A ring-opening mechanism for DNA binding in the central channel of the T7 helicase-primase protein
2000; Springer Nature; Volume: 19; Issue: 13 Linguagem: Inglês
10.1093/emboj/19.13.3418
ISSN1460-2075
Autores Tópico(s)Bacterial Genetics and Biotechnology
ResumoArticle3 July 2000free access A ring-opening mechanism for DNA binding in the central channel of the T7 helicase–primase protein Peter Ahnert Peter Ahnert Department of Biochemistry, Robert Wood Johnson Medical School, Piscataway, NJ, 08854-5635 USA Search for more papers by this author Kristen Moore Picha Kristen Moore Picha Department of Biochemistry, Robert Wood Johnson Medical School, Piscataway, NJ, 08854-5635 USA Search for more papers by this author Smita S. Patel Corresponding Author Smita S. Patel Department of Biochemistry, Robert Wood Johnson Medical School, Piscataway, NJ, 08854-5635 USA Search for more papers by this author Peter Ahnert Peter Ahnert Department of Biochemistry, Robert Wood Johnson Medical School, Piscataway, NJ, 08854-5635 USA Search for more papers by this author Kristen Moore Picha Kristen Moore Picha Department of Biochemistry, Robert Wood Johnson Medical School, Piscataway, NJ, 08854-5635 USA Search for more papers by this author Smita S. Patel Corresponding Author Smita S. Patel Department of Biochemistry, Robert Wood Johnson Medical School, Piscataway, NJ, 08854-5635 USA Search for more papers by this author Author Information Peter Ahnert1, Kristen Moore Picha1 and Smita S. Patel 1 1Department of Biochemistry, Robert Wood Johnson Medical School, Piscataway, NJ, 08854-5635 USA ‡P.Ahnert and K.M.Picha contributed equally to this work *Corresponding author. E-mail: [email protected] The EMBO Journal (2000)19:3418-3427https://doi.org/10.1093/emboj/19.13.3418 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info We have investigated the mechanism of binding single-stranded DNA (ssDNA) into the central channel of the ring-shaped T7 gp4A′ helicase–primase hexamer. Presteady-state kinetic studies show a facilitated five-step mechanism and provide understanding of how a ring-shaped helicase can be loaded on the DNA during the initiation of replication. The effect of a primase recognition sequence on the observed kinetics suggests that binding to the helicase DNA-binding site is facilitated by transient binding to the primase DNA-binding site, which is proposed to be a loading site. The proposed model involves the fast initial binding of the DNA to the primase site on the outside of the helicase ring, a fast conformational change, a ring-opening step, migration of the DNA into the central channel of the helicase ring, and ring closure. Although an intermediate protein–DNA complex is kinetically stable, only the last species in the five-step mechanism is poised to function as a helicase at the unwinding junction. Introduction Helicases are motor proteins that unwind double-stranded DNA/RNA (dsDNA/RNA) using the free energy from NTP hydrolysis. Helicases from various organisms, including bacteriophages, bacteria, archaea, viruses and eukaryotes, have been shown to form ring-shaped hexamers (Matson and Kaiser-Rogers, 1990; Lohman and Bjornson, 1996; Patel and Picha, 2000). Most of these assemble into rings in the absence of DNA but many require Mg2+ and/or NTP for hexamer formation. Most of the hexameric helicases involved in DNA replication have been proposed to bind only one strand of the duplex DNA within the central channel while excluding the complementary strand (Bujalowski and Jezewska, 1995; Egelman et al., 1995; Yu et al., 1996; Smelkova and Borowiec, 1998; Fouts et al., 1999; Morris and Raney, 1999). The translocation of the helicase along the single-stranded DNA (ssDNA) passing through its central channel may play a major role in DNA unwinding. This mode of DNA binding topologically links the ring-shaped helicase and the DNA and contributes to the processivity of translocation and unwinding (Patel and Picha, 2000). The location of the ssDNA-binding site in the central channel of the ring however poses a problem. Free DNA cannot access the ssDNA-binding site within the central channel of the ring, suggesting that there must be a specific mechanism to load the helicase efficiently onto the DNA during initiation. Using presteady state kinetics, we have elucidated such a mechanism for T7 gp4A′ helicase–primase binding to ssDNA. The kinetic mechanism determined for gp4A′ has provided general insights into how hexameric helicases can be loaded on the DNA during initiation. T7 gp4A′ contains both helicase and primase activities, and it does not require any accessory proteins to bind or unwind DNA. Although the two activities reside on separate domains of the protein, they are dependent on each other and the isolated domains are not fully active (Bird et al., 1997; Guo et al., 1999). The C-terminal domain is mostly responsible for the helicase function and the N-terminal domain for the primase (Patel et al., 1992; Frick et al., 1998). The gp4A′ interacts preferentially with ssDNA with a Kd 18 s−1, since that was the observed rate at the highest concentration of ssDNA used. A series of slower conformational changes followed the fast initial steps. ED2 was converted to ED3 at 5.0 s−1 in the presence of ssM13 DNA and at 3.4 s−1 in the presence of 30mer. The ED3 to ED4 conversion rate was 0.5 s−1 for both ssDNAs. The conversion of ED4 to ED5 occurred at 0.02 s−1 with ssM13 DNA and could not be measured in fluorescence experiments with the 30mer. However, the dTTPase stimulation experiments with both DNAs showed a rate of 0.05–0.08 s−1 for the conversion of ED4 to ED5. Thus, considering the different lengths of the substrate ssDNAs, the overall mechanisms of ssDNA binding as determined from the stopped-flow and presteady state ssDNA-stimulated dTTPase experiments are very similar for both 30mer and ssM13 DNAs. The kinetic data presented above established the requirement of a five-step ssDNA-binding mechanism for the formation of a competent helicase. The nature of the intermediate species in this mechanism is unknown, but the final species is well characterized. Studies in the presence of Mg-dTMP-PCP (deoxythymidine [β, γ, methylene] triphosphate) have shown that ssDNA is stably bound within the central channel at equilibrium (Egelman et al., 1995; Yu et al., 1996), and such a species has a half-life of dissociation >60 min (data not shown; Matson and Richardson, 1985). We therefore used kinetic stability as a criterion to determine when the ssDNA migrates into the central channel during the ssDNA-binding process. The 30mer binding mechanism (Table II) shows that the first irreversible step is the conversion of ED3 to ED4, suggesting that ED4 is the first stable helicase–ssDNA complex. This was tested experimentally by the following chase filter-binding experiments. Table 2. DNA-binding mechanism with Mg-dTMP-PCP The measurement of 30mer ssDNA binding by the chase filter-binding assay The rationale for this experiment was to generate the various helicase–ssDNA intermediates as a function of time with a radiolabeled 30mer ssDNA. Then excess nonradiolabeled ssDNA was added as a chase, and the amount of radiolabeled ssDNA bound to the protein determined. These experiments were carried out with Mg-dTMP-PCP rather than Mg-dTTP, because a stable helicase–ssDNA complex is formed only with the nonhydrolyzable analog, and this complex can be quantified by the nitrocellulose-DEAE membrane binding assay (Patel and Hingorani, 1995; Hingorani and Patel, 1996). The stopped-flow kinetic studies have shown that the helicase–ssDNA intermediates are formed in the millisecond time-scale (Picha et al., 2000). Therefore, the gp4A′ hexamer was mixed with radiolabeled 30mer ssDNA in a rapid quenched-flow instrument from 5 ms to several seconds. An excess of chase was added, and after 25 s the radioactive ssDNA that remained bound to the hexamer was quantified by the membrane-binding assay. The resulting 30mer binding kinetics, determined by this chase filter-binding assay, were biphasic (Figure 4). The kinetics were analyzed as described in the Materials and methods to determine when a kinetically stable helicase–ssDNA complex was formed (a species that does not dissociate in the presence of the chase). The models in which ED2 or ED3 were assumed to be kinetically stable did not fit the data (short-dashed and dashed lines, Figure 4B and C). The observed kinetics fit well when we assumed that ED4 was a kinetically stable species. In fact, the chase data could be described with essentially the same intrinsic rate constants as determined from the stopped-flow experiments (Table II). Only the rate constant for the conversion of ED3 to ED4 varied somewhat. The chase data at 400 nM and 1.1 μM 30mer ssDNA were described best with rates of 0.09 and 0.23 s−1, respectively, in comparison with 0.21 s−1 as determined in the stopped-flow experiments (Picha et al., 2000). These experiments, in addition to verifying the kinetic mechanism of 30mer ssDNA binding obtained by fluorescence measurements, indicated that ED4 is the first species in the ssDNA-binding pathway, where the ssDNA is stably bound to the gp4A′ hexamer. Figure 4.The kinetics of 30mer ssDNA binding measured by chase-filter DNA-binding assay. (A) Gp4A′ (142 nM hexamer, final concentration), Mg-dTMP-PCP (2 mM), and MgCl2 (7 mM free) was mixed with 5′-[32P]30mer ssDNA (400 nM or 1.1 μM) for varying times in a rapid quench–flow instrument at 18°C. The amount of radiolabeled 30mer bound to gp4A′ hexamer, after 25 s of chase addition (4 or 11 μM non-radiolabeled 30mer ssDNA, in buffer H with 2 mM Mg-dTMP-PCP, 7 mM MgCl2) was determined. (B) and (C) show the chase kinetics with 400 nM and 1.1 μM ssDNA, respectively. The kinetics of ssDNA binding fit to the sum of two exponentials (Equation 1). The amplitudes and rates at 400 nM 30mer were 51.9 nM and 11.7 s−1 for the first phase and 90.4 nM and 0.094 s−1 for the second phase, respectively. The amplitudes and rate at 1.1 μM 30mer were 65.4 nM and >30 s−1 for the first phase and 61.3 nM and 0.4 s−1 for the second phase, respectively. The solid black lines are predicted chase kinetics curves from the 30mer ssDNA-binding mechanism, shown in Table II (formation of ED4 is irreversible). The short- and long-dashed lines represent the predicted formation ED2 or ED3, respectively, assuming that they are stable protein–DNA complexes. Download figure Download PowerPoint The above experiments raise the next question. Where is the ssDNA bound in the ED1, ED2 and ED3 complexes? Both ED1 and ED2 are formed at very fast rates compared with the rest of the species. ED1 is formed at a close to diffusion-limited rate and its conversion to ED2 is fast. We reasoned that the fast ssDNA-binding event can not occur at the ssDNA-binding site located within the central channel because that site would be inaccessible to free ssDNA. We propose that the fast encounter of the ssDNA occurs at a site that is readily accessible and located on the outside of the ring. A known ssDNA-binding site that was proposed to be accessible and located on the outside, is the primase ssDNA-binding site present on the N-terminal domain of gp4A′ (Kusakabe et al., 1998). The interactions of the ssDNA with the primase ssDNA-binding site have been measured to be weak with a Kd in the range of 10 μM (Frick and Richardson, 1999). In fact, the Kd for ED1 is weak as well (2.5 μM). To investigate the hypothesis that the initial interactions of the ssDNA occur at the primase ssDNA-binding site of gp4A′, we have designed the following stopped-flow kinetic experiments. The effect of the primase recognition sequence on the kinetics of ssDNA binding The primase site of T7 gp4A′ recognizes sequences such as 3′-CTGGT on ssDNA, which it uses as a template to synthesize RNA primers when ATP or ApC and CTP are provided (Scherzinger et al., 1977; Tabor and Richardson, 1981). It has been shown that the primase domain interacts more tightly with the ssDNA that contains a primase recognition sequence (Frick and Richardson, 1999). Therefore, if the first interaction between the helicase and the ssDNA occurred at the primase ssDNA-binding site, then the presence of a primase recognition sequence (and the nucleotide substrates for primer synthesis) may affect the overall kinetics of ssDNA binding. On the other hand, if the initial encounter were to occur with the helicase ssDNA-binding site directly, then having a primase sequence in the ssDNA should have no effect on the kinetics of ssDNA binding. A primase ssDNA recognition sequence (3′-CTGGT) was introduced by changing only one base in the 30mer ssDNA at position 19 from the 5′ end (A to G change). This minimal change allowed a direct comparison to be made between the kinetic mechanisms of binding with the 30mer ssDNA (without the primase sequence) and the pri30mer ssDNA (with the primase sequence). The 30mer and the pri30mer ssDNA-binding kinetics were determined in the presence of dTMP-PCP by monitoring the transient changes in gp4A′ fluorescence in a stopped-flow instrument. Three phases were observed, and their rates were dependent on the ssDNA concentrations (data not shown). The rate of the first phase increased linearly with increasing ssDNA, the rate of the second phase increased in a hyperbolic manner, and the rate of the third phase remained constant with increasing ssDNA. These dependencies indicated that the ssDNA-binding mechanism was essentially unaltered even though the ssDNA contained a primase recognition sequence. However, the intrinsic rate constants were affected (Table II), and these were determined by the method of numerical integration and global fitting, as described previously (Picha et al., 2000). To visualize how the altered intrinsic rate constants affect the overall kinetics of ssDNA binding, we simulated the time-dependent formation and decay of the helicase–ssDNA species for the two ssDNAs, using the respective intrinsic rate constants (Table II). This analysis showed that the ED2 was converted more slowly to ED3 in the pri30mer binding pathway (Figure 5A). This effect was more pronounced when the nucleotide substrates for RNA synthesis, ApC and CTP, were present. Similarly, the formation of ED4 was slower when the ssDNA contained a primase sequence (Figure 5B). The same experiments were carried out with a second set of ssDNAs. These ssDNAs were of a different sequence from the ones used in the above experiments. Again, in this second set, a primase recognition sequence was introduced into one of the two ssDNAs by a single nucleotide change. The same trends in the accumulation of ED2 and ED4 were observed for this second set of 30mer ssDNAs (data not shown). These results support the notion that the ssDNA first binds to the primase ssDNA-binding site to form ED1 and then ED2. It appears that when the ssDNA contains a primase recognition sequence and when the substrates for RNA synthesis are present, then the ssDNA is retained longer at the primase ssDNA-binding site and migrates into the central channel at a lower rate. Figure 5.Kinetics of formation and decay of the various helicase–ssDNA species in the 30mer ssDNA-binding pathways. The formation and decay of ED2 (A) and ED4 (B) in the 30mer ssDNA-binding pathways was simulated using the mechanism derived from the stopped-flow experiments (Table II). The concentrations
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