Mechanism of strand passage by Escherichia coli topoisomerase I. The role of the required nick in catenation and knotting of duplex DNA

We studied the interaction between topoisomerase I and a nicked DNA substrate to determine how the nick permits Escherichia coli topoisomerase I to catenate and knot duplex DNA rings. The presence of just a single nick in a 6600-base pair DNA increased the amount of DNA bound to topoisomerase I by 6-fold. The enzyme acts at the nick, as shown by linearization of nicked circles and covalent attachment of an enzyme molecule opposite the nick. DNA breaks are also introduced by the enzyme at sites not opposite to a nick, but three orders of magnitude less efficiently. The break induced by the enzyme is within several base pairs of the nick and on the complementary strand, but the exact site cut is dictated by DNA sequence requirements. Because these sequence requirements are identical to those for cutting of single-stranded DNA, we conclude that the enzyme stabilizes a denatured region at the nick. Breaks in single-stranded DNA occur 98% of the time when a C residue is four bases to the 5' side unless G is adjacent and 5' to the break. For a DNA circle nicked at a unique location, the efficiency of DNA breakage opposite the nick correlates with the rate of catenation. We present a unified model for the relaxation, catenation, and knotting reactions of topoisomerase I in which the enzyme induces a break in a single-stranded region, but bridges that break with covalent and noncovalent interactions and allows passage of one duplex or single-stranded DNA segment.     

Patterns of digestion of human chromosomes by restriction endonucleases demonstrated by in situ nick translation

Summary A restriction enzyme-nick translation procedure has been developed for localizing sites of restriction endonuclease action on chromosomes. This method involves digestion of fixed chromosome preparations with a restriction enzyme, nick translation with DNA polymerase I in the presence of biotinylated-dUTP, detection of the incorporated biotin label with streptavidinalkaline phosphatase, and finally staining for alkaline phosphatase. Results obtained on human chromosomes using a wide variety of restriction enzymes are described, and compared with results of Giemsa and Feulgen staining after restriction enzyme digestion. Results of nick translation are not in general the opposite of those obtained with Giemsa staining, as might have been expected. Although the nick translation procedure is believed to give a more accurate picture of the distribution of restriction enzyme recognition sites on chromosomes than Giemsa staining, it is clear that the results of the nick translation experiments are affected by accessibility to the enzymes of the chromosomal DNA, as well as by the extractability of the DNA.     

The PcrA3 mutant binds DNA and interacts with the RepC initiator protein of plasmid pT181 but is defective in its DNA helicase and unwinding activities

Plasmid rolling-circle replication initiates by covalent extension of a nick generated at the plasmid double-strand origin (dso) by the initiator protein. The RepC initiator protein binds to the plasmid pT181 dso in a sequence-specific manner and recruits the PcrA helicase through a protein-protein interaction. Subsequently, PcrA unwinds DNA at the nick site followed by replication by DNA polymerase III. The pcrA3 mutant of Staphylococcus aureus has previously been shown to be defective in plasmid pT181 replication. Suppressor mutations in the repC initiator gene have been isolated that allow pT181 replication in the pcrA3 mutant. One such suppressor mutant contains a D57Y change in the RepC protein. To identify the nature of the defect in PcrA3, we have purified this mutant protein and studied its biochemical activities. Our results show that while PcrA3 retains its DNA binding activity, it is defective in its helicase and RepC-dependent pT181 DNA unwinding activities. We have also purified the RepC D57Y mutant and shown that it is similar in its biochemical activities to wild-type RepC. RepC D57Y supported plasmid pT181 replication in cell-free extracts made from wild-type S. aureus but not from the pcrA3 mutant. We also demonstrate that both wild-type RepC and its D57Y mutant are capable of a direct physical interaction with both wild-type PcrA and the PcrA3 mutant. Our results suggest that the inability of PcrA3 to support pT181 replication is unlikely to be due to its inability to interact with RepC. Rather, it is likely that a defect in its helicase activity is responsible for its inability to replicate the pT181 plasmid.     

The generation of a single nick per plasmid molecule using restriction endonucleases with multiple recognition sites

Some restriction endonucleases generate a single-stranded nick at their recognition sequences in the presence of ethidium bromide (EtBr). This nick can then be extended to a single-stranded gap in which mutations can be introduced by a variety of techniques. To date, the templates used in these studies have largely contained a single recognition site for a given enzyme. Therefore, we have extended these studies to twelve enzymes for which multiple recognition sites exist in the template and show that, under appropriate conditions, one single-stranded nick is introduced per plasmid molecule.     

Two nicking enzyme systems specific for mismatch-containing DNA in nuclear extracts from human cells.

We have identified two novel enzyme systems in human HeLa nuclear extracts that can nick at specific sites of DNA molecules with base mismatches, in addition to the T/G mismatch-specific nicking enzyme system (Wiebauer, K., and Jiricny, J. (1989) Nature 339, 234-236). One enzyme (called all-type) can nick all eight base mismatches with different efficiencies. The other (A/G-specific) nicks only DNA containing an A/G mismatch. The all-type enzyme can be separated from the T/G-specific and A/G-specific nicking enzymes by Bio-Rex 70 chromatography. Further purification on a DEAE-5PW column separated the A/G-specific nicking enzyme from the T/G-specific nicking enzyme. Therefore, at least three different enzyme systems are able to cleave mismatched DNA in HeLa nuclear extracts. The all-type and A/G-specific enzymes work at different optimal salt concentrations and cleave at different sites within the mismatched DNA. The all-type enzyme can only cleave at the first phosphodiester bond 5' to the mispaired bases. This enzyme shows nick disparity to only one DNA strand and may be involved in genetic recombination. The A/G-specific enzyme simultaneously makes incisions at the first phosphodiester bond both 5' and 3' to the mispaired adenine but not the guanine base. This enzyme may be involved in an A/G mismatch-specific repair similar to the Escherichia coli mutY (or micA)-dependent pathway.     

Kinetics and thermodynamics of nick sealing by T4 DNA ligase.

T4 DNA ligase is an Mg2+-dependent and ATP-dependent enzyme that seals DNA nicks in three steps: it covalently binds AMP, transadenylates the nick phosphate, and catalyses formation of the phosphodiester bond releasing AMP. In this kinetic study, we further detail the reaction mechanism, showing that the overall ligation reaction is a superimposition of two parallel processes: a 'processive' ligation, in which the enzyme transadenylates and seals the nick without dissociating from dsDNA, and a 'nonprocessive' ligation, in which the enzyme takes part in the abortive adenylation cycle (covalent binding of AMP, transadenylation of the nick, and dissociation). At low concentrations of ATP (<10 microM) and when the DNA nick is sealed with mismatching base pairs (e.g. five adjacent), this superimposition resolves into two kinetic phases, a burst ligation (approximately 0.2 min(-1)) and a subsequent slow ligation (approximately 2x10(-3) min(-1)). The relative rate and extent of each phase depend on the concentrations of ATP and Mg2+. The activation energies of self-adenylation (16.2 kcal.mol(-1)), transadenylation of the nick (0.9 kcal.mol(-1)), and nick-sealing (16.3-18.8 kcal.mol(-1)) were determined for several DNA substrates. The low activation energy of transadenylation implies that the transfer of AMP to the terminal DNA phosphate is a spontaneous reaction, and that the T4 DNA ligase-AMP complex is a high-energy intermediate. To summarize current findings in the DNA ligation field, we delineate a kinetic mechanism of T4 DNA ligase catalysis.     

Mapping of eukaryotic DNA topoisomerase I catalyzed cleavage without concomitant religation in the vicinity of DNA structural anomalies

Sensitive sites for covalent trapping of eukaryotic topoisomerase I at DNA structural anomalies were mapped by a new method using purified enzyme and defined DNA substrates. To insure that the obtained topoisomerase I trapping patterns were not influenced by DNA sequence variations, a single DNA imperfection was placed centrally within a homonucleotide track. Mapping of topoisomerase I-mediated irreversible cleavage sites on homopolymeric DNA substrates containing mismatches showed trapping of the enzyme in several positions in close vicinity of the DNA imperfection, with a strong preference for the 5' junction between the duplex DNA and the base-pairing anomaly. On homopolymeric DNA substrates containing a nick, sites of topoisomerase I-mediated cleavage on the intact strand were located just opposite to the nick and from one to ten nucleotides 5' to the nick. Sites of enzyme-mediated cleavage next to a nick and an immobile single-stranded branch were located 5' to the strand interruption in distances of two to six nucleotides and two to ten nucleotides, respectively. Taken together these findings suggest that covalent trapping of topoisomerase I proceeds at positions adjacent to mismatches, nicks and single-stranded branches, where the cleavage reaction is allowed and the ensuing ligation reaction prevented. In principle, the developed interference method might be of general utility to define topoisomerase-DNA interactions relative to different types of structural anomalies.     

Improving the fidelity of Thermus thermophilus DNA ligase.

The DNA ligase from Thermus thermophilus (Tth DNA ligase) seals single-strand breaks (nicks) in DNA duplex substrates. The specificity and thermostability of this enzyme are exploited in the ligase chain reaction (LCR) and ligase detection reaction (LDR) to distinguish single base mutations associated with genetic diseases. Herein, we describe a quantitative assay using fluorescently labeled substrates to study the fidelity of Tth DNA ligase. The enzyme exhibits significantly greater discrimination against all single base mismatches on the 3'-side of the nick in comparison with those on the 5'-side of the nick. Among all 12 possible single base pair mismatches on the 3'-side of the nick, only T-G and G-T mismatches generated a quantifiable level of ligation products after 23 h incubation. The high fidelity of Tth DNA ligase can be improved further by introducing a mismatched base or a universal nucleoside analog at the third position of the discriminating oligonucleotide. Finally, two mutant Tth DNA ligases, K294R and K294P, were found to have increased fidelity using this assay.     

Biochemical characterization of mutant forms of DNA polymerase I from Escherichia coli. I. The polA12 mutation.

DNA polymerase I has been purified to greater than 90% homogeneity from a strain of Escherichia coli K12 that bears the temperature-sensitive DNA polymerase I mutatation, polA12. The mutant enzyme has a reduced electrophoretic mobility and sedimentation rate. It is abnormally thermolabile and is rapidly inactivated at low salt concentrations. Its polymerase and 5' leads to 3' exonuclease activities are not grossly defective at 30 degrees, yet its capacity to promote the concerted 5' leads to 3' polymerization and the 5' leads to 3' exonucleolytic hydrolysis of nucleotides at a nick ("nick translation") is decreased 10-fold. These effects are probably the result of a significant alteration in the tertiary structure of the enzyme.     

Footprinting of Chlorella virus DNA ligase bound at a nick in duplex DNA.

The 298-amino acid ATP-dependent DNA ligase of Chlorella virus PBCV-1 is the smallest eukaryotic DNA ligase known. The enzyme has intrinsic specificity for binding to nicked duplex DNA. To delineate the ligase-DNA interface, we have footprinted the enzyme binding site on DNA and the DNA binding site on ligase. The size of the exonuclease III footprint of ligase bound a single nick in duplex DNA is 19-21 nucleotides. The footprint is asymmetric, extending 8-9 nucleotides on the 3'-OH side of the nick and 11-12 nucleotides on the 5'-phosphate side. The 5'-phosphate moiety is essential for the binding of Chlorella virus ligase to nicked DNA. Here we show that the 3'-OH moiety is not required for nick recognition. The Chlorella virus ligase binds to a nicked ligand containing 2',3'-dideoxy and 5'-phosphate termini, but cannot catalyze adenylation of the 5'-end. Hence, the 3'-OH is important for step 2 chemistry even though it is not itself chemically transformed during DNA-adenylate formation. A 2'-OH cannot substitute for the essential 3'-OH in adenylation at a nick or even in strand closure at a preadenylated nick. The protein side of the ligase-DNA interface was probed by limited proteolysis of ligase with trypsin and chymotrypsin in the presence and absence of nicked DNA. Protease accessible sites are clustered within a short segment from amino acids 210-225 located distal to conserved motif V. The ligase is protected from proteolysis by nicked DNA. Protease cleavage of the native enzyme prior to DNA addition results in loss of DNA binding. These results suggest a bipartite domain structure in which the interdomain segment either comprises part of the DNA binding site or undergoes a conformational change upon DNA binding. The domain structure of Chlorella virus ligase inferred from the solution experiments is consistent with the structure of T7 DNA ligase determined by x-ray crystallography.     

DNA strand breaks in ejaculated human spermatozoa: comparison of susceptibility to the nick translation and terminal transferase assays

The nick translation and terminal transferase assays have been compared to test their relative efficiency in detecting DNA breakage in ejaculated human spermatozoa. The results have been correlated with the percentage of chromomycin A3 positive sperm, a fluorochrome that is indicative of the protamination state of sperm. Examination of the ejaculated sperm of 30 subjects revealed that the percentage of positivity to the nick translation and terminal transferase assays did not differ, even when using different fixatives. It is concluded that the inability of the two assays to distinguish the type of DNA damage, as is possible in somatic nuclei, is most probably linked to the unique nature of sperm chromatin. It is proposed that the presence of the damaged DNA may be the remnants of an imperfect spermiogenesis, probably related to an inadequate protamine deposition. This is supported by the strong correlation between the presence of DNA damage and underprotamination as evidenced by chromomycin A3. © Chapman & Hall     

The role of single-strand breaks in the catenation reaction catalyzed by the rat type I topoisomerase

The type I topoisomerase from rat cells produces true catenanes from circular SV40 DNA in a reaction which is dependent on the presence of a single-strand break in at least one member of a pair of reacting molecules. The role of the single-strand break in the reaction was examined. Molecules containing a nick with a 3'-hydroxyl and 5'-phosphate or a nick with a 3'-phosphate and 5'-hydroxyl and molecules with single-stranded gaps were all found to be equally effective in the catenation reaction. It was found that the enzyme could, at a low frequency, break DNA by acting opposite a pre-existing single-strand break. Thus, incubation of nicked circular DNA in the presence of the topoisomerase, polynucleotide kinase, and [gamma-32P]ATP led to the production of a low level of labeled linear molecules containing covalently attached protein. Nicked linear molecules treated with topoisomerase in the absence of polynucleotide kinase generated fragments of sizes consistent with breakage in the opposite strand near the pre-existing nick. Based on these results, we propose that the catenation reaction may involve the transient production of linear intermediates by the action of the topoisomerase opposite a pre-existing nick in the DNA. Rejoining of the two ends by the enzyme could lead to the interlocking of two or more circular DNAs. In addition, these results suggest a possible role for the type I topoisomerase in illegitimate recombination.     

In situ fluorescent nick translation procedure for plant chromosomes

Pectinase and cellulase, which are used to macerate plant material, always show traces of DNase activities that result in DNA nicking. Moreover, the DNA polymerase I usually applied in the in situ nick translation techniques shows both 5' to 3' and 3' to 5' exonuclease activities. As a result, significant nonspecific labeling appears in control preparations that are not digested by a restriction endonuclease. Our procedure includes blocking nonspecific nick labeling before incubation with restriction enzymes (HpaII and HaeIII). This is achieved by incorporation of ddGTP into DNA by the Taq polymerase which lacks 3' to 5' exonuclease activity. This method gives satisfactory results because it eliminates nonspecific nick translation signals that are present after applying the methods described for animal material.     

Nick sensing by vaccinia virus DNA ligase requires a 5' phosphate at the nick and occupancy of the adenylate binding site on the enzyme.

Vaccinia virus DNA ligase has an intrinsic nick-sensing function. The enzyme discriminates at the substrate binding step between a DNA containing a 5' phosphate and a DNA containing a 5' hydroxyl at the nick. Further insights into nick recognition and catalysis emerge from studies of the active-site mutant K231A, which is unable to form the covalent ligase-adenylate intermediate and hence cannot activate a nicked DNA substrate via formation of the DNA-adenylate intermediate. Nonetheless, K231A does catalyze phosphodiester bond formation at a preadenylated nick. Hence, the active-site lysine of DNA ligase is not required for the strand closure step of the ligation reaction. The K231A mutant binds tightly to nicked DNA-adenylate but has low affinity for a standard DNA nick. The wild-type vaccinia virus ligase, which is predominantly ligase-adenylate, binds tightly to a DNA nick. This result suggests that occupancy of the AMP binding pocket of DNA ligase is essential for stable binding to DNA. Sequestration of an extrahelical nucleotide by DNA-bound ligase is reminiscent of the base-flipping mechanism of target-site recognition and catalysis used by other DNA modification and repair enzymes.     

Surface-located trypsin-activated Streptococcus sanguis strain Wicky endonuclease

A new Streptococcus sanguis strain Wicky endonuclease was isolated, purified and partially characterized. This nuclease acts preferentially on thermally denatured DNA, is not inhibited by RNA and is activated 3-5 times by trypsin. This activation is accompanied by the reduction of molecular weight of the enzyme. These features distinguish the new S, sanguis nuclease from the 3 previously described S. sanguis endonucleases. With covalently closed circular plasmid DNA, the enzyme causes first the appearance of a single stranded nick, then the second nick on the opposite DNA strand, resulting in plasmid DNA linearization. This nuclease most likely is located at the cell surface. The possible relationship of the described nuclease with ability of S. sanguis cells to take up DNA in genetic transformation is discussed.     

Deinococcus radiodurans RNA ligase exemplifies a novel ligase clade with a distinctive N-terminal module that is important for 5'-PO4 nick sealing and ligase adenylylation but dispensable for phosphodiester formation at an adenylylated nick

Deinococcus radiodurans RNA ligase (DraRnl) is a template-directed ligase that seals nicked duplexes in which the 3′-OH strand is RNA. DraRnl is a 342 amino acid polypeptide composed of a C-terminal adenylyltransferase domain fused to a distinctive 126 amino acid N-terminal module (a putative OB-fold). An alanine scan of the C domain identified 9 amino acids essential for nick ligation, which are located within nucleotidyltransferase motifs I, Ia, III, IIIa, IV and V. Seven mutants were dysfunctional by virtue of defects in ligase adenylylation: T163A, H167A, G168A, K186A, E230A, F281A and E305A. Four of these were also defective in phosphodiester formation at a preadenylylated nick: G168A, E230A, F281A and E305A. Two nick sealing-defective mutants were active in ligase adenylylation and sealing a preadenylylated nick, thereby implicating Ser185 and Lys326 in transfer of AMP from the enzyme to the nick 5′-PO₄. Whereas deletion of the N-terminal domain suppressed overall nick ligation and ligase adenylylation, it did not compromise sealing at a preadenylylated nick. Mutational analysis of 15 residues of the N domain identified Lys26, Gln31 and Arg79 as key constituents. Structure–activity relationships at the essential residues were determined via conservative substitutions. We propose that DraRnl typifies a new clade of polynucleotide ligases. DraRnl homologs are detected in several eukaryal proteomes.     

Two DNA-binding and nick recognition modules in human DNA ligase III

Human DNA ligase III contains an N-terminal zinc finger domain that binds to nicks and gaps in DNA. This small domain has been described as a DNA nick sensor, but it is not required for DNA nick joining activity in vitro. In light of new structural information for mammalian ligases, we measured the DNA binding affinity and specificity of each domain of DNA ligase III. These studies identified two separate, independent DNA-binding modules in DNA ligase III that each bind specifically to nicked DNA over intact duplex DNA. One of these modules comprises the zinc finger domain and DNA-binding domain, which function together as a single DNA binding unit. The catalytic core of ligase III is the second DNA nick-binding module. Both binding modules are required for ligation of blunt ended DNA substrates. Although the zinc finger increases the catalytic efficiency of nick ligation, it appears to occupy the same binding site as the DNA ligase III catalytic core. We present a jackknife model for ligase III that posits conformational changes during nick sensing and ligation to extend the versatility of the enzyme.     

Chi-stimulated patches are heteroduplex, with recombinant information on the phage lambda r chain

Generalized recombination in Escherichia coli is elevated near Chi sites. In vitro, RecBCD enzyme can nick Chi a few nucleotides 3' of the terminal GG of the Chi sequence (5'-GCTGGTGG). The simplest model in which this nick at Chi participates in Chi function predicts that in phage lambda, Chi-stimulated recombinants not crossed-over for flanking markers (patches) should be heteroduplex, with recombinant information on the lambda I chain. I report here that patches are heteroduplex, but that recombinant information occurs primarily on the lambda r chain. This result rules out the simplest model in which the nick at Chi promotes initiation of recombination, forces reconsideration of Chi's role in recombination, and bears on molecular models for Rec-mediated recombination.     

Mechanism of the reaction catalyzed by the DNA untwisting enzyme: attachment of the enzyme to 3'-terminus of the nicked DNA.

The rat liver DNA untwisting enzyme introduces a transient nick into duplex DNA. The enzyme has been shown to be covalently attached to one of the ends of the broken strand in the nicked intermediate (Champoux, 1977). The broken strand containing bound enzyme is shown to be susceptible to phosphorylation by polynucleotide kinase. Therefore, the DNA untwisting enzyme must be attached to the strand at the 3′-phosphate terminus, and this linkage probably conserves the energy required for resealing the single-strand break.     

极端嗜热古菌———芝田硫化叶菌DNA 连接酶的生化性质

极端嗜热古菌———芝田硫化叶菌 DNA 连接酶 (Ssh 连接酶 ) 的最适辅因子为 ATP ,在 dATP 存在时,该酶也能表现出较弱的连接活性 . ATP 或 dATP 都能够使该酶发生腺苷化,腺苷化的 Ssh 连接酶能够将腺苷基团转移至含切刻的 DNA 上 . 电泳迁移率改变实验表明, Ssh 连接酶能够结合双链 DNA ,且与含切刻及不含切刻的 DNA 结合的亲和力相同,但不结合单链 DNA. 酵母双杂交实验显示,硫磺矿硫化叶菌 ( 与芝田硫化叶菌亲缘关系很近 ) 的 DNA 连接酶,与该菌所含的 3 个增殖细胞核抗原 (PCNA) 同源蛋白中的一个 (PCNA-1) 有相互作用,而与另外 2 个同源蛋白 (PCNA-like 和 PCNA-2) 则无相互作用 . 在古菌中高度保守的 Sac10b 蛋白家族成员 Ssh10b 能够激活 Ssh 连接酶的活性,而硫化叶菌中的主要染色体蛋白——— 7 ku DNA 结合蛋白 (Ssh7) 则对该酶活性没有影响 .