The DNA restriction endonuclease of Escherichia coli B. I. Studies of the DNA translocation and the ATPase activities

Electron microscopic examination of DNA intermediates formed by the restriction endonuclease of Escherichia coli B revealed supercoiled loops that are presumably formed during an ATP-dependent DNA translocation process in which the enzyme remains bound to the recognition site while tracking along the DNA helix to a cleavage site. The rate of DNA translocation during this process is at least 5000 base pairs/min at 37 degrees C. Even after all cleavages have been completed, complexes are seen that contain terminal loops or loop plus tail structures. During this later phase of the reaction, ATP is hydrolyzed at a rate which is dependent upon the size of the largest possible loop (or loop plus tail); this ATP hydrolysis can be terminated by one double-strand cleavage within the loop region between the recognition site and the terminus. To explain these results, it is hypothesized that after cleavage the enzyme cycles between a tracking (and possibly back-tracking) mode which is fueled by ATP hydrolysis and a relatively long static period in which ATP hydrolysis does not occur. While tracking, the enzyme would be bound both to the recognition site and to a distal site but, while static, the enzyme would be bound only at the recognition site of nonlooped molecules. This post-nuclease phase of the reaction is hypothesized to reflect a reaction whereby the enzyme initially scans DNA molecules before making a strand cleavage.     

Proton translocation by ATPase and bacteriorhodopsin.

Stable membrane proteins and lipids are convenient to study biomembranes. Two stable proton translocating proteins were purified and reconstituted into vesicles capable of proton translocation. One was a thermostable ATPase (TF0-F1) of thermophilic bacterium PS3 and the other was rhodopsin of Halobacterium halobium. TF0-F1 was composed of a proton pump moiety (TF1) and a proton channel moiety (TF0). TF1 was the first membrane ATPase which was crystallized and reconstituted from its five polypeptides. Like TF0 and TF1, the rhodopsin in purple membrane was highly stable against dissociating agents, acids and alkali. Phospholipids of these biomembranes were also stable and contained no unsaturated fatty acyl groups. The molecular species of the phospholipids of PS3 were determined by mass chromatography. Measurements were made of the difference in electrochemical potential of protons (deltamicronH+) across the membrane of the reconstituted vesicles. The deltamicronH+ attained was 312 mV in TF0-F1 vesciles and was 230 mV in the rhodopsin vesicles. To conclude that electron transport components are not necessary for ATP synthesis in energy yielding biomembranes, two experiments were performed: The ATP synthesis was observed i) on acid-base treatment of TF0-F1 vesicles, and ii) on illumination of the rhodopsin-TF0-F1 vesicles.     

Inhibition of DNA helicase II unwinding and ATPase activities by DNA-interacting ligands. Kinetics and specificity.

Although DNA helicases play important roles in the processing of DNA, little is known about the effects of DNA-interacting ligands on these helicases. Therefore, the effects of a wide variety of DNA-binding ligands on the unwinding and ATPase reactions catalyzed by Escherichia coli DNA helicase II were examined. DNA minor groove binders and simple DNA intercalators did not inhibit helicase II. However, DNA intercalators, such as mitoxantrone and nogalamycin, which position functionalities in the major groove upon binding duplex DNA, were potent inhibitors of helicase II. To determine the mechanism by which mitoxantrone inhibited helicase II, the unwinding and DNA-dependent ATPase activities of helicase II were measured using a spectrum of double- and single-stranded DNA substrates. Using either a 71-base pair (bp) M13mp7 partially duplexed DNA substrate or a 245-bp bluntended, fully duplexed DNA substrate, the apparent Ki value for inhibition by mitoxantrone of both the unwinding and ATPase reactions was approximately 1 microM for both substrates, suggesting that the mechanism of inhibition of helicase II by mitoxantrone is the same for both substrates and requires the presence of double-stranded structure. To strengthen this conclusion, the ability of mitoxantrone to inhibit the DNA-dependent ATPase activity of helicase II was determined using two single-stranded substrates, poly(dT) and the 245-bp substrate after heat denaturation. Using either substrate, mitoxantrone inhibited the ATPase activity of helicase II far less effectively. Thus, these results indicate that the intercalation of mitoxantrone into double-stranded DNA, with accompanying placement of functionalities in the major groove, generates a complex that impedes helicase II, resulting in both inhibition of ATP hydrolysis and unwinding activity. Furthermore, we report here that DNA-binding ligands inhibit the unwinding activity of helicases I and IV and Rep protein from E. coli, demonstrating that the inhibition observed for helicase II is not unique to this enzyme.     

Differential inhibition of the DNA translocation and DNA unwinding activities of DNA helicases by the Escherichia coli Tus protein.

Binding of the Escherichia coli Tus protein to its cognate nonpalindromic binding site on duplex DNA (a Ter sequence) is sufficient to arrest the progression of replication forks in a Ter orientation-dependent manner in vivo and in vitro. In order to probe the molecular mechanism of this inhibition, we have used a strand displacement assay to investigate the effect of Tus on the DNA helicase activities of DnaB, PriA, UvrD (helicase II), and the phi X-type primosome. When the substrate was a short oligomer hybridized to a circular single-stranded DNA, strand displacement by DnaB, PriA, and the primosome (in both directions), but not UvrD, was blocked by Tus in a polar fashion. However, no inhibition of either DnaB or UvrD was observed when the substrate carried an elongated duplex region. With this elongated substrate, PriA helicase activity was only inhibited partially (by 50%). On the other hand, both the 5'----3' and 3'----5' helicase activities of the primosome were inhibited almost completely by Tus with the elongated substrate. These results suggest that while Tus can inhibit the translocation of some proteins along single-stranded DNA in a polar fashion, this generalized effect is insufficient for the inhibition of bona fide DNA helicase activity.     

ATPase activities in peroxisome-proliferating yeast.

Preliminary studies on yeast peroxisomes have suggested that the membrane of these organelles may contain a proton-pumping ATPase. It has been reported that peroxisome-associated activity is similar to the F0-F1 mitochondrial type ATPase in its sensitivity to azide at pH 9.0, but characteristics of the plasma membrane type ATPase are also evident in peroxisomal preparations in that they exhibit pH 6.5 activity that is sensitive to vanadate. A comparative study of the prominent organellar ATPase activities was undertaken as a probe into the existence of an enzyme that is unique to the peroxisome, and biochemical properties of yeast mitochondrial, plasma membrane, together with peroxisomally-associated H(+)-ATPases are presented. Enzyme marker analysis of sucrose gradient fractions revealed a high degree of correlation between the amount of azide-sensitive pH 9.0 ATPase activity and that of the mitochondrial membrane marker, cytochrome c oxidase, in peroxisomal preparations. Purified mitochondrial and peroxisomally-associated activities were highly sensitive to the presence of sodium azide, N,N' -dicyclohexylcarbodiimide (DCCD) and venturicidin when measured at pH 9.0. Comparisons of peroxisomal activities with those of the purified plasma membrane at pH 6.0 in the presence of azide showed similar sensitivity profiles with respect to inhibitors of yeast plasma membrane ATPases such as vanadate and p-chloromercuriphenyl-sulfonic acid (CMP). Purified peroxisomal membranes, furthermore, reacted with antibody to the mitochondrial F1 subunit (as revealed by Western blot analysis), and [35S] methionine-labeled, glucose-grown cells processed with unlabeled methanol-grown cells, yielded sucrose gradient fractions that were radioactive in bands that were also recognized by F1 antibody. Isolated fractions in these experiments had similar ratios of cpm:pH 9.0 ATPase activities, suggesting that this activity is mitochondrial in origin. The data presented for the characteristics of the peroxisomally-associated activity strongly suggest that the majority of the ATPase activity found in peroxisomal preparations is derived from other organelles.     

ATPase and DNA helicase activities of the Saccharomyces cerevisiae anti-recombinase Srs2

Saccharomyces cerevisiae SRS2 encodes an ATP-dependent DNA helicase that is needed for DNA damage checkpoint responses and that modulates the efficiency of homologous recombination. Interestingly, strains simultaneously mutated for SRS2 and a variety of DNA repair genes show low viability that can be overcome by inactivating homologous recombination, thus implicating inappropriate recombination as the cause of growth impairment in these mutants. Here, we report on our biochemical characterization of the ATPase and DNA helicase activities of Srs2. ATP hydrolysis by Srs2 occurs efficiently only in the presence of DNA, with ssDNA being considerably more effective than dsDNA in this regard. Using homopolymeric substrates, the minimal DNA length for activating ATP hydrolysis is found to be 5 nucleotides, but a length of 10 nucleotides is needed for maximal activation. In its helicase action, Srs2 prefers substrates with a 3' ss overhang, and approximately 10 bases of 3' overhanging DNA is needed for efficient targeting of Srs2 to the substrate. Even though a 3' overhang serves to target Srs2, under optimized conditions blunt-end DNA substrates are also dissociated by this protein. The ability of Srs2 to unwind helicase substrates with a long duplex region is enhanced by the inclusion of the single-strand DNA-binding factor replication protein A.     

Nuclear translocation contributes to regulation of DNA excision repair activities

DNA mutations are circumvented by dedicated specialized excision repair systems, such as the base excision repair (BER), nucleotide excision repair (NER), and mismatch repair (MMR) pathways. Although the individual repair pathways have distinct roles in suppressing changes in the nuclear DNA, it is evident that proteins from the different DNA repair pathways interact [Y. Wang, D. Cortez, P. Yazdi, N. Neff, S.J. Elledge, J. Qin, BASC, a super complex of BRCA1-associated proteins involved in the recognition and repair of aberrant DNA structures, Genes Dev. 14 (2000) 927–939; M. Christmann, M.T. Tomicic, W.P. Roos, B. Kaina, Mechanisms of human DNA repair: an update, Toxicology 193 (2003) 3–34; N.B. Larsen, M. Rasmussen, L.J. Rasmussen, Nuclear and mitochondrial DNA repair: similar pathways? Mitochondrion 5 (2005) 89–108]. Protein interactions are not only important for function, but also for regulation of nuclear import that is necessary for proper localization of the repair proteins. This review summarizes the current knowledge on nuclear import mechanisms of DNA excision repair proteins and provides a model that categorizes the import by different mechanisms, including classical nuclear import, co-import of proteins, and alternative transport pathways. Most excision repair proteins appear to contain classical NLS sequences directing their nuclear import, however, additional import mechanisms add alternative regulatory levels to protein import, indirectly affecting protein function. Protein co-import appears to be a mechanism employed by the composite repair systems NER and MMR to enhance and regulate nuclear accumulation of repair proteins thereby ensuring faithful DNA repair.     

Indecisive M13 procoat protein mutants bind to SecA but do not activate the translocation ATPase

The M13 procoat protein serves as the paradigm for the Sec-independent membrane insertion pathway. This protein is inserted into the inner membrane of Escherichia coli with two hydrophobic regions and a central periplasmic loop region of 20 amino acid residues. Extension of the periplasmic loop region renders M13 procoat membrane insertion Sec-dependent. Loop regions with 118 or more residues required SecA and SecYEG and were efficiently translocated in vivo. Two mutants having loop regions of 80 and 100 residues, respectively, interacted with SecA but failed to activate the membrane translocation ATPase of SecA in vitro. Similarly, a procoat mutant with two additional glutamyl residues in the loop region showed binding to SecA but did not stimulate the ATPase. The three mutants were also defective for precursor-stimulated binding of SecA to the membrane surface. Remarkably, the mutant proteins act as competitive inhibitors of the Sec translocase. This suggests that the region to be translocated is sensed by SecA but the activation of the SecA translocation ATPase is only successful for substrates with a minimum length of the translocated region.     

Potent inhibition of DNA unwinding and ATPase activities of pea DNA helicase 45 by DNA-binding agents

Pea DNA helicase 45 (PDH45) is an ATP-dependent DNA unwinding enzyme, with intrinsic DNA-dependent ATPase activity [Plant J. 24 (2000) 219]. We have determined the effect of various DNA-binding agents, such as daunorubicin, ethidium bromide, ellipticine, cisplatin, nogalamycin, actinomycin C1, and camptothecin on the DNA unwinding and ATPase activities of the plant nuclear DNA helicase PDH45. The results show that all the agents except actinomycin C1, and camptothecin inhibited the helicase (apparent K(i) values ranging from 1.5 to 7.0 microM) and ATPase (apparent K(i) values ranging from 2.5 to 11.9 microM) activities. This is the first study to show the effect of various DNA-binding agents on the plant nuclear helicase and also first to demonstrate inhibition of any helicase by cisplatin. Another striking finding that the actinomycin C1 and ellipticine act differentially on PDH45 as compared to pea chloroplast helicase suggests that the mechanism of DNA unwinding could be different in nucleus and chloroplast. These results suggest that the intercalation of the inhibitors into duplex DNA generates a complex that impedes translocation of PDH45, resulting in both the inhibitions of unwinding activity and ATP hydrolysis. This study would be useful to obtain a better understanding of the mechanism of plant nuclear DNA helicase unwinding and the mechanism by which these agents can disturb genome integrity.     

Calcium binding and ATPase activities of heart sarcolemma.

Rat heart sarcolemma prepared by the hypotonic shock-LiBr treatment method was found to bind calcium by a concentration-dependent and saturable process. The calcium binding values at 50 muM and 1.25 mM Ca2+ concentrations were about 30 and 250 nmoles/mg protein, respectively. Both Mg2+ and ATP inhibited calcium binding and no evidence for energy-linked calcium binding with sarcolemmn was found. z sn the other hand, maximal ATP hydrolysis by heart sarcolemma was seen at 4 mM Mg2+ or Ca2+. The Ca2+-ATPase LEO) of Ca2+ failed to stimulate ATP hydrolysis in the presence of various concentrations of Mg-ATP. These results indicate the absence of a "calcium pump" mechanism in the heart sarcolemmal membrane preparation employed in this study.     

Proton translocation in cytochrome-deficient mutants of Escherichia coli.

Cytochrome-deficient cells of a strain of Escherichia coli lacking 5-amino-levulinate synthetase have been used to study proton translocation associated with the reduced nicotinamide adenine dinucleotide (NADH) dehydrogenase region of the electron transport chain. Menadione was used as electron acceptor, and mannitol was used as the substrate for the generation of intracellular NADH. The effects of iron deficiency on NADH- and D-lactate-menadione reductase activities were studied in iron-deficient cells of a mutant strain unable to synthesize the iron chelator enterochelin; both activities were reduced. The NADH- menadione reductase activity in cytochrome-deficient cells was associated with proton translocation and could be coupled to the uptake of proline. However proton translocation associated with the NADH-menadione reductase activity was prevented by a mutation in an unc gene. It was concluded that there is no proton translocation associated with the NADH-dehydrogenase region of the electron transport chain in E. coli and that the proton translocation obtained with mannitol as substrate is due to the activity of membrane-bound adenosine triphosphatase.     

The cytochrome b and ATPase genes of honeybee mitochondrial DNA.

The gene sequences for honeybee cytochrome b, ATPase 6, and ATPase 8 are presented, along with the inferred amino acid sequences of the proteins. These mitochondrial genes are in the same relative positions as are their counterparts in Drosophila mitochondrial mtDNA and have evolved at a significantly greater overall rate than have those of Drosophila. Comparisons using both amino acid identity and the proportion of conservative replacements between the inferred Apis and vertebrate cytochrome b sequences shows the two highly conserved sections reported by Howell, but his recognition of five conserved regions is not well supported. A very high AT bias is reflected in very high codon biases. The best predictors of the number of occurrences of an amino acid in honeybee cytochrome b are the T and G contents of its codon family--unlike the case for vertebrate cytochrome b, in which the codon family size and AT bias are the strongest predictors; protein function, at least as judged by hydrophilicity characteristics, appears to be unaffected by these differing influences on amino acid composition.     

The role of the mature domain of proOmpA in the translocation ATPase reaction.

The export of proOmpA, the precursor of outer membrane protein A from Escherichia coli, requires preprotein translocase, which is comprised of SecA, SecY/E, and acidic phospholipids. Previous studies of proOmpA translocation intermediates (Schiebel, E., Driessen, A. J. M., Hartl, F.-U., and Wickner, W. (1991) Cell 64, 927-939) suggested that the "slippage" of the translocating polypeptide chain and the high level of ATP hydrolysis, characteristic of the "translocation ATPase," were part of a futile cycle. To examine the role of the mature domain of proOmpA in its translocation-dependent ATP hydrolysis, we used chemical cleavage to generate NH2-terminal fragments of this preprotein. Each fragment contained the 21-residue leader region and either 53 or 228 residues of the mature domain (preproteins P74 and P249, respectively). As observed with full-length proOmpA, the translocation of each fragment requires ATP and both the SecA and SecY/E domains of translocase and is stimulated by the transmembrane proton electrochemical gradient. The apparent maximal velocities of P74 and proOmpA translocation are similar. While the translocation of P74 and of proOmpA show the same apparent Km for ATP, far less ATP is hydrolyzed during the translocation of P74. Thus, the mature carboxyl-terminal domain of proOmpA has a major role in supporting the translocation ATPase.     

Genetic and physical studies of restriction-deficient mutants of the Inc FIV plasmids R124 and R124/3

Summary R124 and R124/3 are R plasmids that carry the genes for two different restriction and modification systems. The phenotype of strains carrying either of these plasmids along with the F'lac ⁺ plasmid, is restriction-deficient (Res^-). The Res^- phenotype is not due to selection of preexisting mutants but rather to a complex mutational event caused by the F plasmid. Restriction-deficient mutants carry extensive deletions and other DNA rearrangements. Tn7 insertion is used to locate the restriction gene. Many of the Res^- mutants are genetically unstable and revert at exceptionally high frequencies. Reversion is accompanied by DNA rearrangements which result in a net gain of 9 kb of DNA. F derivates of F⁺ which do not cause restriction-deficiency but do cause deletion were used to distinguish between the DNA rearrangements associated with restriction-deficiency and those associated with deletion. From Res⁺ revertants of strains carrying F'lac ⁺ and R124 or R124/3 we have isolated F plasmids that now carry the genes for the R124 or R124/3 restriction and modification systems. It is suggested that interaction between part of the F plasmid and that segment of the R plasmid which controls the switch in Res-Mod specificity which has been observed (Glover et al. 1983) is responsible for the production of restriction-deficiency.     

The role of bacterial pili in protein and DNA translocation.

Gram-negative bacteria have surface appendages that assemble via different secretion machineries. Recently, new experimental approaches have contributed to a better understanding of the molecular mechanisms of flagellar and pilus assembly, and protein secretion. These findings can be applied to plant pathogenic bacteria, which probably transfer effector proteins directly into their eukaryotic host cells. Here, it is suggested that assembly of Hrp pili occurs in the periplasm and that unfolded effector proteins attach to pilins within the pili, thus effecting protein translocation. A two-domain structure for the HrpA pilin from Pseudomonas syringae is also predicted.     

UPF1 mutants with intact ATPase but deficient helicase activities promote efficient nonsense-mediated mRNA decay

The conserved RNA helicase UPF1 coordinates nonsense-mediated mRNA decay (NMD) by engaging with mRNAs, RNA decay machinery and the terminating ribosome. UPF1 ATPase activity is implicated in mRNA target discrimination and completion of decay, but the mechanisms through which UPF1 enzymatic activities such as helicase, translocase, RNP remodeling, and ATPase-stimulated dissociation influence NMD remain poorly defined. Using high-throughput biochemical assays to quantify UPF1 enzymatic activities, we show that UPF1 is only moderately processive (<200 nt) in physiological contexts and undergoes ATPase-stimulated dissociation from RNA. We combine an in silico screen with these assays to identify and characterize known and novel UPF1 mutants with altered helicase, ATPase, and RNA binding properties. We find that UPF1 mutants with substantially impaired processivity (E797R, G619K/A546H), faster (G619K) or slower (K547P, E797R, G619K/A546H) unwinding rates, and/or reduced mechanochemical coupling (i.e. the ability to harness ATP hydrolysis for work; K547P, R549S, G619K, G619K/A546H) can still support efficient NMD of well-characterized targets in human cells. These data are consistent with a central role for UPF1 ATPase activity in driving cycles of RNA binding and dissociation to ensure accurate NMD target selection.     

Bacillus subtilis F0F1 ATPase: DNA sequence of the atp operon and characterization of atp mutants.

We cloned and sequenced an operon of nine genes coding for the subunits of the Bacillus subtilis F0F1 ATP synthase. The arrangement of these genes in the operon is identical to that of the atp operon from Escherichia coli and from three other Bacillus species. The deduced amino acid sequences of the nine subunits are very similar to their counterparts from other organisms. We constructed two B. subtilis strains from which different parts of the atp operon were deleted. These B. subtilis atp mutants were unable to grow with succinate as the sole carbon and energy source. ATP was synthesized in these strains only by substrate-level phosphorylation. The two mutants had a decreased growth yield (43 and 56% of the wild-type level) and a decreased growth rate (61 and 66% of the wild-type level), correlating with a twofold decrease of the intracellular ATP/ADP ratio. In the absence of oxidative phosphorylation, B. subtilis increased ATP synthesis through substrate-level phosphorylation, as shown by the twofold increase of by-product formation (mainly acetate). The increased turnover of glycolysis in the mutant strain presumably led to increased synthesis of NADH, which would account for the observed stimulation of the respiration rate associated with an increase in the expression of genes coding for respiratory enzymes. It therefore appears that B. subtilis and E. coli respond in similar ways to the absence of oxidative phosphorylation.     

Inhibition of DNA helicase, ATPase and DNA-binding activities of E. coli RecQ helicase by chemotherapeutic agents

RecQ helicases play an essential role in maintaining genetic integrity in all organisms from Escherichia coli to humans. Defects to these enzymes are responsible for three distinct human diseases: Werner syndrome, Bloom syndrome and Rothmund-Thomson syndrome. All three diseases are characterized by a predisposition to cancer due to increased genomic instability. Previous studies on the effects of non-covalent DNA modifications on the catalytic activity of purified Werner and Bloom DNA helicases have shown that both enzymes have similar sensitivity profiles to these DNA-binding agents and are most strongly inhibited by the minor groove binder distamycin A. In this study, we show that the sensitivity profiles of E. coli RecQ to a number of DNA-binding ligands are different to those observed for WRN and Bloom helicases. These observations may give insights into the differences in molecular mechanisms underlying efficient motor function of RecQ helicases.