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A method is described that makes use of a polyclonal antiserum to measure repair of the principal photoproducts induced in DNA by short-wave ultraviolet light (UVC)–pyrimidine-pyrimidone 6-4 photoproducts ([6-4]PPs) and cyclobutane pyrimidine dimers (CPDs). DNA extracted from irradiated cells is applied to a nitrocellulose dot-blot and quantitated using an enzyme-conjugated secondary antibody and a color assay. Although the polyclonal antiserum contains antibodies to both [6-4]PPs and CPDs, repair of these lesions can be measured separately by differential destruction or repair of one or other photoproduct. The method is useful for measuring repair in total genomic DNA, and is sufficiently sensitive to measure repair of damage induced by doses of 10 J/m2 of UVC and less. The method is very versatile and has been used to measure repair in human cells, yeasts, plants, archaea, bacteria, and filamentous fungi. [ABSTRACT FROM AUTHOR]

The unscheduled DNA synthesis (UDS) assay measures the ability of a cell to perform global genomic nucleotide excision repair (NER). This chapter provides instructions for the application of this technique by creating 6-4 photoproducts and pyrimidine dimers using UV-C irradiation. This procedure is designed specifically for quantification of the 6-4 photoproducts. Repair is quantified by the amount of radioactive thymidine incorporated during repair synthesis after this insult, and radioactivity is evaluated by grain counting after autoradiography. The results are used to clinically diagnose human DNA repair deficiency disorders and provide a basis for investigation of repair deficiency in human tissues or tumors. No other functional assay is available that directly measures the capacity to perform NER on the entire genome without the use of specific antibodies. Since live cells are required for this assay, explant culture techniques must be previously established. Host cell reactivation (HCR), as discussed in Chapter 37, is not an equivalent technique, as it measures only transcription-coupled repair (TCR) at active genes, a small subset of total NER. [ABSTRACT FROM AUTHOR]

32P-Postlabeling analysis is an ultra-sensitive method for the detection of DNA adducts, such as those formed directly by the covalent binding of carcinogens and mutagens to bases in DNA and other DNA lesions resulting from modification of bases by endogenous or exogenous agents (e.g., oxidative damage). The procedure involves four main steps: enzymatic digestion of the DNA sample; enrichment of the adducts; radiolabeling of the adducts by T4 kinase-catalyzed transference of 32P-orthophosphate from [γ-32P]ATP; chromatographic separation of labeled adducts; and detection and quantification by means of their radioactive decay. Using 10 µg of DNA or less, it is capable of detecting adduct levels as low as 1 adduct in 109–1010 normal nucleotides. It is applicable to a wide range of investigations, including monitoring human exposure to environmental or occupational carcinogens, determining whether a chemical has genotoxic properties, analysis of the genotoxicity of complex mixtures, elucidation of the pathways of activation of carcinogens, and monitoring DNA repair. [ABSTRACT FROM AUTHOR]

The plastid genome (plastome) has proved a valuable source of data for evaluating evolutionary relationships among angiosperms. Through basic and applied approaches, plastid transformation technology offers the potential to understand and improve plant productivity, providing food, fiber, energy and medicines to meet the needs of a burgeoning global population. The growing genomic resources available to both phylogenetic and biotechnological investigations are allowing novel insights and expanding the scope of plastome research to encompass new species. In this chapter we present an overview of some of the seminal and contemporary research that has contributed to our current understanding of plastome evolution and attempt to highlight the relationship between evolutionary mechanisms and tools of plastid genetic engineering. [ABSTRACT FROM AUTHOR]

In meiotic prophase, homologous chromosomes pair and recombine, which ensures correct segregation of the homologs at the metaphase to anaphase transition of the first meiotic division. As a first step, early in meiotic prophase, DNA double-strand breaks (DSBs) are introduced by SPO11 at hundreds of sites throughout the genome. In mammals, formation and repair of these breaks is essential for the pairing process. The sex chromosomes, X and Y, are largely heterologous and pair only in the small pseudoautosomal homologous regions. The large heterologous regions show delayed repair of DSBs and asynapsis, which triggers meiotic sex chromosome inactivation (MSCI), leading to the formation of the transcriptionally silenced XY body. Initiation of XY body formation is known to require phosphorylation of H2AX, which is also associated with damage-induced DSB repair in somatic cells. MSCI is considered as a specialized form of a general meiotic silencing mechanism that also silences autosomal unsynapsed chromatin during male and female meiotic prophase: meiotic silencing of unsynapsed chromatin (MSUC). Whereas the XY pair remains always largely unsynapsed and inactivated, autosomal nonhomologous regions frequently show heterologous synapsis and escape from inactivation. Hence, MSUC is less efficient than MSCI. However, if MSUC occurs, MSCI is incomplete, indicating that the two mechanisms are functionally interacting. Herein, we briefly review how X and Y evolved from a pair of autosomes, in relation to X-chromosomal dosage compensation in females and MSCI in males. Next, we describe current ideas about initiation and maintenance of MSCI and MSUC. We also discuss MSCI in birds, a species with female heterogamety. Insight in the mechanism of meiotic silencing is highly relevant for our understanding of male infertility associated with translocations, particularly when these occur between an autosome and the X. Furthermore, we anticipate that dysregulation of MSCI may impact on early embryonic development. [ABSTRACT FROM AUTHOR]

Environmental mutagens are global health hazards and a major risk factor for colon cancer development. Environmental mutagens include a variety of genotoxic carcinogenic compounds including polycyclic aromatic hydrocarbons, heterocyclic amines, nitrosamines, and aromatic amines. Their sources are pollution, diet, alcohol and cigarette smoke. These mutagens come in contact with large bowel through blood circulation or direct ingestion. Once these mutagens are in contact with DNA of the gut epithelial cells, they form DNA-adducts. If the damage exceeds the repair capacity of the epithelial cells, then the persistence of mutations can lead to the transformation of these cells. Mutations in adenomatous polyposis coli (APC) gene is considered to be one of the earliest events in the genesis of colorectal cancer, and its role has been well documented in a broad spectrum of functions ranging from cell adhesion to cell migration, regulation of Wnt/β-catenin-signaling pathway, cell cycle control, apoptosis, and chromosomal segregation at mitosis. The contribution of environmental mutagens on the APC gene mutations is very scanty in the literature. In the past several years, we have initiated studies to examine the interaction of APC with pathways induced by environmental mutagens. We showed that APC can be transcriptionally upregulated after DNA damage subsequent to the exposure of environmental mutagens including cigarette smoking. In addition, we recently discovered a novel role of APC in base excision repair (BER) pathway. APC interacts with DNA polymerase β and Fen-1 and blocks Pol-β-directed BER. Thus, APC-mediated block of BER in response to environmental mutagens, especially the DNA-alkylating agents, can be detrimental to the cell. [ABSTRACT FROM AUTHOR]

Epigenetics and oxidative stress are two cellular mechanisms that appear to be independent from each other and associated with distinct cellular processes. Epigenetics is associated with developmental regulation of gene expression, mainly through the process of methylation of genetic promoter regions. On the other hand, oxidative stress is a cellular process of disruption in the homeostasis between cellular antioxidant systems and reactive oxygen and nitrogen species leading to cell malfunction or death. The oxidative stress process is linked to a myriad of diseases and is a consequence after toxicologic challenges. In this chapter, we summarize results from our laboratories that identify developmental stage and toxicologic challenges that result in a novel mechanism in which toxicologic exposure during development alters the normal pattern in markers of oxidative stress and epigenetic regulation. The concordance of these two alterations results in pathologic changes similar to those seen in Alzheimer΄s disease. Thus, we postulate a novel mechanism in which epigenetics and oxidative stress are at play in disease, and the occurrence of one of these processes will alter the occurrence of the other. [ABSTRACT FROM AUTHOR]

Tumor resistance to chemotherapeutic DNA damaging agents, such as cisplatin, is an obstacle in the treatment of many cancers, including lung and ovarian. Resistance is influenced by nucleotide excision repair (NER) catalyzed removal of cisplatin-DNA lesions. NER is the primary pathway used by the cells in the repair of helix-distorting cisplatin lesions; therefore, inhibition of NER may increase the efficacy of cisplatin treatment. More specifically, the recognition and verification of DNA damage by NER is a critical step in the pathway, making it an ideal target for inhibition. Recognition of DNA damage occurs primarily through two proteins, Xeroderma Pigmentosum Group A (XPA) and replication protein A (RPA). XPA has been shown to have a role exclusively in NER, thus making it a highly specific target for inhibition that will lead to a decrease in NER and an increase in sensitivity to cisplatin treatment. RPA is a single-stranded DNA-binding protein that has roles in NER as well as in other metabolic pathways, including DNA replication and recombination. We have developed a high-throughput (HT) assay for XPA/RPA binding to DNA and screened libraries of small molecules to identify compounds capable of interrupting the protein/DNA interaction, an effort that has lead to the identification of small molecule inhibitors of both RPA and XPA. These inhibitors have been validated in secondary in vitro screens and structure–activity relationships were determined for one class of inhibitors. Further development of this class of compounds is anticipated to display cytostatic/cytotoxic activity and sensitize cells to cisplatin therapy. [ABSTRACT FROM AUTHOR]

The H69CIS200 and H69OX400 cell lines are novel models of low-level platinum drug resistance developed from H69 human small-cell lung cancer cells with eight 4-day treatments of 200 ng/ml cisplatin and 400 ng/ml oxaliplatin, respectively. A recovery period was given between treatments to emulate the cycles of chemotherapy given in the clinic. The resistant cell lines were approximately twofold resistant to cisplatin and oxaliplatin, and were cross-resistant to both drugs. Platinum resistance was not associated with increased cellular glutathione, decreased accumulation of platinum or increased DNA repair capacity. The H69 platinum sensitive cells entered a lengthy 3-week growth arrest in response to low-level cisplatin or oxaliplatin treatment. This is an example of the coordinated response between the cell cycle and DNA repair. In contrast, the H69CIS200 and H69OX400 cells have an alteration in the cell cycle allowing them to rapidly proliferate post drug treatment. The resistant cell lines also have many chromosomal rearrangements most of which are not associated with the resistant phenotype, suggesting an increase in the genomic instability in the resistant cell lines. We hypothesized that there was a lack of coordination between the cell cycle and DNA repair in the resistant cell lines allowing proliferation in the presence of DNA damage which has created an increase in genomic instability. The H69 cells and resistant cell lines have mutant p53 and consequently decrease the expression of p21 in response to platinum drug treatment; promoting progression of the cell cycle instead of increasing p21 to maintain the arrest. A decrease in ERCC1 protein expression and an increase in RAD51B foci activity were observed with the platinum-induced cell-cycle arrest and did not correlate with resistance or altered DNA repair capacity. These changes may, in part, be mediating and maintaining the cell-cycle arrest in place of p21.The rapidly proliferating resistant cells have restored the levels of both these proteins to their levels in untreated cells. We use the term ˵regrowth resistance″ to describe this low-level platinum resistance where cells survive treatment through increased proliferation. Regrowth resistance may play a role in the onset of clinical resistance. [ABSTRACT FROM AUTHOR]

Intraoperative radiotherapy differs from conventional fractionated radiotherapy by giving a large dose in a single fraction. Various studies suggest that biological effects of dose fractions larger than 5-10 Gy may be different from those induced by conventional fraction sizes. Here, we classify the clinical dose response for different patterns of tumor cells left after surgery. Isoeffective doses of single fractions estimated from fractionated radiotherapy by using the linear-quadratic formalism are consistent with clinical experience from tumor control after radiosurgery. Using a model cell line in vitro, we show that the DNA repair response induced by 6-18 Gy differs from that after 0-2 Gy suggesting saturation of the repair machinery. Possible influences of the tumor microenvironment that may contribute to making very large dose fractions more effective than predicted from clonogenic cell inactivation alone are reviewed. [ABSTRACT FROM AUTHOR]

Second primary cancers are a rare but relevant late effect of radiotherapy. However few studies have looked into the molecular mechanisms. In this work, basic mechanisms of carcinogenesis, DNA repair and radiation-induced genetic changes are reviewed. Ionizing radiation does not induce changes in specific genes but can inactivate tumor suppressor genes and, more rarely, activate oncogenes in a stochastic fashion. Radiation-induced genomic instability is likely to play a major role, too. A single mechanism is unlikely to explain all second cancers. [ABSTRACT FROM AUTHOR]

The linear-quadratic model has been widely used to describe tumor survival curves for doses under 10 Gy. The absence of a proposed mechanism [1] behind the linearquadratic model is an important limitation for the proper interpretation of clinical results. Models based on a detailed mechanism have the unpleasant feature of a large number of parameters, which makes also the interpretation and use in quantitative radiobiology a very hard task [2,3,4,5,6]. In this work a simple microscopic model based on reversible and irreversible DNA damage is proposed. The model is able to describe the survival curves at high and low LET for V-79 cells in late S phase of Chinese hamsters [7]. The new set of parameters can be related to the ones of the linear-quadratic model and in this way a connection with DNA damage and repair mechanisms is made. At the same time the evolution equations open the possibility for continuous and fractionated treatment plans. [ABSTRACT FROM AUTHOR]

Nemorubicin is a 3'-deamino-3'[2-(S)-methoxy-4-morpholinyl] derivative of doxorubicin. This derivative has been synthesized in the early 1990s by the Farmitalia Carlo Erba Research Center in Italy. The idea was to develop doxorubicin analogues able to circumvent the emergence of chemoresistance, in particular the multi-drug resistance. The drug was reported to be active in vitro against both murine and human tumor cells resistant to doxorubicin. Similar results were obtained when the drug was administered in vivo to mice bearing multi-drug resistant tumors. The compound retained the same activity also in alkylating agents and topoisomerase II resistant tumors and showed an increased potency relative to the parent drug doxorubicin. It is metabolized via P450 CYP3A enzyme to an extremely cytotoxic derivative. Both nemorubicin and its metabolite have a mechanism of action different from that of doxorubicin, with a key role played by the nucleotide excision repair system. The drug is actively tested in clinics as a single agent or in combination with cisplatin. [ABSTRACT FROM AUTHOR]

The aging of the populations in the industrial world means that we are living much longer with the consequences of sun exposure. The first approaches of screening or filtering the solar rays are chemically and physically effective but ran into resistance from human behaviour. A better understanding of the biochemistry led to the use of a variety of antioxidants which quench or intercept oxygen radicals and ameliorate some of the effects of solar UV. We now have available DNA repair enzymes to actually reverse or repair damage after it happens but before it can have its effects. We should be careful to use these tools properly, so that we do not facilitate greater overall exposure to some or all of the solar spectrum in the mistaken belief that protection is perfect. But in societies where the burden of medical care is becoming overwhelming, we should remember that prevention is preferable, both economically and socially, to treatment. [ABSTRACT FROM AUTHOR]

A definition of the term "xeroderma pigmentosum" (XP) is presented. It is a genetic disease with clinical and cellular hypersensitivity to ultraviolet radiation and defective repair of DNA. Patients of this disease display an increase in the frequency of skin malignancies. XP serves as a model disease to study the relationship between defects in DNA repair and skin cancer.

The article discusses nucleotide excision repair (NER), which refers to a complex biochemical mechanism for the removal of DNA damage induced by a variety of environmental and endogenous factors, the most common of which is ultraviolet light. There are two modes of NER: global genomic repair and transcription-coupled repair. The clinical relevance of NER in the prevention of cancer is explored.

An encyclopedia entry for multistep development is presented. It refers to the stepwise development of malignant tumors by successive changes in multiple characteristics. Tumor development can be regarded as a Darwinian process, based on variation and selection. The four major genes participate in multistep development are: oncogenes; suppressor genes that can arrest the growth of illegitimately driven cells; genes that raise the apoptotic threshold; and DNA repair genes.

A definition of the term "Homologous Recombination Repair" is presented. It refers to a DNA repair process that includes the invasion of an undamaged DNA molecule by a damaged molecule of identical or very similar sequence. Resynthesis of the damaged region is accomplished using the undamaged molecule as a template.

An encyclopedia entry for the term repair of DNA is presented. DNA repair comprises of a group of distinct biochemical pathways by which various types of damage to DNA is repaired. The classifications of DNA repair of base damage, namely, the reversal of base damage and removal of damage are described. The article also discusses the relationship between DNA repair and cancer and repair of DNA strand breaks.

A definition for the term "mismatch repair genes" is presented. Mismatch repair genes encode proteins that are involved in post-replication mismatch repair, eliminating from DNA newly incorporated not-matching bases. They are highly conserved among living organisms and were originally identified in bacteria and yeast.

A definition of the term "transcription repair" is presented. It refers to a subpathway of nucleotide excision repair to remove lesions in a transcribed DNA.

A definition of the term "transcription-coupled repair" is presented. It refers to rapid DNA repair in the transcribed strand of expressing genes.

A definition of the term "TFIIH," is presented. It refers to a protein complex that plays a key role in both transcription and DNA repair.

An encyclopedia entry for the term repair endonucleases is presented. Repair endonucleases, examples of which are formamidopyrimidine glycosylase or endonuclease III, often combine two enzymatic activities. The first is a lesions-specific glycosylase that removes the damaged base and the second is an AP-endonuclease that recognizes the resulting baseless sugar and cuts the DNA.

A definition of the term "Rad50," as used in the cancer field, is presented. It refers to a protein forming a multi-function complex with MRE11 and Nijmegen breakage syndrome, and is involved in the processing of DNA damage for DNA maintenance, including the repair of DNA and recombination.

A definition for the term "postmeiotic segregation increased 2" (PMS2) is presented. PMS2 is a protein of 862 aa and 95 kD that is involved in the mismatch repair of DNA. It also interacts with mutL homolog 1 protein. Mutations in PMS2 are associated with familial hereditary nonpolyposis colon cancer. In PMS2, the gene locus maps to 7p22.

A definition of the term "post-replication repair" is presented. It refers to a DNA repair process in which DNA lesions are bypassed during replication by specialized DNA polymerases.

A definition of the term "PIBIDS" is presented. It refers to a photosensitive form of trichothiodystrophy, a genetic syndrome, which is associated with a defect in a protein complex that plays a role in transcription and DNA repair.

A definition for the term "mismatch repair" is presented. It refers to a molecular process by which erroneously incorporated and not matching bases are removed from DNA double strands and are replaced by the correct base. Mismatch repair is a DNA repair system that is highly conserved from bacteria to humans.

An encyclopedia entry for the term "MLH1," is presented. It is also known as COCA2. It refers to a protein involved in mismatch repair of DNA. It forms heterodimers with PMS2. It is believed that the gene maps to 3p21.3. Hence, mutations are related with familial hereditary nonpolyposis colon cancer.