Types Of DNA Synthesis Research Articles

Fractured genes: a novel genomic arrangement involving new split inteins and a new homing endonuclease family

Inteins are genetic elements, inserted in-frame into protein-coding genes, whose products catalyze their removal from the protein precursor via a protein-splicing reaction. Intein domains can be split into two fragments and still ligate their flanks by a trans-protein-splicing reaction. A bioinformatic analysis of environmental metagenomic data revealed 26 different loci with a novel genomic arrangement. In each locus, a conserved enzyme coding region is broken in two by a split intein, with a free-standing endonuclease gene inserted in between. Eight types of DNA synthesis and repair enzymes have this ‘fractured’ organization. The new types of naturally split-inteins were analyzed in comparison to known split-inteins. Some loci include apparent gene control elements brought in with the endonuclease gene. A newly predicted homing endonuclease family, related to very-short patch repair (Vsr) endonucleases, was found in half of the loci. These putative homing endonucleases also appear in group-I introns, and as stand-alone inserts in the absence of surrounding intervening sequences. The new fractured genes organization appears to be present mainly in phage, shows how endonucleases can integrate into inteins, and may represent a missing link in the evolution of gene breaking in general, and in the creation of split-inteins in particular.

Multiple subphases of DNA repair and poly(ADP-ribose) synthesis in Chinese hamster ovary (CHO-K1) cells

The two types of DNA synthesis as well as poly(ADP-ribose) biosynthesis were measured simultaneously in synchronized intact populations of CHO cells throughout the duration of S phase. Naturally occurring DNA fragmentation was detected by random primed oligonucleotide synthesis (ROPS assay). Fractions of synchronous cell populations were obtained by counterflow centrifugal elutriation. By gradually increasing the resolution of centrifugal elutriation multiple non-overlapping repair and replication peaks were obtained. The elutriation profile of DNA repair peaks corresponded to the DNA fragmentation pattern measured by ROPS assay. The number and position of poly(ADP-ribose) peaks during S phase resembled those seen in the DNA replication profile. Our results indicate that PAR synthesis is coupled to DNA replication serving the purpose of genomic stability.

Requirement of RAD5 and MMS2 for postreplication repair of UV-damaged DNA in Saccharomyces cerevisiae.

UV lesions in the template strand block the DNA replication machinery. Genetic studies of the yeast Saccharomyces cerevisiae have indicated the requirement of the Rad6-Rad18 complex, which contains ubiquitin-conjugating and DNA-binding activities, in the error-free and mutagenic modes of damage bypass. Here, we examine the contributions of the REV3, RAD30, RAD5, and MMS2 genes, all of which belong to the RAD6 epistasis group, to the postreplication repair of UV-damaged DNA. Discontinuities, which are formed in DNA strands synthesized from UV-damaged templates, are not repaired in the rad5Delta and mms2Delta mutants, thus indicating the requirement of the Rad5 protein and the Mms2-Ubc13 ubiquitin-conjugating enzyme complex in this repair process. Some discontinuities accumulate in the absence of RAD30-encoded DNA polymerase eta (Poleta) but not in the absence of REV3-encoded DNA Polzeta. We concluded that replication through UV lesions in yeast is mediated by at least three separate Rad6-Rad18-dependent pathways, which include mutagenic translesion synthesis by Polzeta, error-free translesion synthesis by Poleta, and postreplication repair of discontinuities by a Rad5-dependent pathway. We suggest that newly synthesized DNA possessing discontinuities is restored to full size by a "copy choice" type of DNA synthesis which requires Rad5, a DNA-dependent ATPase, and also PCNA and Poldelta. The possible roles of the Rad6-Rad18 and the Mms2-Ubc13 enzyme complexes in Rad5-dependent damage bypass are discussed.

Relationship of repair and replicative DNA synthesis to cell cycle in Chinese hamster ovary (CHO-K1) cells.

To strengthen the causal association between repair and replicative DNA synthesis, we have simultaneously measured the two types of DNA synthesis in a cell cycle-dependent manner. Synchrony was obtained by counterflow centrifugal elutriation of logarithmic-phase Chinese hamster ovary (CHO) cells kept in suspension cultures. A comparison of cell cycle profiles of ATP-dependent replicative and ATP-independent repair synthesis in permeable cells shows opposite trends. The rates of repair synthesis and replication are inversely correlated.

Modifying effects of benzyl isothiocyanate and benzyl thiocyanate on DNA synthesis in primary cultures of rat hepatocytes.

The effects of benzyl isothiocyanate (BITC) and benzyl thiocyanate (BTC) on two types of DNA synthesis were examined in hepatocyte primary cultures (HPC). Male F344 rats were fed BITC- or BTC-containing diets at a concentration of 400 p.p.m. Using hepatocytes isolated from these rats, DNA repair was measured by unscheduled DNA synthesis (UDS) for some genotoxic carcinogens, e.g. 2-acetylaminofluorene (AAF), methylazoxymethanol (MAM) acetate, 9,10-dimethyl-1,2-benzanthracene (DMBA) and diethylnitrosamine (DEN), and compared with that in the hepatocytes from rats without BITC or BTC treatment. Replicative DNA synthesis (RDS) was also evaluated in the hepatocytes of rats with or without thiocyanate treatment. Both BITC and BTC reduced UDS elicited by these carcinogens. The level of RDS in the hepatocytes of rats exposed to BITC or BTC was markedly lower than in the cells of rats without BITC or BTC exposure. These results indicate that in vivo exposure to BITC and BTC suppressed carcinogen-induced genotoxicity and cell proliferative activity and suggest that this assay may prove useful in detecting chemopreventive agents for cancer and in investigating the properties of carcinogenesis modifiers.

Effects of in vivo exposure to antineoplastic drugs on DNA repair and replication in human lymphocytes

In peripheral blood lymphocytes of 12 nurses and 3 patients exposed to antineoplastic drugs we determined the ability to repair DNA after UV irradiation and DNA replicative synthesis after stimulation by PHA. In nurses the levels of unscheduled DNA synthesis and DNA replication were not different than in a control group, whereas in patients significant changes were observed during and after chemotherapy in the level of both types of DNA synthesis.

Characteristics of the major DNA polymerases found during early and late maize germination

Two types of DNA synthesis have been detected during maize germination (0–24 h). To determine if the difference in these two types of synthesis was due to the presence of distinct DNA polymerases, we partially isolated and characterized the enzymes present at 3 (early) and 24 (late) h of germination. The material used was embryo axes. The result indicates that at both 3 and 24 h, enzymes are similar with regard to optimum pH and temperature, absolute requirement of Mg2+ (12 mM), and stimulation by KCl (50 mM). They are also equally inhibited by N-ethylmaleimide and cytosine-β-D-arabinofuranoside. These facts would classify the enzymes as alpha type; however, the enzymes differ in their sensitivity to aphidicolin and the degree of purification. The nature of the enzymes is discussed.

DNA synthesis in vitro in human fibroblast preparations

When confluent cultures of human fibroblasts were ultraviolet irradiated and either permeabilized or lysed, three types of DNA synthesis were subsequently observed during incubation in vitro: (A) a low level of DNA replication, which ceased after 15–30 min incubation at 37°C; (B) radiation-dependent reparative gap-filling, which also ceased after 15 min at 37°C; and (C) radiation-independent DNA synthesis, which was not semiconservative and proceeded at a linear rate for 1 h at 37°C. Normal and xeroderma pigmentosum fibroblasts displayed different rates of radiation-dependent reparative gap-filling after lysis but similar rates of radiation-independent DNA synthesis. The rates of DNA replication and radiation-independent DNA synthesis were less in the permeable cell system than in the lysed cell system, whereas radiation-dependent reparative gap-filling was the same in both. Preparations of permeable and lysed cells activated radiation-dependent reparative gap-filling at about 15% of the rate estimated for intact cells. No radiation-dependent DNA strand breaks, as assayed by alkaline elution, were observed in the lysed cell preparation. Some radiation-dependent breaks were observed in the permeable cell preparation, but radiation-dependent DNA breakage was less than that seen in intact cells. This inability to incise DNA at damaged sites could account for the low rate of activation of reparative gap-filling in vitro. DNA strand breaks were produced in fibroblast preparations nonspecifically during lysis or permeabilization and incubation in vitro, and this breakage of DNA probably was responsible for the radiation-independent DNA synthesis.

A simplified method for measuring scheduled and unscheduled DNA synthesis in human fibroblasts

A simplified method for the rapid measurement of scheduled and unscheduled DNA synthesis in cultures of human fibroblasts is described. The use of cultures in which DNA synthesis is blocked by hydroxyurea (HU) together with those which are not blocked allows an estimation of the effect of chemicals on both types of DNA synthesis. This facilitates the interpretation of results and the selection of an optimum range of doses. Some results obtained with carcinogens and non-carcinogens are presented of the compounds so far tested. The methodology here described compares well in its validity with other more difficult and time consuming methods.

Synthesis of Herpes Simplex Virus DNA in Preparations of Chromatin from Infected Cell Nuclei

Chromatin prepared from cells infected with Herpes simplex virus type 1 or type 2 can synthesize both virus and cell DNA in vitro. The rate of synthesis is comparable to that of isolated whole nuclei. Incorporation is limited, and both cell and virus DNA synthesis are sensitive to the presence of virus-specific antiserum and phosphonoacetate. In chromatin from cells infected with a phosphonoacetate resistant virus mutant, both types of DNA synthesis are resistant to the presence of the inhibitor.

Different types of DNA -synthesis and their importance in cancer growth and therapy.

A review of the present state of knowledge about the mechanisms by which DNA replicates shows that we have definite knowledge that replication begins at a well-defined point (initiation time) in a specific region of DNA (initiation site), and that the cycle of replication hardly ever begins at a random position or at any other than the programmed time.KeywordsFusic AcidCancer TransformationCancer CausationRepair ReplicationPrereplicative PhaseThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Involvement of DNA polymerases I and III in the replication of bacteriophage M-13.

M‐13 replication was investigated in Escherichia coli mutants deficient in DNA polymerase I (polA) and DNA polymerase III (dnaEts). The following results were obtained: M‐13 infection is abortive in both polA− and dnaEts mutants at 42°C. The conversion of the parental single‐stranded DNA to double‐stranded replicative forms (RF) is only slightly affected by the dnaE mutation. However, no parental double strands are formed in the polA− dnaEts double mutant at the restrictive temperature, indicating a possible involvement of DNA polymerase I in this process. The replication of the replicative‐form molecules as well as the synthesis of progeny single strands are inhibited by thermal inactivation of the dnaE function implying a requirement of DNA polymerase III in both types of DNA synthesis. The presence of the polA mutation leads to an accumulation of open‐circular replicative forms. DNA polymerase I might, therefore, also be involved in the filling of gaps in the newly synthesized DNA molecules.

On the types of DNA synthesis in rabbit macrophages

On the types of DNA synthesis in rabbit macrophages

Radiation-stimulated DNA synthesis in cultured mammalian cells.

A type of DNA synthesis in mammalian cells that is stimulated by ultraviolet light has been studied by means of radioautography and density gradient centrifugation. The characteristics of this synthesis are: (a) it is not semiconservative; (b) it is enhanced by the presence of 5-bromodeoxyuridine in the DNA molecule; (c) the degree of stimulation is dose dependent; (d) there is less variability in the rate of incorporation of H(3)-thymidine during this synthesis than during normal DNA synthesis; (e) it occurs in cells that are not in the normal DNA synthesis phase (G(1) and G(2) cells). This kind of synthesis has been found in cultured cell lines from five different species; however, in some strains, the presence of bromouracil in the DNA is required before it can be demonstrated by radioautography.