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

Single-molecule Basis of Transcription-coupled DNA Repair
Single-molecule Basis of Transcription-coupled DNA Repair

Author: Jun Fan

Publisher:

Published: 2015

Total Pages: 0

ISBN-13:

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The DNA in living cells is constantly threatened by damages from both endogenous and exogenous agents, which can threaten genomic integrity, block processes of replication, transcription and translation and have also genotoxic effects. In response to the DNA damage challenge, organisms have evolved diverse surveillance mechanisms to coordinate DNA repair and cell-cycle progression. Multiple DNA repair mechanisms, discovered in both prokaryotic and eukaryotic organisms, bear the responsibility of maintaining genomic integrity; these mechanisms include nucleotide excision repair (NER), base excision repair (BER), mismatch repair (MMR) and double strand break repair (DSBR). Transcription-coupled DNA repair (TCR) is a specialized NER subpathway characterized by enhanced repair of the template strand of actively transcribed genes as compared to the classical global genome repair (GGR) subpathway of NER which does not distinguish between template and non-template strands. TCR achieves specialization via the involvement of RNA polymerase (RNAP) and the Mfd (Mutation Frequency Decline) protein, also known as TRCF (transcription repair coupling factor). TCR repair initiates when RNAP stalls at a DNA lesion on the transcribed strand and serves as the da mage sensor. The stalled RNAP must be displaced so as to make the lesion accessible to downstream repair components. E. coli Mfd translocase participates in this process by displacing stalled RNAP from the lesion and then coordinating assembly of the UvrAB(C) components at th( damage site. Recent studies have shown that after binding to and displacing stalled RNAP, Mfd remains on the DNA in the form of a stable, translocating complex with evicted RNAP. So as to understand how UvrAB(C) are recruited via the Mfd-RNAP complex, magnetic trapping of individual, damaged DNA molecules was employed to observe-in real-time this multi¬component, multi-step reaction, up to and including the DNA incision reaction by UvrC. It was found that the recruitment of UvrA and UvrAB to the Mfd-RNAP complex halts the translocating complex and then causes dissolution of the complex in a molecular "hand-off" with slow kinetics Correlative single-molecule nanomanipulation and fluorescence further show that dissolution of the complex leads to loss of not only RNAP but also Mfd. Hand-off then allows for enhanced incision of damaged DNA by the UvrC component as compared to the equivalent single-moleculE GGR incision reaction. A global model integrating TCR and GGR components in repair was proposed, with the overall timescales for the parallel reactions provided.

Visualization of DNA Double-Strand Break Repair at the Single-Molecule Level
Visualization of DNA Double-Strand Break Repair at the Single-Molecule Level

Author: Yoshihiko Takeda

Publisher:

Published: 2003

Total Pages: 5

ISBN-13:

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Exposure to low doses of ionizing radiation is universal. The signature injury from ionizing radiation exposure is induction of DNA double-strand breaks (DSBs). The first line of defense against DSBs is direct ligation of broken DNA ends via the nonhomologous end-joining pathway. Because even a relatively high environmental exposure induces only a few DSBs per cell, our current understanding of the response to this exposure is limited by the ability to measure DSB repair events reliably in situ at a single-molecule level. To address this need, we have taken advantage of biological amplification, measuring relocalization of proteins and detection of protein phosphorylation as a surrogate for detection of broken ends themselves. We describe the use of specific antibodies to investigate the kinetics and mechanism of repair of very small numbers of DSBs in human cells by the nonhomologous end-joining pathway.

DNA Damage Recognition
DNA Damage Recognition

Author: Wolfram Siede

Publisher: CRC Press

Published: 2005-09-19

Total Pages: 871

ISBN-13: 0849352681

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Stands as the most comprehensive guide to the subject-covering every essential topic related to DNA damage identification and repair. Covering a wide array of topics from bacteria to human cells, this book summarizes recent developments in DNA damage repair and recognition while providing timely reviews on the molecular mechanisms employe

DNA Repair and Mutagenesis
DNA Repair and Mutagenesis

Author: Errol C. Friedberg

Publisher: American Society for Microbiology Press

Published: 2005-11-22

Total Pages: 2587

ISBN-13: 1555813194

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An essential resource for all scientists researching cellular responses to DNA damage. • Introduces important new material reflective of the major changes and developments that have occurred in the field over the last decade. • Discussed the field within a strong historical framework, and all aspects of biological responses to DNA damage are detailed. • Provides information on covering sources and consequences of DNA damage; correcting altered bases in DNA: DNA repair; DNA damage tolerance and mutagenesis; regulatory responses to DNA damage in eukaryotes; and disease states associated with defective biological responses to DNA damage.

DNA Repair in Cancer Therapy
DNA Repair in Cancer Therapy

Author: Mark R. Kelley

Publisher: Academic Press

Published: 2016-06-07

Total Pages: 466

ISBN-13: 0128035994

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DNA Repair and Cancer Therapy: Molecular Targets and Clinical Applications, Second Edition provides a comprehensive and timely reference that focuses on the translational and clinical use of DNA repair as a target area for the development of diagnostic biomarkers and the enhancement of cancer treatment. Experts on DNA repair proteins from all areas of cancer biology research take readers from bench research to new therapeutic approaches. This book provides a detailed discussion of combination therapies, in other words, how the inhibition of repair pathways can be coupled with chemotherapy, radiation, or DNA damaging drugs. Newer areas in this edition include the role of DNA repair in chemotherapy induced peripheral neuropathy, radiation DNA damage, Fanconi anemia cross-link repair, translesion DNA polymerases, BRCA1-BRCA2 pathway for HR and synthetic lethality, and mechanisms of resistance to clinical PARP inhibitors. Provides a comprehensive overview of the basic and translational research in DNA repair as a cancer therapeutic target Includes timely updates from the earlier edition, including Fanconi Anemia cross-link repair, translesion DNA polymerases, chemotherapy induced peripheral neuropathy, and many other new areas within DNA repair and cancer therapy Saves academic, medical, and pharma researchers time by allowing them to quickly access the very latest details on DNA repair and cancer therapy Assists researchers and research clinicians in understanding the importance of the breakthroughs that are contributing to advances in disease-specific research

The Nucleolus
The Nucleolus

Author: Mark O. J. Olson

Publisher: Springer Science & Business Media

Published: 2011-09-15

Total Pages: 434

ISBN-13: 1461405149

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Within the past two decades, extraordinary new functions for the nucleolus have begun to appear, giving the field a new vitality and generating renewed excitement and interest. These new discoveries include both newly-discovered functions and aspects of its conventional role. The Nucleolus is divided into three parts: nucleolar structure and organization, the role of the nucleolus in ribosome biogenesis, and novel functions of the nucleolus.