Activation of the DNA replication checkpoint through RNA synthesis by primase

When DNA replication is inhibited during the synthesis (S) phase of the cell cycle, a signaling pathway (checkpoint) is activated that serves to prevent mitosis from initiating before completion of replication. This replication checkpoint acts by down-regulating the activity of the mitotic inducer cdc2-cyclin B. Here, we report the relation between chromatin structure and induction of the replication checkpoint. Chromatin was competent to initiate a checkpoint response only after the DNA was unwound and DNA polymerase alpha had been loaded. Checkpoint induction did not require new DNA synthesis on the unwound template strand but did require RNA primer synthesis by primase. These findings identify the RNA portion of the primer as an important component of the signal that activates the replication checkpoint.     

Uninterrupted MCM2-7 function required for DNA replication fork progression

Little is known about the DNA helicases required for the elongation phase of eukaryotic chromosome replication. Minichromosome maintenance (MCM) protein complexes have DNA helicase activity but have only been functionally implicated in initiating DNA replication. Using an improved method for constructing conditional degron mutants, we show that depletion of MCMs after initiation irreversibly blocks the progression of replication forks in Saccharomyces cerevisiae. Like the Escherichia coli dnaB and SV40 T antigen helicases, therefore, the MCM complex is loaded at origins before initiation and is essential for elongation. Restricting MCM loading to the G(1) phase ensures that initiation and elongation occur just once per cell cycle.     

TRANSCRIPTION IN VIVO OF DNA FROM BACTERIOPHAGE SP8

The DNA of bacteriophage SP8, when denatured, yields two components differing in buoyant density in cesium chloride gradients and separable by chromatography on a column of methylated bovine serum albumin and kieselguhr. The denser of the two strands (H) contains more pyrimidines and fewer purines than the lighter (L) strand. Only the H strand forms hybrids with the RNA synthesized by the infected host. The L strand is capable of annealing with complementary RNA synthesized in vitro with it as primer in reactions catalyzed by RNA polymerase. During the vegetative development of phage, host-specific messenger RNA is also synthesized.     

Cleaving DNA at any predetermined site with adapter-primers and class-IIS restriction enzymes

A four-component system has been designed that makes it possible to prepare a double-stranded (ds) DNA fragment; one fragment end is predesigned (by the use of a class-IIS restriction enzyme and adapter-primer), and the other end corresponds to any normal restriction cut. The system is composed of the phage M13mp7 single-stranded (ss) target DNA; the Fok I restriction enzyme; an oligodeoxynucleotide adapter-primer, which permits one to introduce Fok I cuts at any specified site in the target DNA; and DNA polymerase, which converts the ss target into a ds form ready for cloning. In this system, the oligodeoxynucleotide adapter-primer serves several purposes. The 5' hairpin ds domain of the adapter-primer contains the Fok I recognition site. Its 3' ss domain selects a complementary site on the target ss DNA, hybridizes with it to form the ds cleavage site, and serves as a primer to convert the ss M13mp7 target to ds DNA.     

Structure of the RNA polymerase domain of E. coli primase

All cellular organisms use specialized RNA polymerases called "primases" to synthesize RNA primers for the initiation of DNA replication. The high-resolution crystal structure of a primase, comprising the catalytic core of the Escherichia coli DnaG protein, was determined. The core structure contains an active-site architecture that is unrelated to other DNA or RNA polymerase palm folds, but is instead related to the "toprim" fold. On the basis of the structure, it is likely that DnaG binds nucleic acid in a groove clustered with invariant residues and that DnaG is positioned within the replisome to accept single-stranded DNA directly from the replicative helicase.     

An origin unwinding activity regulates initiation of DNA replication during mammalian cell cycle

An in vitro assay was developed to study the positive factors that regulate the onset of DNA replication during the mammalian cell cycle. Extracts prepared from cells at defined positions in the cell cycle were used to examine the replication of SV40 DNA in a cell free system. Extracts prepared from S phase cells were ten times more efficient at initiating replication at the SV40 origin than were extracts from G1 cells, whereas elongation rates were similar in G1 and S reactions. At a discrete point in the cell cycle, just before the cell's entry into S, an activity appeared that was required, in conjunction with SV40 T antigen, for site specific initiation at the SV40 origin. This factor had a role in unwinding DNA at the replication origin.     

Start sites of bidirectional DNA synthesis at the human lamin B2 origin

The initiation sites of bidirectional synthesis at the DNA replication origin located at the 3' end of the human lamin B2 gene were investigated. RNA-primed nascent DNA molecules were subjected to second-strand synthesis with appropriate primers, amplified by ligation-mediated polymerase chain reaction, and size fractionated. Evidence for precise start sites was obtained. Exploration of close to 1 kilobase, coupled to inhibition of Okazaki fragment synthesis, demonstrates that the leading strands initiate at precise nucleotides on either helix, overlapping by three base pairs, within the area bound to a protein complex possibly analogous to the prereplicative complex of yeast.     

SARS-COV结构蛋白N的核酸PCR扩增

[目的]探讨SARS-COV结构蛋白的PCR扩增条件。[方法]采用正交试验对SARS全基因组进行PCR反应筛选目标片段,确定退火温度、模板浓度、聚合酶量、引物浓度P、CR延长时间对于PCR反应的影响。[结果]结果表明,在退火温度55~60°C、模板浓度5 mg/ml加入1μl、DNA聚合酶加入0.25μl、引物浓度25 pmol/LP、CR反应时间为60 s时,DNA回收量最高,为4.37μg。[结论]该研究为SARS冠状病毒核酸的转录和复制提供科学依据。     

Visualization and functional analysis of RNA-dependent RNA polymerase lattices

Positive-strand RNA viruses such as poliovirus replicate their genomes on intracellular membranes of their eukaryotic hosts. Electron microscopy has revealed that purified poliovirus RNA-dependent RNA polymerase forms planar and tubular oligomeric arrays. The structural integrity of these arrays correlates with cooperative RNA binding and RNA elongation and is sensitive to mutations that disrupt intermolecular contacts predicted by the polymerase structure. Membranous vesicles isolated from poliovirus-infected cells contain structures consistent with the presence of two-dimensional polymerase arrays on their surfaces during infection. Therefore, host cytoplasmic membranes may function as physical foundations for two-dimensional polymerase arrays, conferring the advantages of surface catalysis to viral RNA replication.     

A novel nucleoprotein complex at a replication origin

The viral protein p6, required for the protein-primed initiation of replication of Bacillus subtilis phage phi 29, forms a nucleoprotein complex at the viral replication origins that shows novel features. Deoxyribonuclease I and hydroxyl radical footprinting data, as well as the induction of positive supercoiling, support a model in which a DNA right-handed superhelix tightly wraps around a multimeric p6 core. The interaction occurs through the DNA minor groove. The activity of p6 not only requires the formation of the complex but also its correct positioning, indicating that the other proteins involved in the initiation of replication recognize, at a precise position, either the p6 core or the DNA conformational change induced by p6.     

Unwinding of duplex DNA from the SV40 origin of replication by T antigen

The T antigen specified by SV40 virus is the only viral-encoded protein required for replication of SV40 DNA. T antigen has two activities that appear to be essential for viral DNA replication: specific binding to duplex DNA at the origin of replication and helicase activity that unwinds the two DNA strands. As judged by electron microscopy, DNA unwinding is initiated at the origin of replication and proceeds bidirectionally. Either linear or circular DNA molecules containing the origin of replication are effective substrates; with closed circular DNA, a topoisomerase capable of removing positive superhelical turns is required for an efficient reaction. Presence of an origin sequence on duplex DNA and a single-strand DNA-binding protein appear to be the only requirements for T antigen to catalyze unwinding. This reaction mediated by T antigen defines a likely pathway to precise initiation of DNA replication: (i) the sequence-specific binding activity locates the origin sequence, (ii) the duplex DNA is unwound at this site, and (iii) the DNA polymerase and primase begin DNA replication. A similar pathway has been inferred for the localized initiation of DNA replication by bacteriophage lambda and by Escherichia coli in which a sequence-specific binding protein locates the origin and directs the DnaB helicase to this site. Observations with the SV40 system indicate that localized initiation of duplex DNA replication may be similar for prokaryotes and eukaryotes.