DNA Damage Response in Live Yeast Using Single-molecule Microscopy
DNA Damage Response in Live Yeast Using Single-molecule Microscopy

Author: Shubhika Ranjan

Publisher:

Published: 2021

Total Pages:

ISBN-13:

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"The genetic information stored in DNA must be faithfully copied and transmitted to the next generation of cells at every cell cycle. The replication of this genetic information is performed by special multiprotein replication machinery, referred to as "replisome," which synthesizes both daughter duplexes simultaneously. At times, the accurate replication of the genome can go awry, causing mutations that can lead to a collection of diseases. In eukaryotic cells, detection and response to DNA damage during DNA replication is performed by the DNA damage response (DDR) pathway. DDR uses exposed single-stranded DNA as a signal for DNA damage and proceeds by activation of kinases that transmit the signal and change the cell program to respond to the damage. The overall purpose of this work is to understand how cells do this initial detection and how they make the decision to activate the signalling pathway. I used budding yeast (Saccharomyces cerevisiae), a unicellular model organism, to understand the eukaryotic genetic architecture because it provides a framework to develop and optimize methods to standardize the analysis. I focus on the study of dynamics of DNA replication and DDR proteins of cells experiencing DNA damage or replication fork stalling using single-molecule microscopy. Although this technique provides a high signal-to-noise ratio for visualization while still retaining the integral features in the physiological context of biological systems, various factors play a major role in attaining such high-quality data for further analysis. In the first part of this work, I provide an overview for optimizing the single-molecule techniques while considering various factors involved. In the second section, I describe the initial work towards visualizing the proteins involved in DNA damage response in HaloTag labelled S. cerevisiae"--

Technologies for Detection of DNA Damage and Mutations
Technologies for Detection of DNA Damage and Mutations

Author: G.P. Pfeifer

Publisher: Springer Science & Business Media

Published: 2013-11-11

Total Pages: 443

ISBN-13: 1489903011

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Man-made carcinogens, natural genotoxic agents in the environment, as well as ionizing and ultraviolet radiation can damage DNA and are a constant threat to genome integrity. Throughout the evolution oflife, complex DNA repair systems have developed in all living organisms to cope with this damage. Unrepaired DNA lesions can promote genetic alterations (mutations) that may be linked to an altered phenotype, and, if growth-controlling genes are involved, these mutations can lead to cell transformation and the development of malignant tumors. Proto oncogenes and tumor suppressor genes may be critical targets for DNA damaging agents. In a number of animal model systems, correlations between exposure to a carcinogen, tumor develop ment, and genetic changes in tumor DNA have been established. To understand mutagenesis processes in more detail at the molecular level, we need to know the type and frequency of DNA adducts within cells, their distribution along genes and specific DNA sequences, as well as the rates at which they are repaired. We also need to know what types of mutations are produced and which gene positions are most prone to mutagenesis. This book provides a collection of techniques that are useful in mutagenesis research. The book is divided into three parts. In Part I, methods for DNA damage and repair analysis are provided.

In Situ Detection of DNA Damage
In Situ Detection of DNA Damage

Author: Vladimir V. Didenko

Publisher: Springer Science & Business Media

Published: 2008-02-05

Total Pages: 314

ISBN-13: 1592591795

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Detection and analysis of DNA damage is of critical importance in a variety of biological disciplines studying apoptosis, cell cycle and cell di- sion, carcinogenesis, tumor growth, embryogenesis and aging, neu- degenerative and heart diseases, anticancer drug development, environmental and radiobiological research, and others. Individual cells within the same tissue or in cell culture may vary in the extent of their DNA damage and, consequently, can display different re- tions to it. These differences between individual cells in the same cell popu- tion are detected using in situ approaches. In situ is a Latin term meaning “on site” or “in place.” It is used to denote the processes occurring or detected in their place of origin. In mole- lar and cell biology this usually refers to undisrupted mounted cells or tissue sections. In that meaning “in situ” is used as part of the terms “in situ PCR,” “in situ transcription,” “in situ hybridization,” “in situ end labeling,” and “in situ ligation.” Sometimes the “in situ” term is applied at the subcellular level to cells disrupted in the process of analysis, for example, in the detection of specific sequences in chromosomes using fluorescent in situ hybridization (FISH). Historically, the term was used primarily in methods dealing with nucleic acids.

Single-molecule Studies Reveal Mechanisms of Human DNA Double-strand Break Repair
Single-molecule Studies Reveal Mechanisms of Human DNA Double-strand Break Repair

Author: Logan Ross Myler

Publisher:

Published: 2018

Total Pages: 238

ISBN-13:

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DNA damage is ubiquitous to all organisms and very complex pathways have evolved to recognize and repair these lesions. The most deleterious DNA damages are double-strand breaks (DSBs), and a single unrepaired DSB can lead to cell death. In human cells, there exist two canonical pathways of DSB repair: Non-Homologous End Joining (NHEJ) and Homologous Recombination (HR). Two protein complexes that rapidly bind DNA ends coordinate these separate pathways: the Ku70-Ku80 heterodimer (Ku) and the Mre11-Rad50-Nbs1 complex (MRN), respectively. Ku encircles the DNA ends and recruits other factors, such the kinase DNA-PKcs, to bluntly ligate the ends back together. In contrast, MRN along with the long-range nuclease Exo1 and helicase BLM digests the DNA to create long 3’ single-stranded DNA overhangs, which are rapidly bound by the single-stranded DNA binding protein RPA. Next, Rad51 replaces RPA and facilitates strand exchange into a homologous chromosome to resynthesize the missing information in a largely error-free way. Despite the importance of DSB repair, many of the underlying mechanisms by which these molecular machines dynamically assemble and carry out the repair process have remained unknown. Here, I use a combination of ensemble biochemical assays as well as high-throughput single-molecule microscopy to visualize the repair process. I have observed two main steps of the repair process: initiation of HR by MRN and long-range resection by Exo1. I have found that MRN locates DSBs by a sliding mechanism that allows it to load on Ku-blocked ends. Then, once it reaches the end, MRN removes DNA-PK and recruits Exo1 and BLM in order to promote long-range digestion of the DNA. Finally, the Exo1/BLM resectosome is attenuated by phosphorylation of RPA. Overall, I have characterized the initiation and regulation of DSBR. This will lead to a new understanding of the ways in which these deleterious lesions are repaired and will contribute to understanding cancer as well as techniques for genetic manipulation