细胞技术

Early Drosophila development begins as a syncytium with 13 rapid nuclear divisions without cytokinesis (1). These divisions provide an excellent system in which to study the effects of DNA damage on the mitotic cycle. Because the effects of ionizing radiation can be assessed in both wild-type ...

The organization of eukaryotic DNA is increasingly well understood at each extreme of chromatin organization. Advances in understanding the organization of DNA at the level of nucleosome and chromosome have outstripped knowledge of the intermediate states and function of DNA. Most w ...

Gene targeting, defined as homologous recombination or genetic exchange between an introduced DNA sequence and its endogenous chromosomal locus, or “target,” is a powerful approach for genetic manipulation. Gene-targeting strategies for both yeast (1) and mammalian cells (2–4) ha ...

DNA double-strand break (DSB) repair in mammalian cells has been demonstrated to be complex, involving both homologous and nonhomologous processes. Although manipulation of chromosomal DSBs and analysis of their repair are possible (1; see Chapters 37–39), this is usually time-cons ...

A large number of studies suggests that double-strand breaks (DSBs) induced in DNA by ionizing radiation or chemical agents are critical lesions, which if unrepaired or misrepaired may kill a cell, or cause its transformation to a cancer cell. Cells have developed efficient repair mechanis ...

The introduction of restriction endonucleases into mammalian cells in culture provides a unique method for introducing double-strand breaks (DSBs) into the DNA of the host cell. Restriction enzymes recognize, bind, and cleave specific DNA sequences to produce a DNA DSB in the absence of ot ...

Until recently, investigators interested in analyzing the repair of chromosomal double-strand breaks (DSBs) in mammalian cells have been limited by the inability to introduce defined DSBs within the genome. Traditional methods of introducing breaks have included irradiation or ...

Double strand-breaks (DSBs) are key intermediates in DNA recombination reactions. The possibility of inducing DSBs at specific sites in the genome by the expression of rare-cutting endonucleases has resulted in a tremendous increase in our knowledge on the mechanisms of DSB repair, esp ...

Generation of double-strand breaks (DSBs) in chromosomal DNA induces repair machinery of a cell, and is also a necessary step for recombination events. A system for the directed introduction of DSBs into a genome could substantially facilitate progress in understanding DSB repair mecha ...

Targeted manipulation of the genome is used to analyze gene expression, genome structure and protein structure. In yeast it has long been possible to introduce sequences into predetermined sites in the genome by double-strand break (DSB) repair (1). Until recently, such specificity has el ...

The repair of chromosomal double-strand breaks (DSBs) in Saccharomyces cerevisiae occurs most efficiently by homologous recombination. Homothallic mating-type (MAT) switching provides the most well-characterized system to study DSB repair by recombination in mitotic ce ...

Nucleotide excision repair (NER) activity can be directly measured in whole-cell extracts by quantifying either the incorporation of radiolabeled deoxynucleotide during the repair synthesis step in damaged plasmid DNA (1; see Chapters 25–27, 29) or the excision of a previously label ...

DNA repair pathways must include proteins that recognize and bind to damaged DNA. The search for such proteins has been facilitated by the use of electrophoretic mobility shift assays (EMSAs), which were first used to detect transcription factors that bind to specific DNA sequences (1,2). To st ...

Cells remove a wide array of potentially toxic and mutagenic lesions from their genomes by a major repair pathway called nucleotide excision repair (NER). This repair process involves a multiprotein nuclease complex that incises a damaged DNA strand on the 5′- and 3′-sides of a lesion (1). In humans, ...

Mammalian cells remove carcinogenic damage caused to DNA by ultraviolet (UV) light and certain other mutagens mainly by using the pathway known as nucleotide excision repair (NER). This involves damage recognition, unwinding of the DNA around the site of damage, incision on either side of the ...

Limited nucleotide excision repair (NER) requires at least ∼40 proteins in extracts from purified proteins (1,2) although perhaps hundreds of proteins may influence DNA repair in cells. For efficient DNA repair in extracts, it is important to utilize a system containing large quantities ...

Nucleotide excision repair (NER) is one of the most important systems in eukaryotes for overcoming DNA damage caused by environmental agents, such as ultraviolet (UV) radiation and chemical mutagens. NER in eukaryotes has been studied by genetic analyses of mainly human, rodent, and yeast r ...

The measurement of DNA excision repair activity in vitro, originally developed by Wood and co-workers (1), utilizes transcriptionally active protein extracts obtained from mammalian cells by the method of Manley et al. (2) (see Chapter 29). Nucleotide excision repair (NER), which requir ...

Nucleotide excision repair (NER) is a particularly versatile pathway of DNA repair capable of removing a broad spectrum of DNA lesions in both prokaryotes and eukaryotes (1–3). NER involves steps of damage recognition, incision and excision of the lesion and its flanking DNA, and repair DNA syn ...

Base excision repair (BER) is a major cellular repair mechanism that corrects a broad range of DNA lesions (for a review, see 1). BER deals with DNA damage generated not only by environmental genotoxins, like ionizing radiation, alkylating agents and oxidative reagents, but also by endogeneou ...