Báo cáo khoa học: Formation of nucleoprotein RecA filament on single-stranded DNA Analysis by stepwise increase in ligand complexity potx

Formation of nucleoprotein RecA filament on single-stranded DNA Analysis by stepwise increase in ligand complexity Irina P. Bugreeva, Dmitry V. Bugreev and Georgy A. Nevinsky Institute of Chemical Biology and Fundamental Medicine, Siberian Division of Russian Academy of Sciences, Novosibirsk, Russia Homologous recombination, required for the mainten- ance of genetic diversity and DNA repair, is one of the most important molecular genetic processes. In Escheri- chia coli, a pivotal role in homologous recombination is played by RecA protein, which is responsible for search for homologous DNA sequences and strand transfer between them [1]. RecA is an ATP-dependent DNA- binding protein consisting of 352 amino acids (37.8 kDa) [1]. Binding of RecA to DNA occurs in three stages: first, the presynapsis, when RecA is polymerized on ssDNA forming a right-handed nucleoprotein filament; second, the synapsis, when the presynaptic complex binds dsDNA and actively searches for homo- logy with the ssDNA; and third strand exchange, when a new DNA duplex is formed and one of the strands formerly in dsDNA is released as ssDNA. Thus, a RecA filament assembles on DNA at the first stage; this process is more efficient with ssDNA. Binding of RecA to ssDNA must be nonspecific, but the protein displays some preferences for binding poly(dT) and GT-rich sequences [2–5]. In the presence of ATP or its nonhydrolysable thio analog (ATPcS), RecA forms a right-handed filament of 100 A ˚ diameter and 95 A ˚ pitch [6]. The filament is assembled cooperatively in the 5¢)3¢ direction (in respect to the ssDNA) [7]. DNA in such complex is stretched by % 50%, with the internucleotide distance increasing to 5.1 A ˚ [8]. If RecA binds to dsDNA, the Keywords RecA; DNA recognition mechanism Correspondence G. A. Nevinsky, Laboratory of repair enzymes, Institute of Chemical Biology and Fundamental Medicine, 8, Lavrentieva Ave., 630090, Novosibirsk, Russia Fax: +7 3832 333677 Tel: +7 3832 396226 E-mail: nevinsky@niboch.nsc.ru (Received 8 January 2005, revised 24 February 2005, accepted 31 March 2005) doi:10.1111/j.1742-4658.2005.04693.x RecA protein plays a pivotal role in homologous recombination in Escheri- chia coli. RecA polymerizes on single-stranded (ss) DNA forming a nucleo- protein filament. Then double-stranded (ds) DNA is bound and searched for segments homologous to the ssDNA. Finally, homologous strands are exchanged, a new DNA duplex is formed, and ssDNA is displaced. We report a quantitative analysis of RecA interactions with ss d(pN) n of var- ious structures and lengths using these oligonucleotides as inhibitors of RecA filamentation on d(pT) 20 . DNA recognition appears to be mediated by weak interactions between its structural elements and RecA monomers within a filament. Orthophosphate and dNMP are minimal inhibitors of RecA filamentation (I 50 ¼ 12–20 mm). An increase in homo-d(pN) 2)40 length by one unit improves their affinity for RecA (f factor) approximately twofold through electrostatic contacts of RecA with internucleoside phos- phate DNA moieties (f % 1.56) and specific interactions with T or C bases (f % 1.32); interactions with adenine bases are negligible. RecA affinity for d(pN) n containing normal or modified nucleobases depends on the nature of the base, features of the DNA structure. The affinity considerably increa- ses if exocyclic hydrogen bond acceptor moieties are present in the bases. We analyze possible reasons underlying RecA preferences for DNA sequence and length and propose a model for recognition of ssDNA by RecA. Abbreviations EMSA, electrophoretic mobility shift assay; ODN, deoxyribooligonucleotide(s); SILC, stepwise increase in ligand complexity; ss-, single-stranded; ds-, double-stranded. 2734 FEBS Journal 272 (2005) 2734–2745 ª 2005 FEBS parameters of the resulting filament are the same as for ssDNA, and the DNA duplex in the filament is unwound as compared with B-DNA [9,10]. In the absence of ATP RecA forms a more compact inactive filament of 64 A ˚ pitch and 2.1 A ˚ internucleotide dis- tance [11]. After the filament is formed, the second DNA bind- ing site of RecA can bind dsDNA. In addition, ssDNA can be bound there, even more efficiently than dsDNA. After strand exchange, the second RecA DNA binding site binds the displaced strand following new DNA duplex formation [12]. Binding of dsDNA to a RecA filament is followed by search of homology between the appropriate strands and then by strand exchange. The mechanism of this process is still unclear. It was hypothesized that ssDNA could invade through the minor or major groove of the duplex and displace the respective strand [1]; if the inva- sion occurs through the major groove, formation of a peculiar DNA triplex (R-form DNA) was proposed [13,14]. An alternative mechanism (melting–annealing model) for the homology search involves only formation of canonical Watson–Crick pairs after melting of the duplex and annealing of its appropriate strand to the incoming strand [15]. As DNA in the filament is consid- erably stretched and unwound, the bases could be easily extruded from the helix to be ‘examined’ for homology with the incoming strand. Howard-Flanders proposed a triple helix as a tran- sient, or even a stable, intermediate in the reaction [16]. However, all recent efforts have failed to detect such a structure as a stable intermediate. Instead, several groups have described a stable synaptic complex con- sisting of three strands and RecA, in which strand exchange has already taken place [17,18]. In this com- plex, the incoming ssDNA is part of the new duplex and the leaving strand has not yet been released. Leaving aside such an early triplex, one can jump forward and ask what are the steps leading to such a poststrand exchange intermediate? One can envision several slower confor- mational changes, such as homology recognition via base flipping (melting) and switching (annealing) [19]. DNA binding by RecA is thought to be mediated by amino-acid residues from two protein loops, L1 (resi- dues 157–164) and L2 (residues 195–209) [1,20]. Both these regions are rather conserved among bacterial RecA proteins but not between bacterial, archaean and eukaryotic RecA homologs. In addition, DNA could interact with several RecA tyrosine residues (Tyr65, Tyr103, Tyr264) [21–23], as well as with Lys183 [23,24], Arg243 [22] and residues 233–242 [24]. This list all but exhausts the available information regarding RecA–DNA interactions. To our knowledge, there have been no quantitative studies on general parameters of and individual contacts within the forming nucleo- protein filament. Our laboratory has designed a novel approach to analysis of protein ⁄ nucleic acid interactions, based on stepwise increase in ligand complexity (SILC approach). SILC produces quantitative estimates of the contributions of individual structural elements of DNA or RNA molecules into the affinity of enzymes to such extended ligands [25–27]. We have applied SILC to analyze DNA binding by a number of DNA polymerases [25–27], DNA repair enzymes [28– 31], EcoRI restriction endonuclease [32], HIV-1 integ- rase [33], and type I DNA topoisomerases [34,35]. In all these instances, virtually every nucleotide unit within the DNA binding cleft (10–20 base pairs cov- ered by the protein globule) interacts with the enzyme through weak additive electrostatic, hydro- phobic or van der Waals contacts to various struc- tural elements of the ligands, with electrostatic interactions of internucleoside phosphate moieties contributing most to the affinity (reviewed in [25– 27]). These nonspecific contacts provide high affinity (K d ¼ 10 )5 )10 )8 m) of all enzymes for specific and nonspecific DNA. A transition from nonspecific to spe- cific DNA usually leads to formation of specific contacts and increase of the affinity by 1–2 orders of magnitude (up to K d ¼ 10 )8 )10 )10 m), while the reaction rate (k cat ) is enhanced by 5–8 orders. Thus, specificity of DNA-dependent enzymes is not of thermodynamic nature (the enzyme-substrate complex formation) but mostly originates from the following stage of enzyme- induced adjustment of DNA conformation and from chemical steps (k cat ) of catalysis [25–27]. Quantitative studies concerning the efficiency of interactions between a RecA filament and DNA are a prerequisite for understanding the nature of RecA filamentation; however, no such information is available so far. SILC is a very promising approach for obtaining the appropriate data. Here we present a SILC analysis of RecA interactions with ssODN of different structures and lengths and estimate the contribution of individual DNA elements in its affinity for a RecA filament. Results Filamentation of RecA on ssDNA and its inhibition In the presence of ATP or ATPcS RecA is polymer- ized on DNA forming a nucleoprotein filament. We have studied the stability of a RecA filament formed with different individual 5¢-[ 32 P]d(pN) n (n ¼ 2–20) by I. P. Bugreeva et al. RecA filament interaction with DNA FEBS Journal 272 (2005) 2734–2745 ª 2005 FEBS 2735 electrophoretic mobility shift assay (EMSA). The com- plexes between RecA and short individual d(pN) n (n ¼ 2–15) were easily disassembled, confirming the lit- erature data on their low stability [36,37]. Individual 5¢-[ 32 P]d(pN) 16)20 formed detectable complexes with RecA under the condition used (data not shown), and the best of them d(pT) 20 was used for the rest of the study. At the RecA monomer: ODN ratio of 10 : 1, almost all d(pT) 20 was in the filament, in agreement with the known RecA monomer interaction with three nucleotide units of ssDNA [1]. As the interactions with short ODNs are of low affin- ity, they are undetectable by EMSA and many other widely used physicochemical techniques [27]. However, interactions of enzymes with low-affinity ligands can be easily followed by observing inhibition of appropriate enzymatic activity by these ligands (reviewed in [25–27]). In the case of short ODN interacting with RecA mono- mers or forming short unstable filaments, the respective ODN ligands should inhibit RecA filamentation on d(pT) 20 . In addition, at high concentration short ODN can compete with d(pT) 20 for the filament formed on this substrate. We have shown that the addition of any short ODN causes a decrease in the amount of 5¢-[ 32 P]d(pT) 20 detectable in the RecA filament complex. Concentration dependencies of RecA-d(pT) 20 complex formation on the inhibitor concentration had regular hyperbolic shapes (Fig. 1), indicating that RecA filamentation on d(pT) 20 and its inhibition by short ODN, including orthophosphate (I 50 ¼ 0.5 m) and various dNMPs (I 50 ¼ 12–20 mm) as minimal ligands, obey formally canonical steady-state equations of complex formation. The apparent values of I 50 (Fig. 1) were used to charac- terize the relative efficiency of RecA interactions with various ODN; these data are summarized in Table 1. The Gibbs free energy characterizing enzyme-ligand complex formation can be presented as a sum of DG° values for each individual contact: DG 0 ¼ DG 0 1 þ DG 0 2 þ ::: þ DG 0 n with DG 0 i ¼ÀRT ln K di ð1Þ where K di is the contribution of an individual contact to the overall affinity [38]. It follows from the additi- vity of Gibbs free energies that the overall K d (K d ¼ K I ) value characterizing complex formation is the product of the K d values for individual contacts: DG 0 ¼ÀRT ln K d ¼ÀRT ln½K d1 K d2 :::K dn ; and K d ¼ K d1 K d2 :::K dn ð2Þ To assess possible additivity of the interactions of ODN with RecA filament, the data from Table 1 were analyzed as logarithmic dependencies of I 50 for d(pN) n on the number (n) of mononucleotide units (0 £ n £ 20, n ¼ 0 corresponds to orthophosphate, P i ). Affinity of d(pN) n ligands to RecA increased mono- tonously in the d(pN) 2 –d(pN) 20 interval, d(pT) n and d(pC) n producing nearly identical results (Fig. 2). Dependencies of lgI 50 on n were linear at 2 £ n £ 20 (Fig. 2), indicating that the affinity of RecA to each of the nucleotide units of d(pN) 20 is additive. Interestingly, experimentally estimated affinities of dNMP (I 50 ¼ 12–20 mm) were somewhat higher than that for corresponding d(pN) 2 (40–47 mm, Table 1). This phenomenon, also observed for some other enzymes, arises from greater conformational freedom of individual dNMP (or short ODN) compared with the same ligands as elements of long DNA [25–27]. Considerable stretching and unwinding of DNA in a RecA filament is associated with energetic costs required for sugar-phosphate backbone deformation and stacking disruption [6]. Mononucleotides are not subject to such restrictions and thus can bind RecA more efficiently. Extrapolation of the log dependencies for d(pT) n and d(pC) n to n ¼ 1 (Fig. 2) gives lower A B Fig. 1. Dependence of the relative level of inhibition of RecA filam- entation on [ 32 P]d(pT) 20 on the concentration of d(pT) 10 inhibitor. (A) Reaction products separated by EMSA in polyacrylamide gel. (B) Band intensities in (A) quantified by Cherenkov counting and plotted against inhibitor concentrations. Lane 1, filamentation without the inhibitor; lane 10, reaction mixture without RecA; d(pT) 10 inhibitor added at 0.05 m M (lane 2), 0.1 mM (lane 3), 0.2 mM (lane 4), 0.3 m M (lane 5), 0.5 mM (lane 6), 0.6 mM (lane 7), 0.8 mM (lane 8) and 1 m M (lane 9). The upward shift in free oligonucleotide position appears due to a time lag in loading different reaction mixtures onto a running gel. RecA filament interaction with DNA I. P. Bugreeva et al. 2736 FEBS Journal 272 (2005) 2734–2745 ª 2005 FEBS affinity values for RecA binding single d(pT) and d(pC) units (I 50 ¼%63 mm) within longer d(pN) n (Fig. 2; Table 1). Thus, this value of I 50 ¼%63 mm is a better parameter to characterize RecA affinity for the higher-affinity nucleotide unit of d(pN) n in com- parison with the remaining (n–1) nucleotide units within d(pT) n or d(pC) n , which have lower affinity for the filament (490 mm, see below). I 50 values are usually related to the K I values [38]. For example, in the case of competitive inhibition, they are related through the equation I 50 ¼ aK I (a ¼ 1 + [S]⁄ K S ; K S is K M or K d for substrate), where the coefficient a depends on the affinity and concentration of a substrate, d(pT) 20 in our case. Therefore, the ratio of K I values for two different inhibitors, K I (2) ⁄ K I (1), is equal to the ratio of apparent I 50 values for these inhibitors, I 50 (2) ⁄ I 50 (1), and the ratio of these values gives the K d value characterizing a difference of the enzyme contacts between the first and the second inhibitors (Eqns 1 and 2) [38]. From the slope of the lgI 50 vs n dependency (Fig. 2) the factor (f) reflecting an increase in affin- ity of the enzyme for d(pN) n upon a one-unit increase in the ligand length can be calculated as: f ¼ 10 –[lgI 50 (n ¼ 20) ⁄ –lgI 50 (n ¼ 2)] ⁄ 18 (exact average values of lgI 50 were calculated using the log curves). From the slopes of the curves for d(pT) n and d(pC) n (Fig. 2), the value f ¼ 2.04 was calculated for the f factor. As 1 ⁄ f(n) ¼ I 50 (n) ⁄ I 50 (n +1)¼ K I (n) ⁄ K I (n +1)¼ K d (n) ⁄ K d (n + 1), interaction of a RecA filament with any of the 19 units of d(pT) 20 or d(pC) 20 is characterized by K d ¼ K I ¼ 1 ⁄ f ¼ 0.49 m. Comparison of this value with I 50 for free dNMP determined experimentally I 50(experimental) ¼ 12–20 mm, Table 1 or for a dNMP unit within d(pN) n by extra- polation to n ¼ 1 I 50(extrapolated) ¼%63 mm; Fig. 2 shows that the affinity of RecA for one of the units or d(pT) 20 or d(pC) 20 is 8–41-fold higher than for any of the remaining 19 units. Extrapolation of the log dependencies for d(pT) n and d(pC) n to n ¼ 0 gives I 50(extrapolated) ¼ 15 mm for a single internucleo- side phosphate group of d(pN) n , approximately 3.3-fold lower than the experimental I 50 ¼ 0.5 m for free orthophosphate. Overall, the affinity of a RecA filament for d(pT) n and d(pC) n at 2 £ n £ 20 may be described as I 50 [d(pN) n ] ¼ I 50 (d(pN) 2 ) · (1 ⁄ f) n)2 ¼ Table 1. I 50 values for interactions of different ligands with the high-affinity DNA-binding center of E. coli RecA filament. Ligand (inhibition of d(pT) 20 ) I 50 , M* –lgI 50 Ligand (inhibition of d(pT) 20 ) I 50 , M –lgI 50 PO 4 3– 0.5 0.30 d(pR)§ 0.6 0.22 One internucleoside phosphate within d(pC) n and d(pT) n ** 0.15 0.63 One internucleoside phosphate within d(pA) n ** 0.23 0.82 dTMP 2.0 · 10 )2 1.70 dCMP 1.3 · 10 )2 1.89 One (pT)-unit of d(pT) n ** % 6.3 · 10 )2 1.20 One (pC)-unit of d(pC) n ** % 6.3 · 10 )2 1.20 d(pT) 2 4.0 · 10 )2 1.40 d(pC) 2 4.7 · 10 )2 1.33 d(pT) 3 1.75 · 10 )2 1.76 d(pC) 4 1.1 · 10 )2 1.96 d(pT) 4 1.0 · 10 )2 2.00 d(pC) 6 2.5 · 10 )3 2.60 d(pT) 5 5.0 · 10 )3 2.30 d(pC) 8 5.7 · 10 )4 3.24 d(pT) 6 2.5 · 10 )3 2.60 d(pC) 10 5.0 · 10 )4 3.30 d(pT) 8 5.0 · 10 )4 3.30 d(pC) 12 4.3 · 10 )5 4.37 d(pT) 10 2.0 · 10 )4 3.70 d(pC) 16 1.8 · 10 )6 5.74 d(pT) 12 3.5 · 10 )5 4.45 d(pC) 20 1.6 · 10 )7 6.80 d(pT) 14 8.3 · 10 )6 5.08 dAMP 1.24 · 10 )2 1.90 d(pT) 16 1.5 · 10 )6 5.82 One (pA)-unit of d(pA) n ** % 10.0 · 10 )2 1.20 d(pT) 20 1.0 · 10 )7 7.00 d(pA) 2 4.5 · 10 )2 1.35 d(Tp) 7 T 4.8 · 10 )3 2.32 d(pA) 4 7.0 · 10 )3 2.15 d(Tp) 8 2.5 · 10 )3 2.60 d(pA) 6 1.04 · 10 )3 2.98 d(pTT(pR) 17 T)*** 9.5 · 10 )6 5.02 d(pA) 8 5.2 · 10 )4 3.28 d[p(ethyl)T] 10 5.0 · 10 )3 2.30 d(pA) 10 2.5 · 10 )4 3.60 d(pR) 20 ** 2.1 · 10 )5 4.68 d(pA) 12 1.3 · 10 )4 3.89 I 50 determined using d(pT) 40 as substrate d(pA) 14 8.0 · 10 )5 4.10 d(pT) 20 1.0 · 10 )7 7.00 d(pA) 16 5.4 · 10 )5 4.27 d(pT) 30 2.3 · 10 )8 7.64 d(pA) 18 3.2 · 10 )5 4.50 d(pT) 40 7.0 · 10 )9 8.15 d(pA) 20 2.4 · 10 )5 4.62 *Error in I 50 values was 10–30%; means of 3–4 measurements are given; **The values of I 50 determined by extrapolation of lg-curves to n ¼ 0 for Pi and n ¼ 1 for dNMPs (Fig. 2); §d(pR), deoxyribosephosphate; ***R is a tetrahydrofuran analog of abasic deoxyribose. I. P. Bugreeva et al. RecA filament interaction with DNA FEBS Journal 272 (2005) 2734–2745 ª 2005 FEBS 2737 I 50 (d(pN) 2 ) · ( f) 2–n , when at 1 £ n £ 20 as I 50 [d(pN) n ] ¼ I 50 (dNMP, extrapolated) · (1⁄ f) n)1 , where I 50 (dNMP, extrapolated) ¼%63 mm reflects the contribution of the high-affinity nucleotide init within longer d(pN) n , and f (2.04) describes an increase in affinity due to a one-unit increase in d(pN) n length. The logarithmic dependence for d(pA) n (Fig. 2) can be broken in two nearly linear segments with different slopes at 2 £ n £ 6–7 and 6–7 £ n £ 20. For the first segment, f ¼ 2.12 (K d % 0.47 m), and for the second, f ¼ 1.32 (K d % 0.76 m). Interestingly, the affinity of RecA for d(pA) 20 is % 240-fold lower than for d(pT) 20 (Table 1, Fig. 2). This observation agrees well with lower stability of a RecA ⁄ d(pA) 20 complex during EMSA. Extrapolation of the logarithmic dependency for d(pA) n towards higher n suggests that only for d(pA) 40)45 the I 50 value will be comparable with that for d(pT) 20 ; empirically, complexes of RecA with d(pA) n are stable during electrophoresis from this length onward (data not shown). It can be clearly seen in Fig. 2 that the nature of protein–DNA interactions was nearly the same for different d(pN) n at 1 £ n £ 10. The next 10 DNA units were bound better in pyrimidine ODN. A decrease in the interaction efficiency at n > 7–8 for d(pA) n could mean that the structure of DNA complex with the first three RecA monomers may be important for the assembly of the next monomers. The data shown in Fig. 2 suggest that the further elongation of d(pT) n (n > 20) should also be accom- panied with a monotonous increase in their affinity. To investigate this possibility, we used a 5¢-[ 32 P]d(pT) 40 substrate and analyzed inhibition of RecA filamenta- tion by d(pT) 20)40 (Table 1). The apparent I 50 values for d(pT) 20 determined with [ 32 P]d(pT) 20 and [ 32 P]d(pT) 40 as substrates were nearly the same (Table 1). Figure 2 (inset) shows that the lgI 50 values for d(pT) 20 , d(pT) 30 , and d(pT) 40 apparently fall on a straight line. This is consistent with an increase in RecA filament affinity with increasing ssDNA length. The shallowing of the log dependence slope at n ¼ 20–40 can be due to two reasons. First, it cannot be excluded that correct determination of I 50 values for d(pN) 30)40 may be unreliable and the observed I 50 val- ues are higher than real I 50 values. On the other hand, the change in the slope of the log dependencies may reflect a decrease in the efficiency of RecA filament interaction with very long DNA due to ‘polymeric effect’ usually associated with increased mobility and flexibility of long polymeric structures with high con- formational freedom. Nature of RecA interactions with nucleic acids It has been shown for many DNA-depending enzymes that strongest contacts they form with ssDNA are those with the internucleoside phosphate moieties; some enzymes also can interact with nucleobases [25– 27]. Introduction of 5¢-or3¢-terminal phosphate moiet- ies in ODN increased their affinity for RecA. For instance, the apparent I 50 value for d(Tp) 7 T (4.8 · 10 )3 m) was about an order of magnitude higher than that for d(pT) 8 (5.0 · 10 )4 m) and twofold higher than for d(Tp) 8 (2.5 · 10 )3 m). Whereas the introduct- ion of a 3¢-phosphate moiety had an effect similar to that of the f factor nature (f ¼ 2.04) for pyrimidine ODN, the effect of a 5¢ phosphate was much more pronounced. Although the negative charge at the ter- minal phosphates is one negative charge higher than at internucleotide phosphate moieties, this increase seems to influence the filament affinity for the 5¢-terminal ODN phosphate to a larger extent than to the 3¢-ter- minal phosphate. It is possible that the 5¢-terminal phosphate of ODN has more conformational freedom and can form additional contacts with the filament. As Fig. 2. Affinity of RecA (logarithmic dependencies of apparent I 50 ) to homo-ODN of different lengths (n) determined using inhibition of RecA filamentation on [ 32 P]d(pT) 20 . The I 50 values for different d(pN) n (1 £ n £ 20) and oligonucleotides containing abasic units or ethylated internucleoside phosphates are obtained using [ 32 P]d(pT) 20 and for d(pT) 20)40 [ 32 P]d(pT) 40 (20 £ n £ 40, see the inset): d(pT) n (open sircules; including the inset), d(pA) n (cross), d(pC) n (triangles), Positions of –lgI 50 values for ethylated d[(pEt)T] 10 and d[(pT) 2 (pR) 17 (pT)] (pR is a tetrahydrofuran analog of abasic deoxyribose) are shown. RecA filament interaction with DNA I. P. Bugreeva et al. 2738 FEBS Journal 272 (2005) 2734–2745 ª 2005 FEBS the filament assembly on ssDNA occurs cooperatively in the 5 ¢)3¢ direction [8], the increased affinity of RecA to the 5¢-terminal phosphate of ODN may be import- ant for better anchoring of ODN on the first RecA monomer during the initiation of filamentation. Ethylation of internucleoside phosphate moieties neutralizes their charges. The affinity of a RecA fila- ment for d(pT) 10 (I 50 ¼ 2.0 · 10 )4 m) was % 25-fold lower than for ethylated d[p(Et)T] 10 (I 50 ¼ 5.0 · 10 )3 m) (Table 1), indicative of an important role of negative charges of internucleoside phosphates for RecA complexation with DNA. The affinity of RecA to d(pT) 20 (I 50 ¼ 1.0 · 10 )7 m) was % 100-fold higher than to d[(pT) 2 (pR) 17 pT] (I 50 ¼ 9.5 · 10 )6 m), a 20-mer lacking 17 out of 20 nucleo- bases (R is a tetrahydrofuran analog of abasic deoxy- ribose). As was shown earlier [25–27], deoxyribose moieties of DNA have little effect on its affinity for proteins, while internucleoside phosphate groups make the main contribution. Taking this into account and assuming that the lack of the bases did not influence the filament interactions with the backbone, the increase in affinity due to a single internucleoside phosphate residue (electrostatic factor e) can be esti- mated as e ¼ (I 50 ¼ 1.75 · 10 )2 m for d(pT) 3 ) ⁄ (I 50 ¼ 9.5 · 10 )6 m for d[(pT) 2 (pR) 17 pT]) 1 ⁄ 17 ¼ 1842 1 ⁄ 17 ¼ 1.56 (K d ¼ 0.64 m). As an increase in the affinity for one (pT) unit (f ¼ 2.04) is a product of its increase due to an internucleoside phosphate group (factor e ¼ 1.56) and a T base (factor f T ), f T can be calculated as a ratio f ⁄ e ¼ 1.31 K d (T base) ¼ 1 ⁄ 1.31 ¼ 0.76 (m). The same value of f T can be calculated directly: f T ¼ (I 50 ¼ 9.5 · 10 )6 m for d[(pT) 2 (pR) 17 pT]) ⁄ (I 50 ¼ 1.0 · 10 )7 m for d[(pT) 20 ]) 1 ⁄ 17 ¼ 95 1 ⁄ 17 ¼ 1.31. The affinity increases due to one C base (f C ¼ 1.31) and one T base (f T ¼ 1.31) are the same. Thus, RecA in the filament forms weak additive contacts with each internucleoside phosphate moiety and each base of pyrimidine ODN, with the phosphates contribution into the affinity being % 1.2-fold more than that of C or T bases. As the filament affinity for d(pA) 20 (I 50 ¼ 2.4 · 10 )5 m) was very similar to that for d[(pT) 2 (pR) 17 pT] (I 50 ¼ 9.5 · 10 )6 m) or for the affin- ity calculated for a totally abasic oligomer d(pR) 20 (I 50 % 2.1 · 10 )5 m), the filament probably interacts with adenine bases in DNA very weakly if at all. RecA interactions with nucleobases To evaluate the importance of exocyclic acceptor moi- eties, we have compared the efficiency of RecA filam- entation on d(pA) 20 and d(pI) 20 , where in the latter, the O6 acceptor moiety of hypoxanthine base substi- tutes for the exocyclic amino group of adenine. The amount of d(pI) 20 incorporated in the filament was less than with d(pT) 20 but d(pI) 20 formed a stronger com- plex with RecA than did d(pA) 20 (Fig. 3). RecA is a DNA-dependent ATPase, with the effi- ciency of ATP hydrolysis correlating with the stability and length of the RecA filament [36]. Figure 4 shows that the extent of ATP hydrolysis correlates well with the efficiency of RecA filamentation on various d(pN) 20 , allowing us to use ATP hydrolysis to estimate the RecA filamentation efficiency and the stability of the resulting nucleoprotein filaments for a variety of DNA substrates. The highest values of ATP hydrolysis rate (expressed as percentage of initial ATP) in the presence of differ- ent polynucleotides are summarized in Table 2. The results show that DNA substrates can be divided into three classes according to the efficiency of ATP hydro- lysis stimulation (Table 2). Although both guanine and hypoxanthine have an acceptor O6 and a donor NH1 moiety, poly(dG) was similar to poly(dA) in poor sti- mulation of ATP hydrolysis. Deamination of poly(dG) and poly(dA) significantly increased the rate of ATP hydrolysis and the efficiency of filamentation. Similar A B Fig. 3. Efficiency of RecA filamentation on 32 P-labeled d(pT) 20 , d(pA) 20 , and d(pI) 20 : electrophoretic mobility shift after 5 min of incubation (A) and time course of filamentation (B). d(pT) 20 (m), d(pI) 20 (d), d(pA) 20 (j). I. P. Bugreeva et al. RecA filament interaction with DNA FEBS Journal 272 (2005) 2734–2745 ª 2005 FEBS 2739 increase in ATP hydrolysis accompanied a switch from poly(dAG) to a mixed deoxy(inosine ⁄ xanthine) poly- mer. DNA containing both purines and pyrimidines displayed wide variations in its interactions with RecA. For instance, poly(dAC) and poly(dTG) were efficiently bound by RecA, poly(dAT) fell between poly(dA) and poly(dT) ligands, and poly(dCG) promo- ted very little ATP hydrolysis. The data in Table 2 indicate that purine poly(dN), even those containing exocyclic hydrogen bond acceptors, generally interacts with RecA and stimulates ATP hydrolysis less effi- ciently than pyrimidine polymers. Perhaps the reason is larger size of purine bases compared with pyrimi- dines, hindering binding of the former by RecA. In addition, contacts formed by RecA could be important not only for the complex formation but also for con- formational changes in individual RecA monomers and their ATPase activity. Deamination of mixed polynucleotides with forma- tion of dI from dA, dX from dG, and dU from dC, caused an increase in the efficiency of interactions with RecA, especially for the poly(dCG) fi poly(dUX) transition. Interestingly, RecA interaction with purine ligands was also improved by replacement of adenine exocyclic amino group with a halogen atom, also a hydrogen bond acceptor due to its lone electron pairs. Discussion We have previously shown that the interaction of dif- ferent sequence-specific DNA enzymes (repair, topo- isomerization, restriction, integration enzymes) with each nucleotide unit of nonspecific ss- or ds-ODNs is usually a superposition of weak electrostatic and hydrophobic or van der Waals interactions with the individual structural elements [25–27]. The interaction can be described by the power law: K d ½dðpNÞ n ¼K d ½ðP i ÞðeÞ Àn ðh C Þ Àc ðh T Þ Àt ðh G Þ Àg ðh A Þ Àa ; where K d [(P i )] is the K d for the minimal orthophos- phate ligand (or sometimes dNMPs), e is a factor reflecting an increase of affinity due to one internucleo- side phosphate group; h N are coefficients of increase in affinity due to hydrophobic and ⁄ or van der Waals interactions of the enzyme with one of the bases: C, T, G and A, the numbers of which in d(pN) n are equal to c, t, g and a, respectively. In addition, factor f reflect- ing increase in affinity due to one (pN)-unit is equal to (h N · e). When passing from one enzyme to another only the values of e (1.35–2.0) and h N (1.0–1.4) factors and K d for orthophosphate (10 -3 -10 -1 m) or dNMP as minimal ligands are changed [25–27]. As shown above, a similar algorithm I 50 [d(pN) n ] ¼ I 50 (dNMP) · f 1–n can be used for description of RecA filament interaction with ssODNs. Protein globules of various enzymes usually cover from 10 to 20 nucleotides of DNA and the affinity of the enzyme active center (or its specific site) for one Fig. 4. Time course of RecA filamentation on 32 P-labeled d(pT) 20 , d(pC) 20 , and d(pA) 20 (A) and RecA-dependent [ 32 P]ATP[cP] hydro- lysis stimulated by the same ODN (B). d(pT) 20 (m), d(pC) 20 (d), d(pA) 20 (j). Table 2. Highest levels of RecA-catalyzed ATP hydrolysis in the presence of various poly(dN). DNA ATP hydrolyzed (%) DNA ATP hydrolyzed (%) poly(dA) 1.6 poly(dG) 3.5 poly(dAT) 33.2 poly(dIT) 63.4 poly(dAC) 59.4 poly(dIX) 43.4 poly(dAG) 1.3 poly(dTX) 63.6 poly(dC) 63.1 poly(dXU) 62.1 poly(dGC) 1.5 poly(dU) 63.0 poly(dT) 61.0 poly(dI) 24.3 poly(dTG) 58.5 poly(dX) 28.4 RecA filament interaction with DNA I. P. Bugreeva et al. 2740 FEBS Journal 272 (2005) 2734–2745 ª 2005 FEBS nucleotide of d(pN) 10)20 is usually significantly higher (K d ¼ 10 )3 )10 )1 m) th an for the remaining 9–19 nucleo- tides of DNA (K d ¼ 0.5–0.8 m) [25–27]. RecA was no exception, accepting free orthophosphate (I 50 ¼ 0.5 m) and various dNMPs (I 50 ¼ 12–20 mm) as minimal lig- ands (Table 1). These experimental I 50 values for free minimal ligands of RecA do not coincide with K d val- ues reflecting the affinity of a single internucleoside phosphate (I 50 ¼ 0.15–0.23 m ) or a single d(pN) unit (I 50 ¼ 0.063–0.1 m) when they are structural elements of longer d(pN) n (Table 1). Similarly to some other enzymes [25–27], the latter I 50 values were determined by extrapolation of lg dependencies to n ¼ 0orn ¼ 1, respectively (Fig. 2; Table 1). Interestingly, the affinity of a single internucleoside phosphate or a single d(pN) unit of d(pN) n for RecA is comparable with the affinity of these DNA structural elements in the case of other enzymes [25–27]. Usually interactions of various enzymes with mono- nucleotides of d(pC) n , d(pT) n , d(pG) n and d(pA) n are additive and elongation of these d(pN) n by one nucleo- tide unit results in an increase in the affinity by a factor f of 1.4–2.0 [25–27]. In principle, similar results were observed for RecA in the case of all d(pN) n (see above). The affinity of some enzymes for d(pN) n does not always depend on the relative hydrophobicity of the bases (f ¼ 1). However, if the enzyme interacts with the bases, the increase in affinity for such ODNs usually follows the same order as the increase in the relative hydrophobicity of the bases: C < T < G <A(h N ¼ 1.1–1.4) [25–27]. The likely reason for this correlation is the formation of very weak hydrophobic and ⁄ or van der Waals contacts of different efficiency and different free energy gain upon transfer of these bases from water to more hydrophobic DNA-binding sites of the enzymes. In a deviation from this empirical rule, RecA binds more hydrophobic d(pA) 20 approximately 240- fold less efficiently than d(pT) 20 and d(pC) 20 (Table 1). A % 25-fold decrease in the affinity of d[p(Et)T] 10 as compared with d(pT) 10 (Table 1) has shown that inter- nucleoside phosphate groups are important for RecA filament interaction with ssDNA. From the comparison of I 50 for d[(pT) 2 (pR) 17 pT] and d(pT) 20 (% 100-fold) the increase in affinity due to a single internucleoside phosphate residue was estimated as the factor e ¼ 1.56. The calculated I 50 for totally abasic oligomer d(pR) 20 (% 2.1 · 10 )5 m) was found practically the same as I 50 for d(pA) 20 (2.4 · 10 )5 m) (Fig. 2, Table 1). This data indicate that the filament probably does not or interact very weakly with poly(dA) adenine bases and contacts mostly with its phosphate groups. The factor e (1.56) for RecA is comparable with e factors for other enzymes: uracil-DNA glycosylase (1.35), AP endonuclease (1.51), DNA polymerases (1.52), Fpg (1.54), RNA helicase (1.61), topoisomerase I (1.67), EcoRI (2.0) and DNA ligase (2.14) [25–27]. DG° % )0.4 kcalÆmol )1 corres- ponding to factor e ¼ 1.56 is significantly lower than would be expected for strong electrostatic contacts (up to )1.0 kcalÆmol )1 ), but comparable with the values for weak ion-dipole and dipole–dipole interactions [38]. Thus, as in the case of the above-mentioned enzymes, the interaction of negatively charged internucleoside groups of ODNs with the RecA filament likely relies on dipolar electrostatic interactions rather than on electro- static interactions of immediately contacting groups. From the ratio of factor f ¼ 2.04 reflecting the increase in the affinity due to one (pN) unit of d(pT) n and d(pC) n and factor e ¼ 1.56 showing the increase in affinity due to a single internucleoside phosphate of these ODNs, the increases in affinity due to RecA interactions with a single T or C base were estimated as the factors f T ¼ f C ¼ 1.31. Thus, RecA in the fila- ment forms weak additive contacts with each inter- nucleoside phosphate mo iety and each base of pyrimidine ODN, with the phosphates contribution into the affin- ity being % 1.2-fold more than that of C or T bases. As the relative affinity of RecA for d(pC) n , d(pT) n , and d(pA) n does not correlate with the relative hydro- phobicity of their bases and RecA does not interact with the bases of d(pA) n , it is reasonable to suggest that RecA could interact with C and T bases by form- ing specific bonds with appropriate amino acids rather than through nonspecific hydrophobic contacts. Wittung et al. reported that entalpy of Rec A bind- ing to ssDNA in the presence of [ 35 S]ATP[cS] depends on the base sequence with a clear preference to T than to A and C bases [39]. Similar results concerning higher affinity of RecA to poly(dT) than to poly(dA) and poly(dC) were demonstrated in the absence of cofactor [3]. Thus, our data are in agreement with the preferential interaction of RecA with d(pT) n in com- parison with d(pA) n , but not with the data concerning d(pC) n . However, data about interactions of RecA with poly(dC) reported in the literature are quite con- tradictory. Amarahung et al. observed that poly(dC) is a very bad effector of ATPase activity of RecA [2]. In contrast, McEttee and Weistock reported poly(dC) to be the most efficient effector of the RecA ATPase activity [40]. Binding of RecA to poly(dC) under a var- ity of conditions has been found to be worse than to other DNA sequences [40]. Thus, the observed differ- ences for poly(dC) interaction with RecA cannot be easily explained. Unlike C and T bases, adenine possesses no exo- cyclic acceptors suitable for hydrogen bonding with RecA amino-acid residues. Deamination of homo- and I. P. Bugreeva et al. RecA filament interaction with DNA FEBS Journal 272 (2005) 2734–2745 ª 2005 FEBS 2741 mixed polynucleotides with formation of dI from dA, dX from dG or and dU from dC containing C ¼ O exocyclic hydrogen bond acceptors also promote for- mation of more stable RecA ⁄ ssDNA filament com- plexes (Table 2). In addition, RecA had high affinity to poly(dN) containing exocyclic acceptor halogen atom instead amino group and to d(eA) n (eA, 1,N 6 - ethenoadenine), in which a hydrogen bond donor moi- ety at C6 is also replaced with an acceptor group (data not shown). Thus, it can be suggested that the RecA filament monomers possess in special positions of sites for binding nucleobases hydrogen bond-donating groups, which can form contacts with C ¼ O exocyclic acceptor groups at C6 of purines and C4 of pyrimi- dines. Figure 5 demonstrates schematically possible hydrogen bonds of RecA with different DNA bases. Thus, NH 2 groups of G bases of ss poly(dG) can form hydrogen bonds with an appropriate group in RecA (for example, OH groups of Ser, Thr, Tyr, or acidic amino acids). Oxygen atoms of G bases (Fig. 5) can interact, for example, with hydrogen atoms of guanidi- nium groups of Arg residues (or NH 2 groups of Lys residues). Similar hydrogen bonds can be formed by C and T bases, but there is no possibility for A bases to form such bonds (Fig. 5) which may be one reason for the low affinity of RecA for d(A) 20 (Fig. 2). As mentioned above, specific interaction of RecA with one C or T base leads to the increase in d(pN) n affinity by a factor of 1.31 (DG° ¼ )0.16 kcalÆmol )1 ). Interestingly, this DG° value is significantly lower than DG° values (from )1 kcalÆmol )1 up to )6 kcalÆmol )1 ) for strong hydrogen bonds which were observed between enzymes and different small ligands [38]. However, a formation of very weak hydrogen bonds is a common situation at recognition of lengthy DNA by various enzymes [25–27]. During formation of a speci- fic complex of dsDNA with EcoRI, 12 specific hydro- gen bonds are formed, providing in total only about two orders of affinity [32]. This means that the energy of every of these 12 bonds is rather low (DG° % )0.23 kcalÆmol )1 ) and comparable with the energy of weak additive nonspecific interactions (see above). DG° % )0.28 kcalÆmol )1 is characterized each of five pseudo-Watson–Crick hydrogen bonds formed by a uracil residue with uracil DNA glycosylase [28]. Sim- ilar weak specific contacts with nucleotides of DNA were observed for all other investigated sequence speci- fic enzymes [25–35]. Altogether, the efficiency of RecA filament inter- action with any individual nucleotide unit (I 50 ¼ 0.5– 0.76 m) except one (I 50 % 63–100 mm) is very low. Nevertheless, the additivity of RecA filament inter- actions should provide extremely high affinity of the filament to long ssDNA. It is reasonable to suggest that the presence of exocyclic acceptor groups capable of hydrogen bonding to the protein can be a critical factor accounting for the efficiency of ssDNA binding by RecA. Depending on the type of the nucleobase (purine or pyrimidine), the nature of RecA interaction with the bases and the conformation of RecA monomers may differ, which could play a key role in the search for homologous DNA. One cannot exclude that interaction of complex of RecA filament and ssDNA with dsDNA can lead to reorganization of firstly formed hydrogen bonds between protein and bases (Fig. 5) and assist formation of new hydrogen bonds between C and G or T and A bases of new DNA duplex. Experimental procedures Materials ATP, ATPcS, poly(N), and poly(dN) were purchased from Sigma-Aldrich (St. Louis, MO, USA), and [ 32 P]ATP[cP] Fig. 5. Proposed RecA amino-acid residue interactions with G, A, C and T bases of poly(dN). Impossibility of hydrogen bond formation is marked (filled star). RecA filament interaction with DNA I. P. Bugreeva et al. 2742 FEBS Journal 272 (2005) 2734–2745 ª 2005 FEBS (2000 CiÆmmol )1 ), from Amersham Biosciences (Piscataway, NJ, USA). Deaminated oligo- and polynucleotides were synthesized as described in [41,42]. To substitute amino groups of different nucleobases in polynucleotides with halogen atoms, the deamination reactions were performed in the presence of 1 m of respective sodium halides. ODN were synthesized, purified and characterized as described [43]. All ODN were proven homogeneous by ion- exchange and reverse-phase chromatography. Concentra- tions of the ODNs were determined from their absorption at 260 nm using molar extinction coefficients calculated according to [44] ODN were 5¢-labelled using bacteriophage T4 polynucleotide kinase and [ 32 P]ATP[cP]. Electrophoreti- cally homogeneous E. coli RecA protein was prepared as described [45]. RecA filamentation The reaction of RecA filamentation was carried out with 5¢-[ 32 P]d(pT) 20 or 5¢-[ 32 P]d(pT) 40 at 30 °C for 5 min. The standard reaction mixture (10 lL) included 50 mm Tris ⁄ HCl (pH 7.5), 10 mm MgCl 2 ,2mm DTT, 1 mm [ 35 S]ATP[cS], 0.1 lm 5¢-[ 32 P]d(pT) 20 , and 1 lm RecA. dNMP, d(pN) 2 or other individual homogeneous d(pN) n (n ¼ 3–20), and their modified analogs used as filamenta- tion inhibitors were added in various concentrations depending on their affinity. Apparent I 50 values for d(pT) 20)40 were obtained using 5¢-[ 32 P]d(pT) 40 (0.04 lm)as a filamentation substrate. The reactions were initiated by adding RecA into the mixture containing 5¢-[ 32 P]d(pT) 20,40 and one of the inhibitors. Free 5¢-[ 32 P]d(pT) 20,40 was separ- ated from 5¢-[ 32 P]d(pT) 20,40 incorporated in the filament by electrophoresis in 10–20% nondenaturing polyacrylamide gel [12] in TBE buffer. The results were visualized by auto- radiography, the bands were cut out from the gel and their radioactivity determined by Cherenkov counting. Affinity of various ligands for RecA was estimated from their I 50 values (inhibitor concentration producing a 50% decrease in filamentation). DNA-dependent ATPase activity of RecA The efficiency of ATP hydrolysis by RecA in the presence of ssDNA was followed by the decrease in [ 32 P]ATP[cP] and accumulation of 32 P-labelled orthophosphate ([ 32 P]P i ) using TLC on PEI-cellulose plates in 0.3 m KH 2 PO 4 (pH 7.5). The standard reaction mixture (20 lL) included 20 mm Tris ⁄ HCl (pH 8.0), 10 mm MgCl 2 ,30mm NaCl, 1mm DTT, 1 mm ATP, 4 lm RecA, and poly(dN) or poly(N) in the concentration 0.1 mm nucleotides, or 70 lm d(pN) 20 or other individual d(pN) n (n ¼ 2–40). The mix- tures were incubated at 30 °C, 2 lL aliquots were with- drawn and spotted on a TLC plate. Vertical development of the plate was performed in the ascending mode using 0.3 m potassium phosphate (pH 7.5). The plates were auto- radiographed, the spots corresponding to [ 32 P]ATP[cP] and [ 32 P]P i were cut out and their radioactivity determined by Cherenkov counting. Acknowledgements The research was made possible in part by grants from the Program of Basic research of the Presidium of RAS ‘Presidium of the Russian Academy of Sciences (Molecular and Cell Biology Program 10.5)’, from the Russian Foundation for Basic Research, and from the Siberian Division of the Russian Academy of Sciences. References 1 Lusetti SL & Cox MM (2002) The bacterial RecA pro- tein and the recombinational DNA repair of stalled replication forks. Annu Rev Biochem 71, 71–100. 2 Amaratunga M & Benight AS (1988) DNA sequence dependence of ATP hydrolysis by RecA protein. Bio- chem Biophys Res Commun 157, 127–133. 3 Cazenave C, Chabbert M, Toulme JJ & Helene C (1984) Absorption and fluorescence studies of the bind- ing of the recA gene product from E. coli to single- stranded and double-stranded DNA. Ionic strength dependence. Biochim Biophys Acta 781, 7–13. 4 McEntee K, Weinstock GM & Lehman IR (1981) Bind- ing of the recA protein of Escherichia coli to single- and double-stranded DNA. J Biol Chem 256, 8835–8844. 5 Tracy RB & Kowalczykowski SC (1996) In vitro selec- tion of preferred DNA pairing sequences by the Escheri- chia coli RecA protein. Genes Dev 10, 1890–1903. 6 Yu, X & Egelman EH (1992) Structural data suggest that the active and inactive forms of the RecA filament are not simply interconvertible. J Mol Biol 227, 334–346. 7 Register JC, 3rd & Griffith J (1985) The direction of RecA protein assembly onto single strand DNA is the same as the direction of strand assimilation during strand exchange. J Biol Chem 260, 12308–12312. 8 Bork JM, Cox MM & Inman RB (2001) RecA protein filaments disassemble in the 5¢-to 3¢ direction on single- stranded DNA. J Biol Chem 276, 45740–45743. 9 Stasiak A & Di Capua E (1982) The helicity of DNA in complexes with recA protein. Nature 299, 185–186. 10 Pugh BF, Schutte BC & Cox MM (1989) Extent of duplex DNA underwinding induced by RecA protein binding in the presence of ATP. J Mol Biol 205, 487– 492. 11 Heuser J & Griffith J (1989) Visualization of RecA pro- tein and its complexes with DNA by quick-freeze ⁄ deep- etch electron microscopy. J Mol Biol 210, 473–484. 12 Mazin AV & Kowalczykowski SC (1998) The function of the secondary DNA-binding site of RecA protein during DNA strand exchange. EMBO J 17, 1161–1168. I. P. Bugreeva et al. RecA filament interaction with DNA FEBS Journal 272 (2005) 2734–2745 ª 2005 FEBS 2743 [...]... (1996) The DNA binding site (s) of the Escherichia coli RecA protein J Biol Chem 271, 11996–12002 25 Bugreev DV & Nevinsky GA (1999) Possibilities of the method of step -by- step complication of ligand structure in studies of protein–nucleic acid interactions: mechanisms of functioning of some replication, repair, topoisomerization, and restriction enzymes Biochemistry (Mosc) 64, 237–249 26 Nevinsky GA.. .RecA filament interaction with DNA 13 Zhurkin VB, Raghunathan G, Ulyanov NB, CameriniOtero RD & Jernigan RL (1994) A parallel DNA triplex as a model for the intermediate in homologous recombination J Mol Biol 239, 181–200 14 Kim MG, Zhurkin VB, Jernigan RL & Camerini-Otero RD (1995) Probing the structure of a putative intermediate in homologous recombination: the third strand in the parallel DNA. .. GA (2004) The role of specific and nonspecific interactions in enzymatic recognition and conversion of long DNA Mol Biol (Mosc) 38, 636–662 27 Nevinsky GA (2003) Structural, thermodynamic, and kinetic basis of DNA- and RNA-dependent enzymes 2744 I P Bugreeva et al 28 29 30 31 32 33 34 35 36 37 38 functioning Important role of weak nonspecific additive interactions between enzymes and long nucleic acids... RecA protein with DNA and nucleotide cofactors Fluorescence study of engineered proteins Eur J Biochem 228, 779–785 22 Morimatsu K & Horii T (1995) Analysis of the DNA binding site of Escherichia coli RecA protein Adv Biophys 31, 23–48 23 Morimatsu K, Funakoshi T, Horii T & Takahashi M (2001) Interaction of tyrosine 65 of RecA protein with the first and second DNA strands J Mol Biol 306, 189–199 24... interaction with nonspecific oligonucleotides Bioorg Khim (Mosc) 29, 143–154 Bugreev DV, Sinitsyna OI, Buneva VN & Nevinskii GA (2003) The mechanism of supercoiled DNA recognition by eukaryotic type I topoisomerases: II A comparison of the enzyme interaction with specific and nonspecific oligonucleotides Bioorg Khim (Mosc) 29, 249–262 Bianco PR & Weinstock GM (1996) Interaction of the RecA protein of Escherichia... 215–219 17 Roca AI & Cox MM (1997) RecA protein: structure, function, and role in recombinational DNA repair Prog Nucleic Acid Res Mol Biol 56, 129–223 18 Folta-Stogniew E, O’Malley S, Gupta R, Anderson KS & Radding CM (2004) Exchange of DNA Base Pairs that Coincides with Recognition of Homology Promoted by E coli RecA Protein Mol Cell 15, 965–975 19 Voloshin ON & Camerini-Otero RD (2004) Synaptic complex... recognition and transformation In Protein Structures Kaleidoscope of Structural Properties and Functions, (Uversky VN, ed), pp 133–222 Research Signpost, Fort P.O., India Vinogradova NL, Bulychev NV, Maksakova GA, Johnson F & Nevinskii GA (1998) Uracil DNA glycosylase: interpretation of X-ray data in the light of kinetic and thermodynamic studies Mol Biol (Mosc) 32, 489–499 Ishchenko AA, Vasilenko NL, Sinitsina... parallel DNA triplex is in contact with the major groove of the duplex J Mol Biol 247, 874–889 15 Zhou X & Adzuma K (1997) DNA strand exchange mediated by the Escherichia coli RecA protein initiates in the minor groove of double-stranded DNA Biochemistry 36, 4650–4661 16 Howard-Flanders P, West SC & Stasiak A (1984) Role of RecA protein spiral filaments in genetic recombination Nature 309, 215–219 17... (1960) Base specificity in the induction of mutations by nitrous acid in phage T-2 [in German] Z Naturforsch 15B, 304–311 FEBS Journal 272 (2005) 2734–2745 ª 2005 FEBS RecA filament interaction with DNA 43 Ishchenko AA, Bulychev NV, Zharkov DO, Maksakova GA, Johnson F & Nevinskii GA (1997) Isolation of 8-oxoguanine -DNA- glycosylase from Escherichia coli and substrate specificity of the enzyme Mol Biol (Mosc)... Starostin KV, Ishchenko AA, Yamkovoy VI, Zharkov DO, Douglas KT & Nevinsky GA (2004) Thermodynamic, kinetic and structural basis for recognition and repair of abasic sites in DNA by apurinic ⁄ apyrimidinic endonuclease from human placenta Nucleic Acids Res 32, 5134–5146 Kolocheva TI, Maksakova GA, Bugreev DV & Nevinsky GA (2001) Interaction of endonuclease EcoRI with short specific and nonspecific oligonucleotides . Formation of nucleoprotein RecA filament on single-stranded DNA Analysis by stepwise increase in ligand complexity Irina P. Bugreeva, Dmitry V. Bugreev and Georgy A. Nevinsky Institute of Chemical. the efficiency of ssDNA binding by RecA. Depending on the type of the nucleobase (purine or pyrimidine), the nature of RecA interaction with the bases and the conformation of RecA monomers may differ,. than dsDNA. After strand exchange, the second RecA DNA binding site binds the displaced strand following new DNA duplex formation [12]. Binding of dsDNA to a RecA filament is followed by search of