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Hydrolysis of Nucleoside Triphosphates Catalyzed by the recA Protein of Escherichia coli

The DNA-dependent ATPase activity of the recA protein of Escherichia coli shows a complex dependence on ATP concentration. With a single-stranded (SS) DNA cofactor, the Hill coefficient for ATP is 3.3 at pH 8.1 and 1.4 at pH 6.2. With a double-stranded (DS) DNA cofactor, the Hill coefficient is 3.3 at pH 6.2 (no reaction is detectable at pH 8.1). In the presence of SS DNA, the K,,, for ATP is 20 PM, independent of pH, while with DS DNA at pH 6.2, KmATP is 100 e. These and other observations indicate that the interaction of recA protein with ATP is influenced by both pH and DNA cofactor. ADP, UTP, dlTP, and GTP are competitive inhibitors of the ATPase activity of recA protein, indicating that there is a single binding site for nucleoside triphosphates. Nucleoside triphosphates, but not ADP, reduce the Hill coefficient for ATP hydrolysis and thus can contribute to the cooperative effect of ATP.

The recA protein of Escherichia coli can promote the hybridization of complementary DNA sequences (either annealing of two single strands or assimilation of single strands into DS' DNA) (1-3) and the DNA-dependent hydrolysis of nucleoside triphosphates (4). Hybridization is specifically dependent on ATP (or dATP). However, both ATP (dATP) and UTP (dUTP) are hydrolyzed by the recA protein at a common or overlapping site on the enzyme (4). Both ATP(@) and UTP(yS) stabilize recA protein-DNA complexes (5) and, thus, there is less nucleoside triphosphate specificity for the binding of recA protein to DNA than for its annealing activities.
* This work was supported by Grant GM06196 from the National Institutes of Health and Grant PCM74-00865 from the National Science Foundation. The,costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "aduertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
.  However, there are different effects of ATP and UTP on the structure of the recA protein (5).
Hydrolysis of ATP by recA protein is a complex reaction with different characteristics depending upon the DNA cofactor and pH (4, 6). Here we present a comparable characterization of UTP hydrolysis. The UTPase activity of recA protein is also affected by the DNA cofactor and pH and shows a complex dependence on substrate concentration. SS DNAdependent UTP hydrolysis differs from ATP hydrolysis in that KmUTP, V,,,, and the dependence on recA protein concentration are all sensitive to pH. This fiding suggests that one or more steps in the ATP hydrolytic cycle are ATP specific and possibly relate to the ability of ATP, but not UTP, to promote the hybridization reactions.

EXPERIMENTAL PROCEDURES
All reagents and assays were as described in the previous paper (4). Initial velocities of UTP hydrolysis were determined by a time course unless otherwise noted.

Hydrolysis of UTP-Hydrolysis of UTP was measured in
the presence of SS DNA at pH 6.2 and pH > 7 and in the presence of DS DNA at pH 6.2. SS DNA-dependent UTP hydrolysis showed linear kinetics, but the extent was dependent on the initial UTP concentration (Fig. l a ) . This effect was more pronounced at pH 8 than at pH 6.2 (data not shown). DS DNA-dependent UTP hydrolysis showed a brief lag before a linear rate was achieved (Fig. l b ) and also showed a concentration-dependent limitation in extent (Fig. IC). These results contrast with ATP hydrolysis where the extent of hydrolysis in the presence of SS DNA is not limited by the initial ATP concentration and where the extent of DS DNA-dependent ATP hydrolysis is greater (4).
DNA-dependent UTPase activity co-purified with the ATPase activity of recA protein through phosphocellulose, DEAE-cellulose, and hydroxylapatite column chromatography (4). In view of this and other similarities with the ATPase activity, it is clear that the UTPase activity is intrinsic to the recA protein.
Effect of pH on UTP Hydrolysis-SS DNA-dependent UTPase activity showed a pH optimum near 6.5 but there was considerable activity at alkaline pH (Fig. 2a). This finding contrasts with ATP hydrolysis, which is independent of pH in the presence of SS DNA (4). DS DNA-dependent UTPase activity showed a sharper optimum than the corresponding ATPase activity at pH 6.2, with little activity above pH 7 (Fig.  2b). This behavior is similar to DS DNA-dependent ATPase activity (4) although the UTPase optimum appeared somewhat narrower. The optimum for SS DNA-dependent UTPase activity (pH 6.5) appeared to be slightly more alkaline than  incubation was for 20 min. Assays of DS DNA-dependent UTPase were the same except that 37 PM P22 DS DNA was used, recA protein was 8.7 PM, and incubation was for 30 min. In the DNA-independent UTPase assays (20 pl), BSA was omitted, UTP was 50 PM, and Fraction IV (4) recA protein was added to 6 p~; incubation was for 60 min. pH was measured in 20 mM buffer in the presence of 10 m M MgC12 at 25 "C. A-A, Sodium acetate; W, sodium maleate; Tris-HC1; M , glycine-NaOH.
U T P hydrolysis also occurred in the absence of DNA, although a t a greatly reduced rate. This activity showed a pH profile that resembled the DNA-independent ATPase activity (4): an optimum near pH 6, a minimum near pH 7, and reduced activity above pH 7 (Fig. 2c)  Dependence of UTP hydrolysis on recA protein concentration. Reactions in Tris-HCl (pH 8.0) or sodium maleate (pH 6.2) contained 990 p~ UTP and either 84 pm +X174 SS DNA or 103 @ pZ6b DS DNA. recA protein was Fraction 11. was essentially constant from 0.4 to 2 pM recA protein (Fig. 3), indicating that the velocity was proportional to recA protein concentration in a manner similar to the ATPase activity (4).
As with ATPase activity, lower protein concentrations showed disproportionately reduced activity, possibly due to inactivation of recA protein or dissociation of recA protein oligomers (5, 7). At higher protein concentrations, activity also declined, possibly due to protein aggregation (5,7) which is not observed at pH 6.2 (5).
At pH 6.2, the turnover number of both SS and DS DNAdependent UTP hydrolysis depended on the protein concentration below 2 p~ recA protein, indicating an exponential dependence of velocity on enzyme concentration. The reaction with DS DNA was somewhat more dependent on protein concentration than the reaction with SS DNA. This finding contrasts with ATP hydrolysis, which is independent of protein concentration in the presence of SS DNA and is saturated at 0.8 p~ recA protein in the DS DNA-dependent ATPase reaction (4). These effects may be related to the dissociation of recA protein oligomers at pH 6.2 (5).

Dependence of UTP Hydrolysis on UTP Concentration-
UTP hydrolysis showed a complex dependence on UTP concentration, affected by both the pH and the DNA cofactor ( Fig. 4 and Table I). SS DNA-dependent UTP hydrolysis at pH 8 had a Hill coefficient of 3.5 a t U T P concentrations below 100 /JM, while a t p H 6.2, the Hill coefficient was reduced to 1.9. A simiiar effect was observed for ATP hydrolysis (6).
However, unlike ATP hydrolysis, KmUTP also decreased, from 147 p~ at pH 8.0 to 33 p~ at pH 6.2. Furthermore, unlike ATP Reactions were performed as in Fig. 1. Vmax was determined from an Eadie-Hofstee plot.

Steady state kinetic parameters for UTP hydrolysis
Reactions were performed as described in Fig. 4. KmUTP and V , , were determined from an Eadie-Hofstee plot. The H i l l coefficients are from Fig. 4 Inhibition of UTP Hydrolysis by ATP-All of the hydrolytic reactions catalyzed by recA protein show the same nucleotide specificity (4), and hydrolysis of ATP is competitively inhibited by UTP (6). UTP hydrolysis was also competitively inhibited by ATP (data not shown). The KiATP was approximately 23 PM, close to the KmATP for ATP hydrolysis (6 Thus, at alkaline pH, ATP, but not UTP, efficiently induces the conformational states required for hydrolysis while at acid pH, the NTP requirement is obviated. Hence Vmax and K , are independent of pH in the ATPase reactions, but are affected by the low pH bypass in the UTPase reaction. The nonlinear dependence of UTP hydrolysis on enzyme concentration at pH 6.2 demonstrates that oligomerization is likely to be important in these reactions. This follows from the observations that at pH 6.2, the recA protein exists in a lower oligomeric form than at pH 7.5, and this low pH form oligomerizes in the presence of ATP but not UTP (5). Since UTP is also inefficient in promoting the single strand annealing and assimilation reactions, this oligomerization may be an important part of these reactions, for instance in the pairing of DNA chains.
These and other observations demonstrate that UTP interacts efficiently with the recA protein of E. coli. Hydrolysis of UTP is comparable to ATP, and ATP and UTP have similar effects on the binding of recA protein to DNA (5). However, the weak stimulation of SS DNA annealing ( l ) , assimilation (2, 3), and protease (8) activities of recA protein by UTP indicates that, in vivo, UTP must function in a manner different from ATP. Whether the role of UTP is as a regulator of ATP-dependent reactions or as a cofactor for additional activities of the recA protein remains to be clarified.