Discontinuous DNA replication in mouse P-815 cells.

The length of newly synthesized DNA strands from mouse P-815 cells was analyzed after denaturation both by electrophoresis and by sedimentation in alkaline sucrose gradients. [3-H]-Thymidine pulses of 2-8 min at 37 degrees C predominantly label molecules of 20-60 S. With 30-s pulses at 25 degrees C, all the [3-H]thymidine appears in short DNA strands of 50-200 nucleotides. Thus, DNA strand elongation occurs discontinuously via Okazaki fragments at both the 5' end and the 3' end. In dodecylsulfate lysates, only 10% of the Okazaki fragments are found as single-stranded molecules. About 90% are resistant to hydrolysis by the single-strand-specific nuclease S-1 and band in isopycnic gradients at the buoyant density of double-stranded DNA. No evidence for ribonucleotides at the 5' end of Okazaki fragments was obtained either in isopycnic CsCl or Cs2SO4 gradients or after incubation with polynucleotide kinase and [gamma-32P]ATP.     

Biochemical characterization of bacteriophage T4 Mre11-Rad50 complex

The Mre11-Rad50 complex (MR) from bacteriophage T4 (gp46/47) is involved in the processing of DNA double-strand breaks. Here, we describe the activities of the T4 MR complex and its modulation by proteins involved in homologous recombination. T4 Mre11 is a Rad50- and Mn(2+)-dependent dsDNA exonuclease and ssDNA endonuclease. ATP hydrolysis is required for the removal of multiple nucleotides via dsDNA exonuclease activity but not for the removal of the first nucleotide or for ssDNA endonuclease activity, indicating ATP hydrolysis is only required for repetitive nucleotide removal. By itself, Rad50 is a relatively inefficient ATPase, but the presence of Mre11 and dsDNA increases ATP hydrolysis by 20-fold. The ATP hydrolysis reaction exhibits positive cooperativity with Hill coefficients ranging from 1.4 for Rad50 alone to 2.4 for the Rad50-Mre11-DNA complex. Kinetic assays suggest that approximately four nucleotides are removed per ATP hydrolyzed. Directionality assays indicate that the prevailing activity is a 3' to 5' dsDNA exonuclease, which is incompatible with the proposed role of MR in the production of 3' ssDNA ends. Interestingly, we found that in the presence of a recombination mediator protein (UvsY) and ssDNA-binding protein (gp32), Mre11 is capable of using Mg(2+) as a cofactor for its nuclease activity. Additionally, the Mg(2+)-dependent nuclease activity, activated by UvsY and gp32, results in the formation of endonuclease reaction products. These results suggest that gp32 and UvsY may alter divalent cation preference and facilitate the formation of a 3' ssDNA overhang, which is a necessary intermediate for recombination-mediated double-strand break repair.     

The Zea mays glycine-rich RNA-binding protein MA16 is bound to a ribonucleotide(s) by a stable linkage

Expression of the gene encoding the maize glycine-rich RNA-binding protein MA16 is developmentally regulated and it is involved in environmental stress responses. The MA16 protein shows a wide spectrum of RNA-binding activities. On the basis of in vivo labelling, where a [32P]phosphate label was linked to the MA16 protein, Freire and Pages (Plant Mol Biol 29:797-807, 1995) suggested that the protein may be post-translationally modified by phosphorylation. However, further analysis showed that the [32P]phosphate label was sensitive to different treatments, suggesting that modification distinct from protein phosphorylation might occur in the MA16 protein. Biochemical analysis revealed that this [32P]phosphate labelling was resistant to phenol extraction and denaturing SDS-PAGE but sensitive to micrococcal nuclease, RNase A and RNase T1 treatments. The mobility of [3?S] labelled MA16 protein on SDS-PAGE did not significantly changed after the nuclease treatments suggesting that the [32P]phosphate label associated to MA16 protein could be a ribonucleotide or a very short ribonucleotide chain. In addition, immunoprecipitation of labelled extracts showed that the ribonucleotide(s) linked to the MA16 protein was removed by phosphorolytic activity. This activity could be catalysed by a phosphate-dependent ribonuclease. The C-terminus of MA16 protein harbouring a glycine-rich domain was predicted to be an intrinsically disordered region.     

A 32P postlabeling assay for determining the incorporation of bromodeoxyuridine into cellular DNA

Randerath's procedure for 32P postlabeling of 3'-monophosphate deoxyribonucleotides from digests of cellular DNA has been modified. 3'-Monophosphate deoxyribonucleotides are converted to 3',5'-bis[32P]phosphate deoxyribonucleotides with polynucleotide kinase and [32P]ATP; these products are enzymatically converted by P1 nuclease and polynucleotide kinase into 5'-[32P]monophosphate deoxyribonucleotides, which are separated from [32P]ATP on an anion-exchange column eluted with 0.1 M NaH2PO4, pH 6.5. Labeled mononucleotides in the effluent are separated by high-performance liquid chromatography. Values for the base composition of calf thymus DNA determined with this modified assay compare very favorably with reported values. The assay was used to measure the level of incorporation of the clinically useful agent bromodeoxyuridine into the DNA of 9L rat brain tumor cells. The modified assay appears to be a very accurate method for the determination of levels of base analogs incorporated into DNA.     

DNA footprinting studies of the complex formed by the T4 DNA polymerase holoenzyme at a primer-template junction

We have used DNA footprinting techniques to analyze the interactions of five DNA replication proteins at a primer-template junction: the bacteriophage T4 DNA polymerase (the gene 43 protein), its three accessory proteins (the gene 44/62 and 45 proteins), and the gene 32 protein, which is the T4 helix-destabilizing (or single-stranded DNA-binding) protein. The 177-nucleotide-long DNA substrate consisted of a perfect 52-base pair hairpin helix with a protruding single-stranded 5' tail. As expected, the DNA polymerase binds near the 3' end of this molecule (at the primer-template junction) and protects the adjacent double-stranded region from cleavage. When the gene 32 protein binds to the single-stranded tail, it reduces the concentration of the DNA polymerase required to observe the polymerase footprint by 10-30-fold. Periodic ATP hydrolysis by the 44/62 protein is required to maintain the activity of the DNA polymerase holoenzyme (a complex of the 43, 44/62, and 45 proteins). Footprinting experiments demonstrate the formation of a weak complex between the DNA polymerase and the gene 45 protein, but there is no effect of the 44/62 protein or ATP on this enlarged footprint. We propose a model for holoenzyme function in which the complex of the three accessory proteins uses ATP hydrolysis to keep a moving polymerase tightly bound to the growing 3' end, providing a "clock" to measure polymerase stalling.     

ATP nucleotidylation of creatine kinase.

Creatine kinase (CK) will autoincorporate radiolabel from [gamma32P]ATP and has thus been reported to be autophosphorylated. Also, in contrast to normal brain enzyme, CK in Alzheimer-diseased brain homogenate shows greatly decreased activity, abolished photolabeling with [32P]8N3ATP, and no detectable autoincorporation of radiolabel by [gamma32P]ATP. Surprisingly, our studies with both human brain and purified CK showed that [alpha32P]ATP, [gamma32P]ATP, [alpha32P]ADP, [2,8H3]ATP, [gamma32P]2',3'-O-(2,4, 6-trinitrophenyl)-ATP, and [gamma32P]benzophenone-gammaATP all autoincorporate radiolabel into CK with good efficiency. This demonstrates that the gamma-phosphate and the 2' and 3' hydroxyls are not involved in the covalent linkage and that all three phosphates, the ribose and base of the ATP molecule are retained upon autoincorporation (nucleotidylation). Treatment with NaIO3 to break the 2'-3' linkage effected total loss of radiolabel indicating that nucleotidylation resulted in opening of the ribose ring at the C1' position. Nucleotidylation with increasing [alpha32P]ATP at 37 degrees C gives an approximate k0.5 of 125 microM and saturates at 340 microM nucleotide. Modification of 8-10% of the copy numbers occurs at saturation, and CK activity is inhibited to approximately the same degree. Low micromolar levels of native substrates such as ADP, ATP, and phosphocreatine substantially reduce [alpha32P]ATP nucleotidylation. In contrast, AMP, GTP, GMP, NADH, and creatine did not effectively reduce nucleotidylation. When [alpha32P]ATP-nucleotidylated or [alpha32P]8N3ATP-photolabeled CK is treated with trypsin a single, identical radiolabeled peptide (V279-R291) is generated that comigrates on reverse phase HPLC and Tris-tricine electrophoresis. Nucleotidylation into this peptide was prevented 86% by the presence of ATP. We conclude that CK is nucleotidylated within the active site by modification at the C1'position and that autophosphorylation of this enzyme does not occur.     

Translocation of E. coli RecQ helicase on single-stranded DNA

A member of the SF2 family of helicases, Escherichia coli RecQ, is involved in the recombination and repair of double-stranded DNA breaks and single-stranded DNA (ssDNA) gaps. Although the unwinding activity of this helicase has been studied biochemically, the mechanism of translocation remains unclear. To this end, using ssDNA of varying lengths, the steady-state ATP hydrolysis activity of RecQ was analyzed. We find that the rate of ATP hydrolysis increases with DNA length, reaching a maximum specific activity of 38 ± 2 ATP/RecQ/s. Analysis of the rate of ATP hydrolysis as a function of DNA length implies that the helicase has a processivity of 19 ± 6 nucleotides on ssDNA and that RecQ requires a minimal translocation site size of 10 ± 1 nucleotides. Using the T4 phage encoded gene 32 protein (G32P), which binds ssDNA cooperatively, to decrease the lengths of ssDNA gaps available for translocation, we observe a decrease in the rate of ATP hydrolysis activity that is related to lattice occupancy. Analysis of the activity in terms of the average gap sizes available to RecQ on the ssDNA coated with G32P indicates that RecQ translocates on ssDNA on average 46 ± 11 nucleotides before dissociating. Moreover, when bound to ssDNA, RecQ hydrolyzes ATP in a cooperative fashion, with a Hill coefficient of 2.1 ± 0.6, suggesting that at least a dimer is required for translocation on ssDNA. We present a kinetic model for translocation by RecQ on ssDNA based on this characterization.     

Species-dependent isoenzyme subtypes of membrane-bound cyclic AMP-dependent protein kinase in highly purified cardiac sarcolemma.

Highly purified sarcolemma from dog and pig cardiac muscle has been shown to contain significant activities of a membrane-bound cyclic AMP-dependent protein kinase. In addition, these membranes undergo endogenous phosphorylation when incubated with Mg2+ and [gamma-32P]ATP. By comparing 32P-labelled patterns obtained with [gamma-32P]ATP and the photoaffinity label 8-azidoadenosine 3':5'-[32P]monophosphate (8-azido-cyclic [32P]AMP), we have demonstrated that, whereas the major kinase isoenzyme in dog sarcolemma was Type II, that in the pig membrane was the Type I isoenzyme.     

Purification and properties of human DNA helicase VI.

A novel ATP-dependent DNA unwinding enzyme, called human DNA helicase VI (HDH VI), was purified to apparent homogeneity from HeLa cells and characterized. From 327 g of cultured cells, 0.44 mg of pure enzyme was recovered, free of DNA polymerase, ligase, topoisomerase, nicking and nuclease activities. The enzyme behaves as a monomer having an M(r) of 128 kDa, whether determined with SDS-PAGE, or in native conditions. Photoaffinity labelling with [alpha-32P]ATP labelled the 128 kDa protein. Only ATP or dATP hydrolysis supports the unwinding activity for which a divalent cation (Mg2+ > Mn2+) is required. HDH VI unwinds exclusively DNA duplexes with an annealed portion < 32 bp and prefers a replication fork-like structure of the substrate. It cannot unwind blunt-end duplexes and is inactive also on DNA-RNA or RNA-RNA hybrids. HDH VI unwinds DNA unidirectionally by moving in the 3' to 5' direction along the bound strand.     

Sequence analysis of the nicks and termini of bacteriophage T5 DNA.

Bacteriophage T5 DNA, when isolated from mature phage particles, contains several nicks in one of the two strands. The 5'-terminal nucleotides at the nicks were labeled with polynucleotide kinase and [gamma-32P]ATP, and the 3'-terminal nucleotides were labeled with Escherichia coli DNA polymerase I and [alpha-32P]dGTP. The sequences around the nicks were analyzed by partial nuclease digestion followed by homochromatography fractionation of the resulting oligonucleotides. The nicks had at least the sequence -PuOH pGpCpGpC- in common. In addition, the two 5' external termini had the first seven nucleotides in common.     

A method for the enzymatic synthesis and purification of [alpha-32P] nucleoside triphosphates.

A simplified method is described for the enzymatic synthesis and purification of [alpha-32P]ribo- and deoxyribonucleoside triphosphates. The products are obtained at greater than 97% radiochemical purity with yields of 50--70% (relative to 32Pi) by a two-step elution from DEAE-Sephadex. All reactions are done in one vessel as there is no need for intermediate product purifications. This method is therefore suitable for the synthesis of these radioactive compounds on a relatively large scale. The sequential steps of the method involve first the synthesis of [gamma-32P]ATP and the subsequent phosphorylation of nucleoside 3' monophosphate with T4 polynucleotide kinase to yield nucleoside 3', [5'-32P]diphosphate. Hexokinase is used after the T4 reaction to remove any remaining [gamma-32P]ATP. Nucleoside 3',[5'-32P]diphosphate is treated with nuclease P-1 to produce the nucleoside [5'-32P]monophosphate which is phosphorylated to the [alpha-32P]nucleoside triphosphate with pyruvate kinase and nucleoside monophosphate kinase. Adenosine triphosphate used as the phosphate donor for [alpha-32P]deoxynucleoside triphosphate syntheses is readily removed in a second purification step involving affinity chromatography on boronate-polyacrylamide. [alpha-32P]Ribonucleoside triphosphates can be similarly purified when deoxyadenosine triphosphate is used as the phosphate donor.     

Characterization of strand exchange activity of yeast Rad51 protein.

The Saccharomyces cerevisiae RAD51 gene product takes part in genetic recombination and repair of DNA double strand breaks. Rad51, like Escherichia coli RecA, catalyzes strand exchange between homologous circular single-stranded DNA (ssDNA) and linear double-stranded DNA (dsDNA) in the presence of ATP and ssDNA-binding protein. The formation of joint molecules between circular ssDNA and linear dsDNA is initiated at either the 5' or the 3' overhanging end of the complementary strand; joint molecules are formed only if the length of the overhanging end is more than 1 nucleotide. Linear dsDNAs with recessed complementary or blunt ends are not utilized. The polarity of strand exchange depends upon which end is used to initiate the formation of joint molecules. Joint molecules formed via the 5' end are processed by branch migration in the 3'-to-5' direction with respect to ssDNA, and joint molecules formed with a 3' end are processed in the opposite direction.     

嗜热古菌Archaeoglobus fulgidus RecJ核酸酶的表达纯化及酶学特征

[目的]克隆表达嗜热古菌Archaeoglobus fulgidus(A.fulgidus)来源的RecJ核酸酶基因(ORF编号AF_0699,NCBI数据库基因登陆号为AF_RS03550),对该重组蛋白的核酸酶活性及酶学特征进行鉴定和分析.[方法]将A.fulgidus RecJ(AfuRecJ)核酸酶在大肠杆菌中...     

Nicking activity on pBR322 DNA of ribosome inactivating proteins from Phytolacca dioica L. leaves

Ribosome-inactivating proteins isolated from Phytolacca dioica L. leaves are rRNA-N-glycosidases, as well as adenine polynucleotide glycosylases. Here we report that some of them cleave supercoiled pBR322 dsDNA, generating relaxed and linear molecules. PD-L1, the glycosylated major form isolated from the winter leaves of adult P . dioica plants, produces both free 3'-OH and 5'-P termini randomly distributed along the DNA molecule, as suggested by labelling experiments with [alpha- 32P]dCTP and [gamma- 32 P]dATP. Moreover, when the reaction is carried out under low-salt conditions, cleavage is observed mainly at a specific site, located downstream of the ampicillin resistance gene (close to position 3200), ending with the deletion of a fragment of approximately 70 nucleotides. This cleavage pattern is similar to that obtained under the same conditions with mung bean nuclease, a single-strand endonuclease. Furthermore, pBR322 DNA treated with PD-L1 shows reduced transforming activity with E . coli HB101 competent cells in comparison to untreated control plasmid DNA.     

Purification, properties and specificity of an endonuclease from Agropyron elongatum seedlings.

An endonuclease was isolated from 5 days old Agropyron elongatum 8x = Elytrigia turcica McGuire seedlings. The enzyme was purified by means of ammonium sulfate fractionation, DEAE-cellulose and Heparin Sepharose column. The final preparation, named nuclease A, gave a single band after silver staining had followed SDS-electrophoresis that was identified with nuclease activities. The enzyme also showed a single band after activity staining on gel polymerized in the presence of heat denatured DNA (ssDNA)/RNA. The Mr of native enzyme was 36 and the enzyme's moiety consisted of one polypeptide chain. Nuclease A activity was stimulated in the presence of Zn(2+) and was moderately reduced by NaCl yet strongly by spermine. The enzyme had pH optimum 5.5 and isoelectric point (pI) 4.7. It hydrolyzed the nucleic acids in the order ssDNA > dsDNA > or = RNA; hence it was classified as a plant nuclease type I (EC 3.1.30.2). Synthetic homopolyribonucleotides were hydrolyzed in the order polyU > polyI > or = polyA > polyG > polyC. Nuclease A nicked the supercoiled plasmid DNA while it was incapable of hydrolyzing dinucleoside monophosphates. With regard to nuclease A base linkage specificity towards a synthetic 5'-(32)P labeled deoxydecanucleotide [5'-(32)P]CCTGGCAGTT, the enzyme firstly exhibited a preference to Ap downward arrow G bond and then to Gp downward arrow T, Cp downward arrow A and Gp downward arrow G bonds while it was incapable of hydrolyzing the Cp downward arrow C bond. The substrate's products of nuclease A were oligonucleotides with the monoesterified phosphate at the 3' position. Nuclease A may perform a crucial function in the metabolism of nucleic acids during seedling growth and could be used as a biochemical tool for analysis of nucleic acids structure.     

Biochemical characterization of the WRN-FEN-1 functional interaction

Werner Syndrome is a premature aging disorder characterized by chromosomal instability. Recently we reported a novel interaction of the WRN gene product with human 5' flap endonuclease/5'-3' exonuclease (FEN-1), a DNA structure-specific nuclease implicated in pathways of DNA metabolism that are important for genomic stability. To characterize the mechanism for WRN stimulation of FEN-1 cleavage, we have determined the effect of WRN on the kinetic parameters of the FEN-1 cleavage reaction. WRN enhanced the efficiency of FEN-1 cleavage rather than DNA substrate binding. WRN effectively stimulated FEN-1 cleavage on a flap DNA substrate with streptavidin bound to the terminal 3' nucleotide at the end of the upstream duplex, indicating that WRN does not require a free upstream end to stimulate FEN-1 cleavage of the 5' flap substrate. These results indicate that the mechanism whereby WRN stimulates FEN-1 cleavage is distinct from that proposed for the functional interaction between proliferating cell nuclear antigen and FEN-1. To understand the potential importance of the WRN-FEN-1(1) interaction in DNA replication, we have tested the effect of WRN on FEN-1 cleavage of several DNA substrate intermediates that may arise during Okazaki fragment processing. WRN stimulated FEN-1 cleavage of flap substrates with a terminal monoribonucleotide, a long 5' ssDNA tract, and a pseudo-Y structure. The ability of WRN to facilitate FEN-1 cleavage of DNA replication/repair intermediates may be important for the role of WRN in the maintenance of genomic stability.     

DNA helicase III from HeLa cells: an enzyme that acts preferentially on partially unwound DNA duplexes.

Human DNA helicase III, a novel DNA unwinding enzyme, has been purified to apparent homogeneity from nuclear extracts of HeLa cells and characterized. The activity was measured by using a strand displacement assay with a 32P labeled oligonucleotide annealed to M13 ssDNA. From 305 grams of cultured cells 0.26 mg of pure protein was isolated which was free of DNA topoisomerase, ligase, nicking and nuclease activities. The apparent molecular weight is 46 kDa on SDS polyacrylamide gel electrophoresis. The enzyme shows also DNA dependent ATPase activity and moves unidirectionally along the bound strand in 3' to 5' direction. It prefers ATP to dATP as a cofactor and requires a divalent cation (Mg2+ > Mn2+). Helicase III cannot unwind either blunt-ended duplex DNA or DNA-RNA hybrids and requires more than 84 bases of ssDNA in order to exert its unwinding activity. This enzyme is unique among human helicases as it requires a fork-like structure on the substrate for maximum activity, contrary to the previously described human DNA helicases I and IV, (Tuteja et al. Nucleic Acids Res. 18, 6785-6792, 1990; Tuteja et al. Nucleic Acids Res. 19, 3613-3618, 1991).     

Kinetics of ATP-stimulated nuclease activity of the Escherichia coli RecBCD enzyme

The RecBCD enzyme is an ATP-dependent nuclease on both single-stranded and double-stranded DNA substrates. We have investigated the kinetics of the RecBCD-catalyzed reaction with small, single-stranded oligodeoxyribonucleotide substrates under single-turnover conditions using rapid-quench flow techniques. RecBCD-DNA complexes were allowed to form in pre-incubation mixtures. The nuclease reactions were initiated by mixing with ATP. The reaction time-courses were fit to several possible reaction mechanisms and quantitative estimates were obtained for rate constants for individual reaction steps. The relative rates of forward reaction versus dissociation from the DNA, and the fact that inclusion of excess non-radiolabeled single-stranded DNA to trap free RecBCD has no effect on the nuclease reaction, indicates that the reaction is processive. The reaction products show that the reaction begins near the 3'-end of the [5'-32P]DNA substrates and the major cleavage sites are two to four phosphodiester bonds apart. The product distribution is unchanged as the ATP concentration varies from 10 microM to 100 microM ATP, while the overall reaction rate varies by about tenfold. These observations suggest that DNA cleavage is tightly coordinated with movement of the enzyme along the DNA. The reaction time-courses at low concentrations of ATP (10 microM and 25 microM) have a significant lag before cleavage products appear. We propose that the lag represents ATP-dependent movement of the DNA from an initial binding site in the helicase domain of the RecB subunit to the nuclease active site in a separate domain of RecB. The extent of reaction of the substrate is limited (approximately 50%) under all conditions. This may indicate the formation of a non-productive RecBCD-DNA complex that does not dissociate in the 1-2 s time-scale of our experiments.