Mechanism of DNA chain growth: XV. RNA-linked nascent DNA pieces in Escherichia coli strains assayed with spleen exonuclease

A new method for the detection and assay of RNA-linked nascent DNA pieces has been developed. The method relies on selective degradation by spleen exonuclease of radioactive 5′-OH terminated DNA produced from the pulse-labelled nascent pieces upon alkaline hydrolysis. Analysis with this method in wild type Escherichia coli has shown relatively high proportions of the RNA-linked molecules after shorter pulses and in the smaller pieces, supporting the transient nature of the RNA attachment to the nascent pieces. The RNA-linked nascent DNA pieces are accumulated by both E. coli polAex1 (defective in 5′ → 3′ exonuclease of DNA polymerase I) and E. coli polA12 and polA1 (defective in polymerase of DNA polymerase I), suggesting the requirement of the concerted action of both 5′ → 3′ exonuclease and polymerase of DNA polymerase I for the removal of the RNA attached to the nascent pieces. Most of the nascent DNA pieces accumulated by E. coli ligts7 (defective in DNA ligase) are not linked to RNA, as expected from the direct role of DNA ligase in joining of the pieces. The analysis also has shown that a large portion of the nascent DNA pieces present in the cell under the normal steady-state conditions are not linked to RNA and that the level of the RNA-free DNA pieces is also increased in polA mutants. These findings suggest that the removal of RNA from the nascent pieces is a relatively rapid process and the joining reaction is a rate-limiting step that requires the concurrent action of DNA polymerase and DNA ligase.     

RNA-linked nascent DNA pieces in phage T7-infected Escherichia coli. III. Detection of intact primer RNA

RNA-linked DNA fragments of T7-infected Escherichiacoli were labeled with [(32)P]orthophosphate invivo. The RNA segments of the labeled fragments were isolated by degrading the DNA portion with the 3'--> 5' exonuclease intrinsic to bacteriophage T4 DNA polymerase and fractionated according to net charge by a DEAE-Sephadex A-25 column chromatography in the presence of 7 M urea. Tri-, tetra- and pentanucleotides were obtained which have ATP residues at their 5' ends. Most of the pentanucleotides had a single deoxynucleotide at the 3' end but a minor portion was totally an oligoribonucleotide. In the light of prior results, the former is a cooligomer of an intact tetraribonucleotide primer and a monodeoxynucleotide and the latter is an intact pentaribonucleotide primer. Tri- and tetraribonucleotides with ATP at the 5' ends had no deoxynucleotide at the 3' ends, therefore it is not clear if intact triribonucleotide primers are present. The 5'-terminal dinucleotides of the tetra- and pentanucleotides were mostly pppApC and a trace amount of pppApA was present.Images     

RNA-linked nascent DNA pieces in phage T7-infected Escherchia coli. II. Primary structure of the RNA portion.

Short DNA chains were purified from phage T7 infected E. coli cells and 5' ends were labeled with 32P. By an alkali-treatment, pNp's rich in pAp and pCp were liberated from the T7 short DNA chains. After digestion of the [5'-32P] short DNA with the 3' to 5' exonuclease of T4 DNA polymerase, [5'-32P] mono- to pentaribonucleotides tipped with a deoxyribonucleotide residue at their 3' ends were isolated. 5' terminal ribonucleotides were; exclusively AMP in the penta- and the tetraribonucleotides, mostly CMP in the triribonucleotide and mainly CMP and AMP in di- and monoribonucleotides. The 5' terminal dinucleotide of the penta- and the tetraribonucleotides was pApC. The nucleotide sequence of the tetraribonucleotide was mainly pApCpCpN and some pApCpApN, where N was mainly A and C. These results indicate that oligoribonucleotides shorter than trinucleotide may result from in vivo degradation of the tetra- and pentaribonucleotides. A possibility that the tetra- and pentaribonucleotides with a 5' triphosphate terminus are the intact primers for the discontinuous T7 DNA replication is discussed.     

RNA-linked nascent DNA pieces in T7 phage-infected Escherichia coli cells. I. Role of gene 6 exonuclease in removal of the linked RNA.

Summary The presence of RNA-linked nascent DNA pieces in T7 phage-infectedEscherichia coli cells has been shown by the selective degradation of the 5-hydroxyl-terminated nascent DNA, produced by alkali or RNase treatment, with spleen exonuclease. At 43°C, the proportion of RNA-linked DNA pieces in nascent short DNA is 50 to 60% in T7ts136 (ts mutant of gene 6) phage-infectedE. coli, whereas that in T7 wild-type phage-infected cells is less than 6%. Joining of the nascent pieces is greatly retarded in T7ts136-infectedE. coli temperature sensitivepolA mutants at 43° C. These results suggest that gene 6 exonuclease plays a role in removal of the linked RNA during the discontinuous replication of T7 DNA.     

Xrcc4 physically links DNA end processing by polynucleotide kinase to DNA ligation by DNA ligase IV

Nonhomologous end joining (NHEJ) is the major DNA double-strand break (DSB) repair pathway in mammalian cells. A critical step in this process is DNA ligation, involving the Xrcc4-DNA ligase IV complex. DNA end processing is often a prerequisite for ligation, but the coordination of these events is poorly understood. We show that polynucleotide kinase (PNK), with its ability to process ionizing radiation-induced 5'-OH and 3'-phosphate DNA termini, functions in NHEJ via an FHA-dependent interaction with CK2-phosphorylated Xrcc4. Analysis of the PNK FHA-Xrcc4 interaction revealed that the PNK FHA domain binds phosphopeptides with a unique selectivity among FHA domains. Disruption of the Xrcc4-PNK interaction in vivo is associated with increased radiosensitivity and slower repair kinetics of DSBs, in conjunction with a diminished efficiency of DNA end joining in vitro. Therefore, these results suggest a new role for Xrcc4 in the coordination of DNA end processing with DNA ligation.     

Anomalous electrophoretic migration of oligodeoxynucleotides with terminal OH groups: Applications for DNA exonuclease characterization

Oligodeoxynucleotides with a terminal OH group on both the 5′ and 3′ ends migrate anomalously in 23% polyacrylamide-7 m urea gels. This migration anomaly can be exploited to characterize nuclease digestion products. Thus, using specific substrates and the methods described, several types of DNA exonuclease activity can be readily distinguished.     

Characterization of an alpha-like DNA polymerase from Bombyx mori silkglands.

A soluble DNA polymerase has been purified near to homogeneity from Bombyx mori silkglands. The following characteristics were observed: high molecular weight (about 150 000 - 220 00); optimum pH about 8; inhibition by high salt concentrations, sulfhydryl-group blocking agents and polyamines; absence of nuclease activity; preference for magnesium as required divalent cation with all the efficient template-primers tested; and clear template-primer specificity, the purified enzyme being able to copy primed - polydeoxyribonucleotide templates [activated DNA, poly(dA).oligo(dT), poly(dA).oligo(rU)] but not polyribonucleotide chains [poly(rA).oligo(dT), poly(rA).oligo(rU)] in the presence of either Mg++ or MN++. Believed to represent the bulk of silkgland DNA polymerase activity, the purified soluble enzyme most resembles vertebrate DNA polymerases alpha when it is compared to other eukaryotic DNA polymerases as yet characterized.     

Preparation of ligation intermediates and related polynucleotide pyrophosphates

Unprotected oligonucleotides and oligodeoxynucleotides terminated with an unhindered 5′-phosphate group react with nucleoside 5′-phosphorimidazolides in aqueous solution to give ‘capped’ pyrophosphates in at least 70% yield. If adenosine 5′-phosphorimidazolide is used as a substrate in the reaction, ligase intermediates are obtained as products.     

Structure of the RNA portion of the RNA-linked DNA pieces in bacteriophage T4-infected Escherichia coli cells.

A method for the isolation of the RNA portion of RNA-linked DNA fragments has been developed. The method capitalizes on the selective degradation of DNA by the 3′ to 5′ exonuclease associated with bacteriophage T4 DNA polymerase. After hydrolysis of the DNA portion, the RNA of RNA-linked DNA is recovered mostly as RNA tipped with a deoxyribomononucleotide and a small fraction as pure RNA. On the other hand, the 5′ ends of RNA-free DNA are recovered mostly as dinucleotides and a small fraction as mononucleotides.Using this method, we have isolated the primer RNA for T4 phage DNA synthesis. Nascent short DNA pieces were isolated from T4 phage-infected Escherichia coli cells and the 5′ ends of the pieces were dephosphorylated and then phosphorylated with polynucleotide kinase and [γ-³²P]ATP. After selective degradation of the DNA portions, [5′-³²P]oligoribonucleotides (up to pentanucleotide) were obtained with covalently bound deoxymononucleotides at their 3′ ends. More than 40% of the oligoribonucleotides isolated were pentanucleotides with pApC at the 5′-terminal dinucleotide. The 5′-terminal nucleotide of the tetraribonucleotides was AMP, but that of the shorter chains was not unique. The pentanucleotide could represent the intact primer RNA for T4 phage DNA synthesis.     

Non-dependence on native structure of pig liver pyruvate kinase when used as a substrate for cyclic 3',5'-AMP-stimulated protein kinase.

Alkali-inactivated pig liver pyruvate kinase, type L, and a cyanogen bromide fragment from the same enzyme were shown to be phosphorylated by (³²P)ATP and cyclic 3′,5′-AMP-stimulated protein kinase. In both cases the rate of phosphorylation was higher than with the native enzyme. Pyruvate kinases types A and M were not phosphorylated under the same conditions. From the ³²P-labelled cyanogen bromide fragment (³²P)phosphorylserine was isolated. The electrophoretic pattern of (³²P)phosphopeptides obtained on partial acid hydrolysis of the fragment indicated that the phosphorylated site of the fragment was identical with that of the native pyruvate kinase.     

5-Methylthioribose kinase activity in plants

The presence of a previously unidentified plant enzyme, 5-methylthioribose kinase, has been demonstrated to exist in cell-free extracts from several fruit tissues. The enzyme catalyzes the ATP-dependent phosphorylation of 5-methylthioribose to 5-methylthioribose-1-phosphate. Enzyme activity has been found in avocado, pear, apple, strawberry and tomato tissues. The significance of the presence of this enzyme in relation to ethylene biosynthesis is discussed.     

ATM-mediated phosphorylation of polynucleotide kinase/phosphatase is required for effective DNA double-strand break repair

The cellular response to double-strand breaks (DSBs) in DNA is a complex signalling network, mobilized by the nuclear protein kinase ataxia-telangiectasia mutated (ATM), which phosphorylates many factors in the various branches of this network. A main question is how ATM regulates DSB repair. Here, we identify the DNA repair enzyme polynucleotide kinase/phosphatase (PNKP) as an ATM target. PNKP phosphorylates 5'-OH and dephosphorylates 3'-phosphate DNA ends that are formed at DSB termini caused by DNA-damaging agents, thereby regenerating legitimate ends for further processing. We establish that the ATM phosphorylation targets on human PNKP-Ser 114 and Ser 126-are crucial for cellular survival following DSB induction and for effective DSB repair, being essential for damage-induced enhancement of the activity of PNKP and its proper accumulation at the sites of DNA damage. These findings show a direct functional link between ATM and the DSB-repair machinery.     

5′ Phosphorylation of DNA in mammalian cells: Identification of a polymin P-precipitable polynucleotide kinase

Proteins that catayze 5′ phosphorylation of an oligodeozyribonucleotide substrate can be fractionated by polumin P treatment of whole cell extrats of calf thymus glands. Anion exchange chromatography on Q-Sepharose revealed three separable peaks of activity in the polymin P supernatant fraction, and one peak of activity in the Polymin P pellet fraction. The latter activity, polymin P-precipitable polynucleotide kinase (PP-PNK), was futher purified with a 1,500-fold increase of specific activity compared to the crude polymin fraction. Oligonucleotides, a dephosphorylated 2.9-kb EcoRI fragment, and poly(A) were phosphorylated by the enzyme preparation, but thymidine 3′monophosphate was not a substrate. PP-PNk preparations exhibited an apparent K_M of 52 μM for ATP and 8 μM for oligo dT₂₅. The enzyme preparation displayed no detectable 3′ phosphatase or cyclic 2′,3′ phosphohydrolase activities. The sedimentation coefficient of the PP-Pnk activity was 3.85 as determined by sucrose density gradient analysis; the stokes radius was 45 Å, leading to an estimated molecular mass of 72 kDa. The enzyme had a pH optimun in the neutral to alkaline range in several buffer systems and is distinct from the DNA Kinase with an acidic pH optimum previously described in calf thymus. © Wiley-Liss, Inc.     

Biomolecular response of oxanine in DNA strands to T4 polynucleotide kinase, T4 DNA ligase, and restriction enzymes

Oxanine (Oxa), generated from guanine (Gua) by NO- or HNO₂-induced nitrosative oxidation, has been thought to cause mutagenic problems in cellular systems. In this study, the response of Oxa to different enzymatic functions was explored to understand how similarly it can participate in biomolecular reactions compared to the natural base, Gua. The phosphorylation efficiency of the T4 polynucleotide kinase was highest when Oxa was located on the 5′-end of single stranded DNAs compared to when other nucleobases were in this position. The order of phosphorylation efficiency was as follows; Oxa > Gua > adenine (Ade) ∼ thymine (Thy) > cytosine (Cyt). Base-pairing of Oxa and Cyt (Oxa:Cyt) between the ligation fragment and template was found to influence the ligation performance of the T4 DNA ligase to a lesser degree compared to Gua:Cyt. In addition, EcoRI and BglII showed higher cleavage activities on DNA substrates containing Oxa:Cyt than those containing Gua:Cyt, while BamHI, HindIII and EcoRV showed lower cleavage activity; however, this decrease in activity was relatively small.     

Loss of reiterated DNA sequences during serial passage of human diploid fibroblasts

A specific family of tandemly repeated DNA sequences was found to diminish in the human genome after serial passage of three strains of diploid fibroblasts. Eco RI restriction fragments of 340 and 680 bp were significantly reduced in quantity at late passage as determined by autoradiography of 14C-DNA and also by ethidium bromide fluorescence. The reduction in these closely related DNA sequences was confirmed by saturation hybridization to excess 14H-RNA transcribed from a homogeneous restriction fragment recleaved from the 340 bp DNA. The maximal fraction of DNA hybridizing to the 3H-RNA probe declined by 33-50% over 21-41 population doublings. Divergence and/or methylation of such sequences could not account for these results since the thermal stability of cRNA:DNA duplexes actually increased by 0.3 degrees C at late passage. Total highly repetitive sequences assayed by reassociation kinetics were also substantially reduced at late passage, implying that depletion may be common to many repeat families in DNA. The denaturation temperature for such rapidly reassociated duplexes again increased slightly at late passage, possibly reflecting the minor decreases in DNA methylation which were detected in two of the cell strains. Karyotype analyses demonstrated that over 95% euploidy was maintained, with no specific chromosome loss and no visible deletions at late passage. The depletion of reiterated sequences during repeated cell division is thus attributed to numerous small DNA deletions, which may arise from unequal recombination coupled with selection or from a nonreciprocal mechanism such as excision.     

Metastasis suppressor NME1 promotes non-homologous end joining of DNA double-strand breaks

NME1 (also known as NM23-H1) was the first identified tumor metastasis suppressor, which has been reported to link with genomic stability maintenance and cancer. However its underlying mechanisms are still not fully understood. Here we find that NME1 is required for non-homologous end joining (NHEJ) of DNA double-strand breaks (DSBs). Mechanistically, NME1 re-localizes to DNA damage sites in a Ku-XRCC4-dependent manner, and regulates downstream LIG4 recruitment and end joining efficiency. Furthermore, we show that the 3′-5′ exonuclease activity of NME1 is critical for its function in NHEJ. Taken together, our findings identify NME1 as a novel NHEJ factor, and reveal how this metastasis suppressor promotes genome stability.     

Characteristics of enzymatic DNA methylation in cultured cells of human and hamster origin,and the effect of DNA replication inhibition

In mammalian cells, inhibitors of DNA replication have been shown to induce chromosomal aberrations, cell death and changes in gene control. Inhibition of DNA synthesis has been reported to induce hypermethylation of mammalian DNA (enzymatic postsynthetic formation of 5-methylcytosine). These 5-methylcytosines in mammalian DNA have variously been suggested to be important in gene control, DNA repair, and control of DNA replication. In establishing the normal characteristics of enzymatic DNA methylation, we have demonstrated that, in asynchronously growing cells of both human and hamster origin, some cytosine methylation is delayed for several hours after strand synthesis and that this delayed methylation is completed before the DNA strand acts as a template for DNA replication in the next S-phase. Further, in testing whether the deleterious effects on mammalian cells of DNA synthesis inhibitors might be mediated via changes in enzymatic DNA methylation, we have found, contrary to some previous findings, no evidence for any change in the level of DNA methylation in DNA strands synthesized during 6 h of treatment of cells of human origin with high concentrations of four different inhibitors of DNA replication or during the 4 h following the 6 h treatment. Almost totally blocking DNA replication had no effect on the small amount of delayed methylation of DNA strands not involved in semi-conservative replication during the time of the experiment. This lack of effect on DNA methylation was obtained when the labelling medium contained normal, undialysed serum. In contrast, if dialysed serum was used in the labelling medium in order to maximize l-[Me-³H]methionine utilization, highly variable, totally irreproducible patterns of apparent DNA hypermethylation were obtained.