On the Mechanism of the Polynucleotide Joining Reaction

Olivera, Baldomero M.; Hall, Zach W.; Anraku, Yasuhiro; Chien, Janice R.; Lehman, I. R.

doi:10.1101/sqb.1968.033.01.008

Cited by 50 publications

(21 citation statements)

References 9 publications

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“…The detection of ADP-ribosylated histones and peptides in reactions containing 32 P-labeled NAD and acetylated substrates may therefore reflect a low-frequency capture of such an intermediate (3,18). Transient ADP-ribose-substrate or AMP-substrate intermediates have also been detected in the case of a 2Ј-phosphotransferase involved in tRNA splicing (mentioned above) and E. coli DNA ligase, two other enzymes that use NAD as a cofactor for catalysis (25,27). Nonetheless, it remains possible that some of the ADP-ribosylated species detected in Sir2 reactions are the result of nonenzymatic linkage of the released ADP-ribose or acetyl-ADP-ribose to amino acid side chains.…”

Section: Discussionmentioning

confidence: 99%

“…Whereas some precedent exists for coupling of NAD hydrolysis to catalysis of additional chemical reactions (24,27), coupling of amide hydrolysis to additional steps has never been observed and raises questions about the possible biological significance of such a reaction mechanism. The energy of hydrolysis of the nicotinamide ribose glycosidic bond of NAD is Ϫ8.2 kcal͞mol (28,29).…”

Section: Discussionmentioning

confidence: 99%

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Coupling of histone deacetylation to NAD breakdown by the yeast silencing protein Sir2: Evidence for acetyl transfer from substrate to an NAD breakdown product

Tanny¹

2000

Proceedings of the National Academy of Sciences

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The Saccharomyces cerevisiae silencing protein Sir2 is the founding member of a universally conserved family of proteins that have been shown to possess NAD-dependent histone deacetylation and ADP-ribosylation activities. Here we show that histone deacetylation by Sir2 is coupled to cleavage of the high-energy bond that links the ADP-ribose moiety of NAD to nicotinamide. Analysis of the NAD cleavage products revealed the presence of nicotinamide, ADP-ribose, and a third product that appeared to be related to ADP-ribose. With the use of label transfer experiments, we show that the acetyl group in the histone substrate is transferred to this NAD breakdown product during deacetylation, forming a product that we conclude to be O-acetyl-ADP-ribose. Detection of this species strongly argues for obligate coupling of histone deacetylation to NAD breakdown by Sir2. We propose reaction mechanisms that could account for this coupling via acetyl-ADP-ribose formation. The unprecedented coupling of amide bond cleavage to cleavage of a high-energy bond raises the possibility that NAD breakdown by Sir2 plays an important role in silencing that is independent of its requirement for deacetylation.

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Coupling of histone deacetylation to NAD breakdown by the yeast silencing protein Sir2: Evidence for acetyl transfer from substrate to an NAD breakdown product

Tanny¹

2000

Proceedings of the National Academy of Sciences

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“…[For example, it is possible that the 5'-terminal nucleotide is linked through a pyrophosphate bond to another nucleotide. Such a bond occurs at the 5'-ends of mammalian RNA viruses (14) and at the 5'-ends of DNA chains prior to ligation by polynucleotide kinase (15 No doubt, the special structure of 5'-ends of nascent DNA chains as well as its association with protein play a role in the replication process.…”

Section: Discussionmentioning

confidence: 99%

Novel structure at 5'-ends of nascent DNA chains.

Siegmann¹,

Werner²

1976

Proc. Natl. Acad. Sci. U.S.A.

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ABSTRACr Because of their association with protein, short nascent DNA chains in Escherichia coli can be separated from other cellular DNA by chromatography on hydroxylapatite. Protein-free DNA chains of less than 500 nucleotides in length are resistant to degradation from the 5'-end by alkaline phosphatase [orthophosphoric-monoester phosphohydrolase (alkaline optimum); EC 3.1.3.11 and spleen phosphodiesterase (oligonucleate 3'-nucleotidohydrolase; EC 3.1.4.18). In contrast, DNA chains containing more than 500 nucleotides are degradable. From these results we conclude that short nascent DNA chains are structurally modified at their 5'-ends. The nature of this structure and its possible functions are discussed.Okazaki pieces, short DNA chains of about 1000 nucleotides in length, have long been considered the first intermediates in DNA replication (1). Recent evidence, however, shows that Okazaki pieces arise through the joining of many much shorter DNA chains (2, 3). Such short DNA chains accumulate in certain dna mutants (4, 5), suggesting that more than one reaction is involved in the synthesis of Okazaki pieces. The formation of a single Okazaki piece seems to involve many independent initiation events. Since all known DNA polymerases require a primer for the synthesis of a new DNA strand, we decided to study the 5'-ends of the short DNA chains, hoping that their structure might provide some clue to the mechanism of DNA chain initiation. In the course of these studies we found that the shortest replication intermediates, containing fewer than 500 nucleotides, carry an unusual group at-their 5'-ends, which may function in the initiation or elongation of nascent DNA chains. MATERIALS AND METHODSBacterial Strains and Culture Conditions. Escherichla colh strain 15 TAMT (thy-, arg-, met-, trp-) was grown at 140 in LSTL medium supplemented with 5 ,tg/ml of thymine and with 40,g/ml each of arginine, methionine, and tryptophan, as described (2).Labeling and Cell Lysis. In experiments where intracellular thymidine nucleotide pools were depleted, bacteria were incubated for 30 min in the absence of thymine and then pulselabeled with 1 ,gg/ml of [3H]thymine (50 Ci/mmol) for 10 sec as described (2). For steady-state labeling, an equal volume of LSTL medium, containing 5 ,Ag/ml of [3H]thymine, was added to the culture, and incubation was continued for 0.3-10 min (2). Incorporation of label was terminated with phenol-ethanol, and the cells were lysed as described (2)

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“…The ligase reaction proceeds via a covalent enzyme-adenylate intermediate (11,12). Since the formation of this intermediate can be measured independently of the overall joining reaction, we determined whether the ligase defect in ts-7 is also expressed at this step in the catalysis.…”

Section: Thermolability Of Enzyme-adenylate Formation In Ts-7mentioning

confidence: 99%

“…The formation of the enzyme-adenylate intermediate, the first step of the ligase reaction (11,12), was assayed by a method devised by Dr. Richard Gumport. To discharge any ligase already in the adenylylated form, the enzyme fractions were made 4 [32P]DPN, bovine plasma albumin (50 ,ug/ml), and 0.1-0.5 mg of protein from the NMN-treated, dialyzed preparations (the enzyme fractions were incubated at the temperature of the assay for 5 min before the addition of the other components to initiate the reaction).…”

Section: Enzyme Fractionation and Assaysmentioning

confidence: 99%

Enzymatic Characterization of a Mutant of Escherichia coli with an Altered DNA Ligase

Modrich

Lehman

1971

Proc. Natl. Acad. Sci. U.S.A.

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A temperature-sensitive, radiation-sensitive mutant of Escherichia coli has been assayed for DNA ligase activity in vitro. The strain contains a markedly reduced amount of DNA-joining activity, which is thermolabile. The formation of the ligase-adenylate intermediate is also temperature-sensitive in vitro. Two temperature-resistant revertants of the mutant contain normal amounts of a thermostable ligase. The mutant is killed by growth at 42'C, a temperature at which it displays aberrant DNA synthesis. These results suggest that the ligase is necessary for normal DNA metabolism and viability in this strain.Polynucleotide-joining enzymes (ligases) have been implicated in DNA replication (1) and repair (2), and in genetic recombination (2). The function of these enzymes in vivo has generally been studied in T4 phages with amber and temperature-sensitive mutations in the structural gene for the phageinduced ligase (3, 4). An inherent difficulty in interpreting the results obtained with such mutants has been uncertainty in regard to the extent to which the Escherichia coli ligase can substitute for the phage-induced enzyme. We have, in fact, recently found that the specific activity of the phage-induced ligase in extracts of T4-infected cells is only two-to three-fold higher than that of the host enzyme when the two are assayed under optimal conditions (5).Gellert and Bullock (6) have recently characterized several E. coli strains defective in ligase that they isolated by a novel selection technique. The three mutants studied had 40-150% of normal joining activity when analyzed at 300C, but only 4-14% of normal activity when assayed at 420C. Adenylylation of the enzyme was reduced in two of the mutants, but was not temperature sensitive. Except for the case of their iop8 1lg4 mutant (150% of normal joining at 300C, 4% of normal at 420C), which became UV sensitive at 420C, the mutants grew normally on minimal or rich media and showed no obvious defect in their DNA metabolism. These results do not permit unambiguous conclusions concerning the physiological role of the ligase.In 1968, Pauling and Hamm described a conditional-lethal mutant of E. coli that was temperature sensitive in a late step of dark repair (7). They also observed that at restrictive temperatures, the newly replicated DNA accumulated in the form of small (1OS) fragments (8). These properties led them to suggest that the mutant had a defective DNA ligase. Using as an assay the joining of oligo(dT) annealed to poly(dA) (9), they compared partially purified fractions derived from the mutant ts-7 and the parent TAU-bar; they found that the ligase activity in the mutant was significantly reduced compared to that of the parent. However, no difference in the thermolability of the two enzyme fractions could be detected (8). Measurement of ligase activity by the (dA) -(dT) method may have been complicated by the presence of exonucleases in these relatively crude enzyme preparations.We have recently developed a new assay for DNA ligase that measures the...

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On the Mechanism of the Polynucleotide Joining Reaction

Cited by 50 publications

References 9 publications

Coupling of histone deacetylation to NAD breakdown by the yeast silencing protein Sir2: Evidence for acetyl transfer from substrate to an NAD breakdown product

Coupling of histone deacetylation to NAD breakdown by the yeast silencing protein Sir2: Evidence for acetyl transfer from substrate to an NAD breakdown product

Novel structure at 5'-ends of nascent DNA chains.

Enzymatic Characterization of a Mutant of Escherichia coli with an Altered DNA Ligase

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