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At least 19 recordsLinked to original sources
PubMed1979

Metabolism of Okazaki fragments during simian virus 40 DNA replication.

Essentially all of the Okazaki fragments on replicating Simian virus 40 (SV40)DNA could be grouped into one of three classes. Class I Okazaki fragments (about 20%) were separated from longer nascent DNA chains by a single phosphodiester bond interruption (nick) and were quantitatively identified by treating purified replicating DNA with Escherichia coli DNA ligase and then measuring the fraction of Okazaki fragments joined to longer nascent DNA chains. Similarly, class II Okazaki fragments (about 30%) were separated by a region of single-stranded DNA template (gap) that could be filled and sealed by T4 DNA polymerase plus E. coli DNA ligase, and class III fragments (about 50%) were separated by RNA primers that could be removed with E. coli DNA olymerase I, allowing the fragments to be joined with E. coli DNA ligase. These results were obtained with replicating SV40 DNA that had been briefly labeled with radioactive precursors in either intact cells or isolated nuclei. When isolated nuclei were further incubated in the presence of cytosol, all of the Okazaki fragments were converted into longer DNA strands as expected for intermediates in DNA synthesis. However, when washed nuclei were incubated in the abscence of cytosol, both class I and class II Okazaki fragments accumulated despite the excision of RNA primers: class III Okazaki fragments and RNA-DNA covalent linkages both disappeared at similar rates. These data demonstrate the existence of RNA primers in whole cells as well as in isolated nuclei, and identify a unique gap-filling step that is not simply an extension of the DNA chain elongation process concomitant with the excision of RNA primers. One or more factos found in cytosol, in addition to DNA polymerase alpha, are specifically involved in the gap-filling and ligation steps. The sizes of mature Okazaki fragments (class I) and Okazaki fragments whose synthesis was completed by T4 DNA polymerase were measured by gel electrophoresis and found to be broadly distributed between 40 and 290 nucleotides with an average length of 135 nucleotides. Since 80% and 90% of the Okazaments does not occur at uniformly spaced intervals along the DNA template. During the excision of RNA primers, nascent DNA chains with a single ribonucleotide covalently attached to the 5' terminus were identified as transient intermediates. These intermediates accumulated during excision of RNA primers in the presence of adenine 9-beta-D-arabinoside 5'-triphosphate, and those Okazaki fragments blocked by RNA primers (class III) were found to have originated the farthest from the 5' ends of long nascent DNA strands. Thus, RNA primers appear to be excised in two steps with the second step, removal of the final ribonucleotide, being stimulated by concomitant DNA synthesis. These and other data were used to construct a comprehensive metabolic pathway for the initiation, elongation, and maturation of Okazaki fragments at mammalian DNA replication forks.

DNA Ligases↗
PubMed2025

The DNA replication checkpoint limits Okazaki fragment accumulation to protect and restart stalled forks.

Understanding how DNA replication forks stall and restart and how the DNA replication checkpoint prevents irreversible fork collapse in molecular detail are crucial for understanding how cells maintain stable genomes and how they prevent the genetic instability that drives cancer. Here, we describe the reconstitution of fork stalling and restart with purified budding yeast proteins. After nucleotide depletion, leading-strand DNA synthesis quickly stops but CMG helicase continues to unwind, and Okazaki fragments continue to initiate on the lagging strand. Incomplete Okazaki fragments sequester PCNA, RFC, and DNA polymerases δ and ε, which prevents normal DNA synthesis restart and exposes nascent DNA to nuclease attack. The DNA replication checkpoint restrains fork progression, which limits this sequestration, protecting stalled forks from collapse and ensuring restart.

DNA Replication↗
PubMed1978

Formation of Okazaki fragments in polyoma DNA synthesis caused by misincorporation of uracil.

When dUTP replaced dTTP during polyoma DNA replication in isolated cell nuclei, radioactivity from labeled deoxynucleoside triphosphates was almost exclusively recovered in very short Okazaki fragments and incorporation ceased after a short time. Addition of uracil, a known inhibitor of the enzyme uracil-DNA glycosidase (Lindahl et al., 1977), increased total synthesis and shifted the incorporation to longer progeny strands. The presence of as little as 2.5% of dUTP in a dTTP-containing system gave a distinct increase in isotope incorporation into Okazaki pieces accompanied by a corresponding decrease in longer strands. This effect was reversed completely by uracil. The short strands formed from dUTP could be chased efficiently into long strands. Our results suggest that dUTP can be incorporated in place of dTTP into polyoma DNA, and that polyoma-infected nuclei, similar to E. coli (Tye et al., 1977), contain an excision-repair system which by removal of uracil causes strand breakage and under certain circumstances may contribute to the formation of Okazaki fragments.

DNA, Viral↗
PubMed2015

Okazaki fragment maturation involves α-segment error editing by the mammalian FEN1/MutSα functional complex.

During nuclear DNA replication, proofreading-deficient DNA polymerase α (Pol α) initiates Okazaki fragment synthesis with lower fidelity than bulk replication by proofreading-proficient Pol δ or Pol ε. Here, we provide evidence that the exonuclease activity of mammalian flap endonuclease (FEN1) excises Pol α replication errors in a MutSα-dependent, MutLα-independent mismatch repair process we call Pol α-segment error editing (AEE). We show that MSH2 interacts with FEN1 and facilitates its nuclease activity to remove mismatches near the 5' ends of DNA substrates. Mouse cells and mice encoding FEN1 mutations display AEE deficiency, a strong mutator phenotype, enhanced cellular transformation, and increased cancer susceptibility. The results identify a novel role for FEN1 in a specialized mismatch repair pathway and a new cancer etiological mechanism.

Animals↗
PubMed1977

Transient accumulation of Okazaki fragments as a result of uracil incorporation into nascent DNA.

Strains of Escherichia coli with a mutation in the sof (dnaS) locus show a higher than normal frequency of recombination (are hyper rec) and incorporate label into short (4-5S) DNA fragments following brief [3H]thymidine pulses [Konrad and Lehman, Proc. Natl. Acad. Sci. USA 72, 2150 (1975)]. These mutant strains have now been found to be defective in deoxyuridinetriphosphate diphosphohydrolase (dUTPase; deoxyuridinetriphosphatase, EC 3.6.1.23), the enzyme that catalyzes the hydrolysis of dUTP to dUMP and PPi. Reversion of one sof- mutation to sof+ restores dUTPase activity and abolishes the accumulation of labeled 4-5S DNA fragments. Mutants initially isolated as defective in dUTPase (dut-) are also hyper rec and show transient accumulation of short DNA fragments. Both the sof and dut mutations are located at 81 min on the E. coli map, closely linked to the pyrE locus. The sof and dut loci thus appear to be identical. A decrease in dUTPase as a consequence of a sof or dut mutation may result in the increased incorporation of uracil into DNA. Rapid removal of the uracil by an excision-repair process could then lead to the transient accumulation of short DNA fragments. It is possible that at least a portion of the Okazaki fragments seen in wild-type cells may originate in this way.

Chromosome Mapping↗
PubMed1978

Mutator activity of a short Okazaki fragment mutant of Escherichia coli.

A mutant of Escherichia coli (sof) which was previously shown to have increased recombination frequency, to produce abnormally short "Okazaki fragments," and to be deficient in deoxyuridine triphosphatase has now been found also to possess mutator activity for several genes; point mutation rates and deletion rates are affected. The mutational stimulation effects are consistent with the hypothesis that incorporation of uracil into DNA is directly or indirectly responsible for the observed mutator activity.

DNA, Bacterial↗
PubMed2026

DNA polymerase I: structure, activity, and function in bacterial DNA replication and repair.

Faithful replication and repair of the genome are essential processes for all life. Genome maintenance is coordinated by a complex suite of proteins, with bacteria evolving intricate systems despite their relatively simplistic genomes. DNA polymerases are a key class of proteins that mediate genome maintenance. DNA polymerases are all capable of extending nascent strands of DNA but contribute to DNA replication and repair in distinct ways depending on their active site and substrate specificity. The first discovered polymerase, bacterial DNA polymerase I (Pol I), has long been considered the primary enzyme responsible for Okazaki fragment maturation and resynthesis in many DNA repair pathways. These conclusions derive primarily from studies using the gram-negative bacterium, Escherichia coli. Given that some bacterial lineages diverged from E. coli over a billion years ago, these assumptions may not account for evolution in functional diversity. In this review, we examine the structural features of bacterial Pol I and discuss how each of its distinct enzymatic activities contribute to genome maintenance. Throughout, we introduce differences that have been discovered between gram-negative and gram-positive species and explore how activity differences may translate to functional adaptations in replication or repair. We focus on evidence from gram-positive bacteria, particularly Bacillus subtilis and Geobacillus stearothermophilus, that challenges the universality of Pol I's functions and reveals lineage-specific adaptations in replication and repair mechanisms. By synthesizing historical perspectives with recent discoveries, this review underscores both the importance of Pol I and the evolutionary diversification of Pol I in bacterial DNA metabolism.

Bacterial DNA replication↗
PubMed1978

Uracil incorporation: a source of pulse-labeled DNA fragments in the replication of the Escherichia coli chromosome.

Uracil is incorporated into newly synthesized DNA by mutants of Escherichia coli with reduced levels of dUTPase (dUTP nucleotidohydrolase; EC 3.6.1.23). Excision-repair of the incorporated uracil results in the generation of labeled DNA fragments that appear after brief pulses with [(3)H]thymidine [Tye, B-K., Nyman, P.-D., Lehman, I. R., Hochhauser, S. & Weiss, B. (1977) Proc. Natl. Acad. Sci. USA 74, 154-157]. Uracil is also incorporated into the newly synthesized DNA of strains of E. coli that contain normal levels of dUTPase. DNA fragments generated by the postreplication excision-repair of uracil may therefore contribute to the pool of nascent DNA (Okazaki) fragments that normally appear in wild-type strains. Discontinuous DNA replication has been examined in the absence of uracil excision by comparing Okazaki fragments in strains that are defective in DNA polymerase I (polA(-)) and polA(-) strains that are also defective in uracil N-glycosidase, an enzyme required for the excision-repair of uracil in DNA (polA(-)ung(-)). Little or no difference was detected in the level of Okazaki fragments in the polA(-) strain as compared with the polA(-)ung(-) strain. Thus, the uracil-induced cleavage of DNA cannot be the sole mechanism for the generation of Okazaki fragments. Mutants that are defective both in dUTPase and in uracil N-glycosidase incorporate uracil into their DNA with a high frequency (up to 1 per 100 nucleotides). These uracil residues, once incorporated, persist in the DNA without an adverse affect on the growth of the cells.

Chromosomes↗
PubMed1979

[Methylation of newly synthesized DNA in mouse fibroblast culture].

After a 10 min- or more prolonged incubation of transformed mouse fibroblasts (L.-cells) with [3H]-thymidine or [3H-methyl]-methionine and a subsequent centrifugation of cell lysates in an alkaline sucrose gradient the DNA radioactivity is detected in long (28, 33 and 45S) and short (5, 13 and 18S) fragments. An increase in cell concentration in the cultural layer results in inhibition of 5S fragments linkage rather than in inhibition of their synthesis. The blocking of the Okazaki fragment linkage may be regarded as one of the inhibitory molecular mechanisms of cell depletion. Both in the case of normal and suppressed (by 99%) replication by arabofuranosylcytosine [3H]-thymidine and [3H-5-methyl] cytosine are detected in the Okazaki fragments (5S) as well as in some discrete lower molecular weight fractions (lesser than 5S) of newly synthesized DNA. Thus, replicative methylation of DNA in the fibroblasts occurs in the replicative fork during DNA synthesis and the functioning DNA methylase is an indispensable component of the replicative complex. The methylation of Okazaki fragments is non-chaotic and has a specificity other than that of total DNA. This may be due to the multiplicity and different specificity of nuclear DNA-methylases. Thus, there exist in animal cells replicative and post-replicative methylation of DNA, which may differ in the nature of substrates and enzymes, in specificity of recognizable sequences and in their functional significanse.

Animals↗
PubMed2025

DNA lesions can frequently precede DNA:RNA hybrid accumulation.

While DNA:RNA hybrids contribute to multiple genomic transactions, their unscheduled formation is a recognized source of DNA lesions. Here, through a suite of systematic screens, we rather observed that a wide range of yeast mutant situations primarily triggering DNA damage actually leads to hybrid accumulation. Focusing on Okazaki fragment processing, we establish that genic hybrids can actually form as a consequence of replication-born discontinuities such as unprocessed flaps or unligated Okazaki fragments. Strikingly, such "post-lesion" DNA:RNA hybrids neither detectably contribute to genetic instability, nor disturb gene expression, as opposed to "pre-lesion" hybrids formed upon defective mRNA biogenesis, e.g., in THO complex mutants. Post-lesion hybrids similarly arise in distinct genomic instability situations, triggered by pharmacological or genetic manipulation of DNA-dependent processes, both in yeast and human cells. Altogether, our data establish that the accumulation of transcription-born DNA:RNA hybrids can occur as a consequence of various types of natural or pathological DNA lesions, yet do not necessarily aggravate their genotoxicity.

Saccharomyces cerevisiae↗
PubMed1978

Size distribution and maturation of newly replicated DNA through the S and G2 phases of Physarum polycephalum.

The size distribution of newly made DNA and the dynamics of size maturation of progeny DNA molecules were studied in the synchronous S and G2 phases of Physarum polycephalum. Pulse labeling of DNA and analysis of the products on alkaline sucrose gradients showed that synthesis of primary replication units (which will also be referred to as "Okazaki" fragments) occurred throughout the S period. Pulse and pulse-chase experiments revealed a distinct pattern of size maturation. An apparently linear increase in molecular weight of progeny DNA molecules during the first hour of the S phase occurred at a rate of approximately 4-5 X 10(5) daltons per min at 26 degrees C, corresponding to the joining of 6-8 Okazaki fragments. The resulting 35-45S (1.1-2.2 X 10(7) daltons) DNA molecules may correspond to the Physarum "replicon." The further size increases of the newly made DNA appear to occur in steps, possibly reflecting a clustering of isochronous replicons along the chromatide. These observations are discussed with regard to mechanisms of DNA replication and size maturation.

Cell Cycle↗
PubMed1977

Semiconservative DNA replication in vitro. II. Replicative intermediates of mouse P-815 cells.

DNA chain growing during semiconservative replication was studied using both in vitro systems described in the preceding paper (preceding paper, ref 1) 3H-Labeled, 4-S Okazaki fragments synthesized in vivo just prior to permeabilization or lysis with Brij-58 were metabolically stable and quantitatively chased into high molecular weight DNA (20--100 S) during a subsequent incubation in vitro. Thus, DNA replication continued in vitro at the same growing points that were active in vivo. After a 20-s pulse at 30 degress C in vitro, more than 50% of incorporated radioactivity was found in the 4 S region of alkaline sucrose gradients suggesting a totally discontinuous mode of DNA chain growth. If the pulse were followed by a 1-min chase, 4-S molecules were converted into 6--12-S intermediates which upon continued incubation were joined with growing 20--100-S molecules (replicon-sized chains). Formation of all three classes of replicative intermediates, Okazaki fragments, 6--12-S intermediates, and 20--100-S molecules, occurred in vitro at least during the first 20 min. During this time, average rates of DNA chain growth and overall DNA synthesis were reduced to about the same extent, if compared to rates of intact cells. Thus, reduced chain growth rates appear to reflect primary deficiences of our in vitro systems, while initiation of replicative intermediates still occurs.

Animals↗
PubMed1978

Rates of DNA synthesis during the S-phase of HeLa cells.

The rates of DNA synthesis were determined for each of two consecutive journeys through S-phase by highly synchronized HeLa cells. Cells at various times after release from the metabolic block were pulsed with [3H]thymidine. The amount of radioactivity in whole cells, purified DNA, and Okazaki fragments provided indexes of the rates of DNA synthesis. Measurements of the average DNA content per cell by the diphenylamine method and the individual DNA content per cell by DNA:propidium fluorescence provided better estimations of the actual rates of DNA synthesis, independent of thymidine metabolism. Unsynchronized cells that had been pulsed with [3H]thymidine were sorted into early, middle, and late S-phase preparations for estimations of the amount of radioactivity per cell. There were differences in the rates predicted by each of the various methods. Rates estimated by fluorescence measurements of DNA content per cell, or by diphenylamine measurements of average DNA content per cell exhibited a pattern of an initial burst, followed by a decreased rate then a final burst. Similar patterns were obtained for the amount of radioactivity in Okazaki fragments, and in early, middle, and late S-phase cells separated from a log-phase culture by electronic cell sorting. Rates estimated by measurements of the amount of radioactivity in whole cells, and the specific activity of purified DNA exhibited a different pattern of an initial slow rate, followed by a maximal rate then a slow rate.

Cell Cycle↗
PubMed1977

Ficellomycin and feldamycin; inhibitors of bacterial semiconservative DNA replication.

The two peptide-like antibiotics ficellomycin and feldamycin impair semiconservative DNA replication but not DNA repair synthesis in bacteria. Specifically both antibiotics cause the accumulation of a 34S DNA species in toluenized Escherichia coli cells which lacks the capability of being integrated into larger DNA pieces and eventually the complete bacterial chromosome. Novobiocin, a known inhibitor of replicative DNA synthesis, was investigated for comparative purposes. The action of this latter antibiotic differs from the ones exerted by ficellomycin and feldamycin in the novobiocin appears to block an event associated with the initiation of Okazaki fragments. The fact that novobiocin impairs DNA gyrase suggests that this enzyme plays an essential role during the initiation of Okazaki pieces.

Anti-Bacterial Agents↗
PubMed1977

Method for the isolation of Escherichia coli mutants with enhanced recombination between chromosomal duplications.

A method is described for the isolation of Escherichia coli mutants that show increased recombination between a pair of chromosomal duplications. These "hyper-rec" mutants display a variety of secondary phenotypes. I have isolated a large number of hyper-rec mutants and found them useful in screening for mutants that accumulate labeled DNA fragments after short pulses with [3H]thymidine. The mutants so recovered include ones that are defective in deoxyribonucleic acid ligase, deoxyribonucleic acid polymerase I and its associated 5' yields 3' exonuclease, and a group of mutants, dnaS, that accumulate abnormally short Okazaki fragments. Evidence is presented that suggests that the lac-att80 segment of the chromosome cannot be inverted.

Chromosomes, Bacterial↗