general mechanism for all methyltransferases. If no structure is available, the fluorescence of 2-aminopurine is often used as a signal for base flipping as it shows enhanced fluorescence when its environment is perturbed. We find that 2-aminopurine gives enhanced fluorescence emission not only when it is placed at the M.EcoKI methylation sites but also at a location adjacent to the target adenine. Thus it appears that 2-aminopurine fluorescence intensity is not a clear indicator of base flipping but is a more general measure of DNA distortion. Upon addition of the cofactor S-adenosyl-methionine to the M.EcoKI:DNA complex, the 2-aminopurine fluorescence changes to that of a new species showing excitation at 345 nm and emission at 450 nm. This change requires a fully active enzyme, the correct cofactor and the 2-aminopurine located at the methylation site. However, the new fluorescent species is not a covalently modified form of 2-aminopurine and we suggest that it represents a hitherto undetected physicochemical form of 2-aminopurine.     

Probing DNA polymerase-DNA interactions: examining the template strand in exonuclease complexes using 2-aminopurine fluorescence and acrylamide quenching

The bacteriophage T4 DNA polymerase forms fluorescent complexes with DNA substrates labeled with 2-aminopurine (2AP) in the template strand; the fluorescence intensity depends on the position of 2AP. When preexonuclease complexes are formed, complexes at the crossroads between polymerase and exonuclease complexes, 2AP in the +1 position in the template strand is fully free of contacts with the adjacent bases as indicated by high fluorescence intensity and a long fluorescence lifetime of about 10.9 ns. Fluorescence intensity decreases for 2AP in the template strand when the primer end is transferred to the exonuclease active center to form exonuclease complexes, which indicates a change in DNA conformation; 2AP can now interact with adjacent bases, which quenches fluorescence emission. Some polymerase-induced base unstacking for 2AP in the template strand in exonuclease complexes is observed but is restricted primarily to the n and +1 positions, which indicates that the DNA polymerase holds the template strand in a way that forces base unstacking only in a small region near the primer terminus. A hold on the template strand will help to maintain the correct alignment of the template and primer strands during proofreading. Acrylamide quenches 2AP fluorescence in preexonuclease and in exonuclease complexes formed with DNA labeled with 2AP in the template strand, which indicates that the template strand remains accessible to solvent in both complexes. These studies provide new information about the conformation of the template strand in exonuclease complexes that is not available from structural studies.     

Site-specific variations in RNA folding thermodynamics visualized by 2-aminopurine fluorescence

The fluorescent base analogue 2-aminopurine (2-AP) is commonly used to study specific conformational and protein binding events involving nucleic acids. Here, combinations of steady-state and time-resolved fluorescence spectroscopy of 2-AP were employed to monitor conformational transitions within a model hairpin RNA from diverse structural perspectives. RNA substrates adopting stable, unambiguous secondary structures were labeled with 2-AP at an unpaired base, within the loop, or inside the base-paired stem. Steady-state fluorescence was monitored as the RNA hairpins made the transitions between folded and unfolded conformations using thermal denaturation, urea titration, and cation-mediated folding. Unstructured control RNA substrates permitted the effects of higher-order RNA structures on 2-AP fluorescence to be distinguished from stimulus-dependent changes in intrinsic 2-AP photophysics and/or interactions with adjacent residues. Thermodynamic parameters describing local conformational changes were thus resolved from multiple perspectives within the model RNA hairpin. These data provided energetic bases for construction of folding mechanisms, which varied among different folding-unfolding stimuli. Time-resolved fluorescence studies further revealed that 2-AP exhibits characteristic signatures of component fluorescence lifetimes and respective fractional contributions in different RNA structural contexts. Together, these studies demonstrate localized conformational events contributing to RNA folding and unfolding that could not be observed by approaches monitoring only global structural transitions.     

Synthesis and properties of defined DNA oligomers containing base mispairs involving 2-aminopurine.

DNA heptamers containing the mutagenic base analogue 2-aminopurine (AP) have been chemically synthesized and physically characterized. We report on the relative stabilities of base pairs between AP and each of the common DNA bases, as determined from heptamer duplex melts at 275 and 330 nm. Base pairs are ranked in order of decreasing stability: AP.T greater than AP.A greater than AP.C greater than AP.G. It is of interest that AP.A is more stable than AP.C even though DNA polymerase strongly favors the formation of AP.C over AP.A base pairs. Comparisons of melting profiles at 330 nm and 275 nm indicate that AP.T, AP.A, and AP.C base pairs are annealed in heptamer duplexes and melt 2-3 degrees prior to surrounding base pairs, whereas AP.G appears not to be annealed.     

Simultaneous DNA binding, bending, and base flipping: evidence for a novel M.EcoRI methyltransferase-DNA complex

We measured the kinetics of DNA bending by M.EcoRI using DNA labeled at both 5'-ends and observed changes in fluorescence resonance energy transfer. Although known to bend its cognate DNA site, energy transfer is decreased upon enzyme binding. This unanticipated effect is shown to be robust because we observe the identical decrease with different dye pairs, when the dye pairs are placed on the respective 3'-ends, the effect is cofactor- and protein-dependent, and the effect is observed with duplexes ranging from 14 through 17 base pairs. The same labeled DNA shows the anticipated increased energy transfer with EcoRV endonuclease, which also bends this sequence, and no change in energy transfer with EcoRI endonuclease, which leaves this sequence unbent. We interpret these results as evidence for an increased end-to-end distance resulting from M.EcoRI binding, mediated by a mechanism novel for DNA methyltransferases, combining DNA bending and an overall expansion of the DNA duplex. The M.EcoRI protein sequence is poorly accommodated into well defined classes of DNA methyltransferases, both at the level of individual motifs and overall alignment. Interestingly, M.EcoRI has an intercalation motif observed in the FPG DNA glycosylase family of repair enzymes. Enzyme-dependent changes in anisotropy and fluorescence resonance energy transfer have similar rate constants, which are similar to the previously determined rate constant for base flipping; thus, the three processes are nearly coincidental. Similar fluorescence resonance energy transfer experiments following AdoMet-dependent catalysis show that the unbending transition determines the steady state product release kinetics.     

Intrinsic fluorescence as a probe of structure-function relationships in Ca2+-transport ATPases

Applications of intrinsic fluorescence measurements in the study of Ca²⁺-transport ATPases are reviewed. Since the initial reports showing that the fluorescence emission was sensitive to Ca²⁺ binding, a substantial amount of work has focused on the use of both steady-state and time-resolved fluorescence spectroscopy to investigate structure-function relationships in sarcoplasmic reticulum and plasma membrane Ca²⁺-ATPases. These studies have revealed ligand-induced conformational changes, as well as provided information on protein-protein, protein-solvent and/or protein-lipid interactions in different functional states of these proteins. The main results of these studies, as well as possible future prospects are discussed.     

Functions of base flipping in E. coli nucleotide excision repair

UvrB is the main damage recognition protein in bacterial nucleotide excision repair and is capable of recognizing various structurally unrelated types of damage. Previously we have shown that upon binding of Escherichia coli UvrB to damaged DNA two nucleotides become extrahelical: the nucleotide directly 3' to the lesion and its base-pairing partner in the non-damaged strand. Here we demonstrate using a novel fluorescent 2-aminopurine-menthol modification that the position of the damaged nucleotide itself does not change upon UvrB binding. A co-crystal structure of B. caldotenax UvrB and DNA has revealed that one nucleotide is flipped out of the DNA helix into a pocket of the UvrB protein where it stacks on Phe249 [J.J. Truglio, E. Karakas, B. Hau, H. Wang, M.J. DellaVecchia, B. van Houten, C. Kisker, Structural basis for DNA recognition and processing by UvrB, Nat. Struct. Mol. Biol. 13 (2006) 360-364]. By mutating the equivalent of Phe249 (Tyr249) in the E. coli UvrB protein we show that on damaged DNA neither of the extrahelical nucleotides is inserted into this protein pocket. The mutant UvrB protein, however, resulted in an increased binding and incision of undamaged DNA showing that insertion of a base into the nucleotide-binding pocket is important for dissociation of UvrB from undamaged sites. Replacing the nucleotides in the non-damaged strand with a C3-linker revealed that the extruded base in the non-damaged strand is not directly involved in UvrB-binding or UvrC-mediated incision, but that its displacement is needed to allow access for residues of UvrB or UvrC to the neighboring base, which is directly opposite the DNA damage. This interaction is shown to be essential for optimal 3'-incision by UvrC. After 3'-incision base flipping in the non-damaged DNA strand is lost, indicative for a conformational change needed to prepare the UvrB-DNA complex for 5'-incision.