DNA polymerase epsilon: the latest member in the family of mammalian DNA polymerases

DNA polymerase epsilon is a mammalian polymerase that has a tightly associated 3'----5' exonuclease activity. Because of this readily detectable exonuclease activity, the enzyme has been regarded as a form of DNA polymerase delta, an enzyme which, together with DNA polymerase alpha, is in all probability required for the replication of chromosomal DNA. Recently, it was discovered that DNA polymerase epsilon is both catalytically and structurally distinct from DNA polymerase delta. The most striking difference between the two DNA polymerases is that processive DNA synthesis by DNA polymerase delta is dependent on proliferating cell nuclear antigen (PCNA), a replication factor, while DNA polymerase epsilon is inherently processive. DNA polymerase epsilon is required at least for the repair synthesis of UV-damaged DNA. DNA polymerases are highly conserved in eukaryotic cells. Mammalian DNA polymerases alpha, delta and epsilon are counterparts of yeast DNA polymerases I, III and II, respectively. Like DNA polymerases I and III, DNA polymerase II is also essential for the viability of cells, which suggests that DNA polymerase II (and epsilon) may play a role in DNA replication.     

DNA polymerases from Chlamydomonas reinhardii. Further characterization, action of inhibitors and associated nuclease activities.

The properties of three DNA polymerase species A, B and C, purified from Chlamydomonas reinhardii were compared. DNA polymerases A and B have Km values with respect to deoxyribonucleoside triphosphates of 19 micron and 3 micron respectively. DNA polymerase A is most active with activated DNA, but will also use native DNA and synthetic RNA and DNA templates with DNA primers. DNA polymerase B is also most active with activated DNA, but will use denatured DNA and synthetic DNA templates. It is inactive with RNA templates. DNA polymerase B is completely inactive in the presence of 100 micron-heparin, which has no effect on DNA polymerase A activity. Heparin dissociates DNA polymerase B into subunits that are still catalytically active, but which heparin inhibited. DNA polymerase B possesses deoxyribonuclease activity that is inhibited by 5 micron-heparin, suggesting that the deoxyribonuclease is an integral part of the DNA polymerase moiety. DNA polymerase A is devoid of nuclease activity. DNA polymerase C is similar to DNA polymerase B in all these properties, though it is more active with RNA primers and has greater heat-sensitivity.     

Duplication of single stranded DNA catalyzed by calf thymus DNA polymerase alpha.

Purified calf thymus DNA polymerase alpha is inactive with native DNA as template and shows little activity with denatured DNA. DNA synthesis with denatured DNA as template is greatly stimulated by the addition of a nuclease which initially copurifies with DNA polymerase but is separated from the polymerase on DEAE-cellulose chromatography. A limit digest of nuclease treated native DNA which is then denatured is replicated 80-95%; extensive replication is also obtained with native DNA partially degraded by pancreatic DNase and then denatured. The product of the reaction with calf thymus nuclease-treated DNA as template is double-stranded DNA with a hairpin (looped back) structure.     

Regulation of the genes for E. coli DNA gyrase: Homeostatic control of DNA supercoiling

DNA gyrase is the bacterial enzyme responsible for converting circular DNA to a negatively supercoiled form. We show that the synthesis of DNA gyrase is itself controlled by DNA supercoiling; synthesis is highest when the DNA template is relaxed. The rates of synthesis in vivo of both the A and B subunits of DNA gyase are increased up to 10-fold by treatments that block DNA gyrase activity and decrease the supercoiling of intracellular DNA. Similarly, efficient synthesis of both gyrase subunits in a cell-free S-30 extract depends on keeping the closed circular DNA template in a relaxed conformation. The results suggest that DNA supercoiling in E. coli is controlled by a homeostatic mechanism. Synthesis of the RecA protein and several other proteins is also increased by treatments that relax intracellular DNA.     

There exists a distinct stage during mammalian DNA synthesis immediately after joining of replication intermediates.

We describe an approach, using alkaline cell lysis and digestion with nuclease S1, which permits to distinguish between newly ligated DNA and the DNA of mature chromatin. When cells with steady-state labelled DNA (mature DNA) are analyzed, the results show labelled "nucleosomal-sized" DNA. However, when DNA of cells pulse-labelled with thymidine for 45 seconds is examined one can detect only large DNA. The newly ligated DNA is not reduced to "nucleosomal-sized" DNA by nuclease S1. When the large DNA is denatured in formamide one can detect 10 kb DNA fragments. Furthermore in pulse-chase experiments there appear, after formamide-treatment, increasing amounts of "nucleosomal-sized" DNA with a parallel decrease in the amount of 10 kb DNA fragments. Hence the newly ligated, large, DNA differs from mature DNA and represents a distinct stage during DNA replication.     

DNA polymerase I is required for premeiotic DNA replication and sporulation but not for X-ray repair in Saccharomyces cerevisiae.

We have used a set of seven temperature-sensitive mutants in the DNA polymerase I gene of Saccharomyces cerevisiae to investigate the role of DNA polymerase I in various aspects of DNA synthesis in vivo. Previously, we showed that DNA polymerase I is required for mitotic DNA replication. Here we extend our studies to several stages of meiosis and repair of X-ray-induced damage. We find that sporulation is blocked in all of the DNA polymerase temperature-sensitive mutants and that premeiotic DNA replication does not occur. Commitment to meiotic recombination is only 2% of wild-type levels. Thus, DNA polymerase I is essential for these steps. However, repair of X-ray-induced single-strand breaks is not defective in the DNA polymerase temperature-sensitive mutants, and DNA polymerase I is therefore not essential for repair of such lesions. These results suggest that DNA polymerase II or III or both, the two other nuclear yeast DNA polymerases for which roles have not yet been established, carry out repair in the absence of DNA polymerase I, but that DNA polymerase II and III cannot compensate for loss of DNA polymerase I in meiotic replication and recombination. These results do not, however, rule out essential roles for DNA polymerase II or III or both in addition to that for DNA polymerase I.     

DNA ligase I and proliferating cell nuclear antigen form a functional complex

DNA ligase I is responsible for joining Okazaki fragments during DNA replication. An additional proposed role for DNA ligase I is sealing nicks generated during excision repair. Previous studies have shown that there is a physical interaction between DNA ligase I and proliferating cell nuclear antigen (PCNA), another important component of DNA replication and repair. The results shown here indicate that human PCNA enhances the reaction rate of human DNA ligase I up to 5-fold. The stimulation is specific to DNA ligase I because T4 DNA ligase is not affected. Electrophoretic mobility shift assays indicate that PCNA improves the binding of DNA ligase I to the ligation site. Increasing the DNA ligase I concentration leads to a reduction in PCNA stimulation, consistent with PCNA-directed improvement of DNA ligase I binding to its DNA substrate. Two experiments show that PCNA is required to encircle duplex DNA to enhance DNA ligase I activity. Biotin-streptavidin conjugations at the ends of a linear substrate inhibit PCNA stimulation. PCNA cannot enhance ligation on a circular substrate without the addition of replication factor C, which is the protein responsible for loading PCNA onto duplex DNA. These results show that PCNA is responsible for the stable association of DNA ligase I to nicked duplex DNA.     

DNA序列的“双符三阶”图形编码

DNA的图形编码是在几何意义下,在不同位置,用不同的标记符号及不同的方向线段,对DNA的序列进行编码．DNA图形编码相对于DNA的字符编码而言,具有直观、简明、形象和便于比较局部DNA序列的相似性等特点。在分析已知各类：DNA的图形表示模式的基础上,提出一种DNA序列的“双符三阶”图形编码,并以此对一些特异DNA编码序列进行分析。DNA图形编码与DNA字符编码呈一一对应关系,具有简便易行、编译方便、形象丰富、便于比较等优点。适用于DNA短序列的相似性检测与分析,在生物信息学上有一定的应用前景。     

Assaying DNA Damage in Hippocampal Neurons Using the Comet Assay

A number of drugs target the DNA repair pathways and induce cell kill by creating DNA damage. Thus, processes to directly measure DNA damage have been extensively evaluated. Traditional methods are time consuming, expensive, resource intensive and require replicating cells. In contrast, the comet assay, a single cell gel electrophoresis assay, is a faster, non-invasive, inexpensive, direct and sensitive measure of DNA damage and repair. All forms of DNA damage as well as DNA repair can be visualized at the single cell level using this powerful technique.The principle underlying the comet assay is that intact DNA is highly ordered whereas DNA damage disrupts this organization. The damaged DNA seeps into the agarose matrix and when subjected to an electric field, the negatively charged DNA migrates towards the cathode which is positively charged. The large undamaged DNA strands are not able to migrate far from the nucleus. DNA damage creates smaller DNA fragments which travel farther than the intact DNA. Comet Assay, an image analysis software, measures and compares the overall fluorescent intensity of the DNA in the nucleus with DNA that has migrated out of the nucleus. Fluorescent signal from the migrated DNA is proportional to DNA damage. Longer brighter DNA tail signifies increased DNA damage. Some of the parameters that are measured are tail moment which is a measure of both the amount of DNA and distribution of DNA in the tail, tail length and percentage of DNA in the tail. This assay allows to measure DNA repair as well since resolution of DNA damage signifies repair has taken place. The limit of sensitivity is approximately 50 strand breaks per diploid mammalian cell ^1,2. Cells treated with any DNA damaging agents, such as etoposide, may be used as a positive control. Thus the comet assay is a quick and effective procedure to measure DNA damage.     

Conjugal Deoxyribonucleic Acid Replication by Escherichia coli K-12: Stimulation in dnaB(ts) Donors by Minicells

R64-11(+) donor cells that are thermosensitive for vegetative DNA replication will synthesize DNA at the restrictive temperature when recipient minicells are present. This is conjugal DNA replication because it is R64-11 DNA that is being synthesized and there is no DNA synthesis if minicells that cannot be recipients of R64-11 DNA are used. The plasmid DNA present in the donor cells before mating is transferred to recipient minicells within the first 20 min of mating, but additional copies of plasmid DNA synthesized during the mating continue to be transferred for at least 90 min. However, the transfer of R64-11 DNA to minicells is not continuous because the plasmid DNA in minicells is the size of one R64-11 molecule or smaller, and there are delays between the rounds of plasmid transfer. DNA is synthesized in minicells during conjugation, but this DNA has a molecular weight much smaller than that of R64-11. Thus, recipient minicells are defective and are not able to complete the synthesis of a DNA strand complementary to the single-stranded R64-11 DNA received from the donor cell.     

Generation of supercoils in nicked and gapped DNA drives DNA unknotting and postreplicative decatenation

Due to the helical structure of DNA the process of DNA replication is topologically complex. Freshly replicated DNA molecules are catenated with each other and are frequently knotted. For proper functioning of DNA it is necessary to remove all of these entanglements. This is done by DNA topoisomerases that pass DNA segments through each other. However, it has been a riddle how DNA topoisomerases select the sites of their action. In highly crowded DNA in living cells random passages between contacting segments would only increase the extent of entanglement. Using molecular dynamics simulations we observed that in actively supercoiled DNA molecules the entanglements resulting from DNA knotting or catenation spontaneously approach sites of nicks and gaps in the DNA. Type I topoisomerases, that preferentially act at sites of nick and gaps, are thus naturally provided with DNA–DNA juxtapositions where a passage results in an error-free DNA unknotting or DNA decatenation.     

Intracellular Forms of Adenovirus DNA III. Integration of the DNA of Adenovirus Type 2 into Host DNA in Productively Infected Cells

KB cells productively infected with human adenovirus type 2 contain an alkalistable class of viral DNA sedimenting in a broad zone between 50 and 90S as compared to 34S for virion DNA. This type of DNA is characterized as viral by DNA-DNA hybridization. It is extremely sensitive to shear fragmentation. Extensive control experiments demonstrate that the fast-sedimenting viral DNA is not due to artifactual drag of viral DNA mechanically trapped in cellular DNA or to association of viral DNA with protein or RNA. Furthermore, the fast-sedimenting DNA is found after infection with multiplicities between 1 and 1,000 PFU/cell and from 6 to 8 h postinfection until very late in infection (24 h). Analysis in dye-buoyant density gradients eliminates the possibility that the fast-sedimenting viral DNA represents supercoiled circular molecules. Upon equilibrium centrifugation in alkaline CsCl density gradients, the fast-sedimenting viral DNA bands in a density stratum intermediate between that of cellular and viral DNA. In contrast, the 34S virion DNA isolated and treated in the same manner as the fast-sedimenting DNA cobands with viral marker DNA. After ultrasonic treatment of the fast-sedimenting viral DNA, it shifts to the density positions of viral DNA and to a lesser extent to that of cellular DNA. The evidence presented here demonstrates that the 50 to 90S viral DNA represents adenovirus DNA covalently integrated into cell DNA.     

Recognition of cisplatin-DNA interstrand cross-links by replication protein A

Replication protein A (RPA) is a heterotrimeric protein that is required for DNA replication and most DNA repair pathways. RPA has previously been shown to play a role in recognizing and binding damaged DNA during nucleotide excision repair (NER). RPA has also been suggested to play a role in psoralen DNA interstrand cross-link (ICL) repair, but a clear biochemical activity has yet to be identified in the ICL DNA repair pathways. Using HeLa cell extracts and DNA affinity chromatography, we demonstrate that RPA is preferentially retained on a cisplatin interstrand cross-link (ICL) DNA column compared with undamaged DNA. The retention of RPA on cisplatin intrastrand and ICL containing DNA affinity columns is comparable. In vitro electrophoretic mobility shift assays (EMSAs) using synthetic DNA substrates and purified RPA demonstrate higher affinity for cisplatin ICL DNA binding compared with undamaged DNA. The enhanced binding of RPA to the cisplatin ICL is dependent on the DNA length. As the DNA flanking the cisplatin ICL is increased from 7 to 21 bases, preferential RPA binding is observed. Fluorescence anisotropy reveals greater than 200-fold higher affinity to a cisplatin ICL containing 42-mer DNA compared with an undamaged DNA and a 3-4-fold higher affinity when compared with a cisplatin intrastrand damaged DNA. As the DNA length and stringency of the binding reaction increase, greater preferential binding of RPA to cisplatin ICL DNA is observed. These data are consistent with a role for RPA in the initial recognition and initiation of cisplatin ICL DNA repair.     

Three Size-Classes of Intracellular Adenovirus Deoxyribonucleic Acid

When human adenovirus type 2 or 12 infects cells, either productively or non-productively, three classes of viral deoxyribonucleic acid (DNA) are found within the cells: (i) viral DNA which cosediments with DNA extracted from infectious adenovirions at 31.3S for adenovirus type 2 and at 29.0S for adenovirus type 12, (ii) viral DNA which sediments at about 18S, and (iii) viral DNA which sediments at >45S and is apparently integrated into the cellular DNA. A precursor-product relationship is suggested as a working hypothesis; the intact viral DNA is hydrolyzed to slowly sedimenting DNA and the slowly sedimenting DNA is integrated into the cellular DNA. Both the parental and the newly synthesized viral DNA are altered by this route. The intact viral DNA within the cells apparently is cleaved into the slowly sedimenting DNA by a preformed enzyme.     

DNA依赖蛋白激酶研究进展

DNA依赖蛋白激酶由Ku异二聚体和DNA-PKcs组成,结合Ku蛋白后,DNA-PK激酶活性激活,DNA依赖蛋白激酶具有多功能性,参与DNA修复、基因重组以及复制、转录等多种细胞学过程.     

Heterologous deoxyribonucleic acid uptake and complexing with cellular constituents in competent Bacillus subtilis.

With competent cultures of Bacillus subtilis the uptake of Escherichia coli deoxyribonucleic acid (DNA) is about 50% that for homologous DNA. Uptake of phage T6 DNA, if any, is of the order of 7%, while nonglucosylated phage T6 (T6) DNA is taken up almost as effectively as homologous DNA. Both T6 and T4 DNA interfere only minimally with uptake of homologous DNA; by contrast, T6 DNA competes with homologous DNA as effectively as the latter itself. These results indicate that the glucose residues in the T-even phage DNA, located in the large groove of the DNA helix, reduce affinity for cellular receptors, leading to low binding of T6 DNA. The latter DNA is considerably less degraded by extracellular nucleases than homologous DNA, thus excluding enzymatic hydrolysis as the source of poor uptake. Affinity of DNA for competent cells was also evaluated by the formation, and detection in a CsCl density gradient, of complexes of DNA with cellular constituent(s). Such comlexes, similar to those previously observed with transforming DNA, are formed by E. coli DNA and T6 DNA; in reconstruction experiments the denatured forms of these same DNA samples form complexes when added to the cells before lysis. T6 DNA, on the other hand, does not form such a complex. The possible role of such complexes in transport of DNA to the cell interior is discussed.     

Stable integration of foreign DNA into the chromosome of the cyanobacterium Synechococcus R2

The blue-green alga, Synechococcus R2, is transformed to antibiotic resistance by chimeric DNA molecules consisting of Synechococcus R2 chromosomal DNA linked to antibiotic-resistance genes from Escherichia coli. Chimeric DNA integrates into the Synechococcus R2 chromosome by homologous recombination. The efficiency of transformation, as well as the stability of integrated foreign DNA, depends on the position of the foreign genes relative to Synechococcus R2 DNA in the chimeric molecule. When the Synechococcus R2 DNA fragment is interrupted by foreign DNA, integration occurs through replacement of chromosomal DNA by homologous chimeric DNA containing the foreign insert; transformation is efficient and the foreign gene is stable. Mutagenesis in some cases attends integration, depending on the site of insertion. Foreign DNA linked to the ends of Synechococcus R2 DNA in a circular molecule, however, integrates less efficiently. Integration results in duplicate copies of Synechococcus R2 DNA flanking the foreign gene and the foreign DNA is unstable. Transformation in Synechococcus R2 can be exploited to modify precisely and extensively the genome of this photosynthetic microorganism.     

Heterogeneity in premature senescence by oxidative stress correlates with differential DNA damage during the cell cycle

The development of cellular senescence both by replication and by oxidative stress is not homogenous in cultured primary human fibroblasts. To investigate whether this is due to the heterogeneity in the susceptibility of DNA in different phases of the cell cycle, we subjected synchronised cells to oxidative stress and examined the extent of DNA damage and its long-term effects on the induction of cellular senescence. Here, we first show marked heterogeneity in DNA damage as detected by markers of double strand breaks caused by oxidative stress in an asynchronous human fibroblast culture. Cell cycle synchronization followed by oxidative stress demonstrated that DNA in S-phase is most susceptible to oxidative stress whereas DNA in the quiescent phase is most resistant. DNA repair is an ongoing process after sensing DNA damage; reparable DNA damage is repaired even in cells that contain persistent DNA damage. The extent of persistent DNA damage is tightly correlated with permanent cessation of DNA replication and SA-beta-gal activity. Oxidative stress encountered by cells in S-phase resulted in more persistent DNA damage, more permanent cell cycle arrest and the induction of premature senescence.     

Structure and function of the DNA ligases encoded by the mammalian LIG3 gene

Among the mammalian genes encoding DNA ligases (LIG), the LIG3 gene is unique in that it encodes multiple DNA ligase polypeptides with different cellular functions. Notably, this nuclear gene encodes the only mitochondrial DNA ligase and so is essential for this organelle. In the nucleus, there is significant functional redundancy between DNA ligase IIIα and DNA ligase I in excision repair. In addition, DNA ligase IIIα is essential for DNA replication in the absence of the replicative DNA ligase, DNA ligase I. DNA ligase IIIα is a component of an alternative non-homologous end joining (NHEJ) pathway for DNA double-strand break (DSB) repair that is more active when the major DNA ligase IV-dependent pathway is defective. Unlike its other nuclear functions, the role of DNA ligase IIIα in alternative NHEJ is independent of its nuclear partner protein, X-ray repair cross-complementing protein 1 (XRCC1). DNA ligase IIIα is frequently overexpressed in cancer cells, acting as a biomarker for increased dependence upon alternative NHEJ for DSB repair and it is a promising novel therapeutic target.