细胞功能测定

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 ...

Base excision repair is a major mechanism for correcting modified bases. The first step of this repair mechanism is the removal of a modified base by a specific DNA-N-glycosylase to leave an apurinic/apyrimidinic (AP) site. Subsequently, the AP site is repaired through sequential reactions, ...

Apurinic/apyrimidinic (AP) sites are one of the more common lesions formed in DNA that, if left unrepaired, can represent potential sites of mutation (1). To illustrate the importance of these sites, all organisms that have thus far been tested have been found to contain enzymes that incise abasic ...

Several types of DNA lesions are formed on irradiation of cells with ultraviolet (UV) light (1,2). The two most frequent lesions are the cyclobutane pyrimidine dimers (CPDs) and the pyrimidine (6-4) pyrimidone photoproducts ( photoproducts; PPs). In addition, UV irradiation produces, alt ...

Physical and chemical agents in the environment, those used in clinical applications, or encountered during recreational exposures to sunlight, induce damages in DNA. Understanding the biological impact of these agents requires quantitation of the levels of such damages in labora ...

Ultraviolet (UV) radiation that overlaps the absorption spectrum of DNA induces a variety of photoproducts. Because stratospheric ozone screens out shorter UV wavelengths, DNA-damaging solar irradiance at the terrestrial surface is confined to the UV-B band (290–320 nm). However, g ...

Radioimmunoassay (RIA) is a competitive binding assay between an unlabeled and a radiolabeled antigen for binding to antibody raised against that antigen. For the development of this technique, Yalow and Berson (1) received the Nobel Prize in Medicine. For detailed theory and troublesh ...

Measurement of DNA damage can be difficult if the levels of damage are small. For example, the ultraviolet (UV) radiation in sunlight creates cyclobutane pyrimidine dimers (CPDs), but this type of damage is rapidly repaired. The steady-state level of CPDs is thus low, and sensitive methods are req ...

The dot-blot method described here can be used to measure repair of pyrimidine-pyrimidone 6-4 photoproducts (PPs) and cyclobutane pyrimidine dimers (CPDs) in total genomic DNA from any organism. The DNA does not have to be especially intact nor is it necessary to have any sequence information. ...

The adverse biological consequences of unrepaired toxic UV-B-induced photoproducts in DNA, mostly cyclobutane pyrimidine dimers (CPDs) and pyrimidine-(6-4′)-pyrimidone photoproducts , are touched on in Chapters 4, 15, and 18. Enzymes that use UV-A/blue-light energy to reverse CPDs ...