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DNA-Binding Proteins: Proteins which bind to DNA. The family includes proteins which bind to both double- and single-stranded DNA and also includes specific DNA binding proteins in serum which can be used as markers for malignant diseases.

The DNA Helix

JoVE 10784

Deoxyribonucleic acid, or DNA, is the genetic material responsible for passing traits from generation to generation in all organisms and most viruses. DNA is composed of two strands of nucleotides that wind around each other to form a double helix. The discovery of the structure of DNA occurred incrementally over nearly a century, representing one of the most famous and captivating stories in the history of science. Each strand of DNA consists of subunits called nucleotides that contain the sugar deoxyribose, a phosphate group, and one of four nitrogen-containing bases: adenine (A), guanine (G), cytosine (C), and thymine (T). Adenine and guanine are members of a larger class of chemicals called purines that all contain two-ringed structures. Cytosine and thymine belong to a group of single-ringed structures called pyrimidines. Adjacent nucleotides in the same strand are covalently linked by phosphodiester bonds. The two strands of nucleotides are held together by hydrogen bonds, in which the adenines in one strand pair with thymines at the same position in the other strand, and the cytosines in one strand pair with guanines in the same position in the other strand. This hydrogen bonding is made possible by the antiparallel arrangement of the two DNA strands, in which the 5’ and 3’ ends of the strands are oriented in opposite directions. Withou

 Core: DNA Structure and Function

What is Gene Expression?

JoVE 10797

Gene expression is the process in which DNA (i.e., a gene) directs the synthesis of functional products, such as proteins. Cells can regulate gene expression at various stages. It allows organisms to generate different cell types and enables cells to adapt to internal and external factors.

A gene is a stretch of DNA that serves as the blueprint for functional RNAs and proteins. Since DNA is made up of nucleotides and proteins consist of amino acids, a mediator is required to convert the information that is encoded in DNA into proteins. This mediator is the messenger RNA (mRNA). mRNA copies the blueprint from DNA by a process called transcription. In eukaryotes, transcription takes place in the nucleus by complementary base-pairing with the DNA template. The mRNA is then processed and transported into the cytoplasm where it serves as a template for protein synthesis during translation. In prokaryotes, which lack a nucleus, the processes of transcription and translation occur at the same location and almost simultaneously since the newly-formed mRNA is susceptible to rapid degradation. Every cell of an organism contains the same DNA, and consequently the same set of genes. However, not all genes in a cell are “turned on” or use to synthesize proteins. A gene is said to be “expressed” when the protein it encodes is produced by the cel

 Core: Gene Expression

Protein Crystallization

JoVE 5689

Protein crystallization, obtaining a solid lattice of biomolecules, elucidates protein structure and enables the study of protein function. Crystallization involves drying purified protein under a combination of many factors, including pH, temperature, ionic strength, and protein concentration. Once crystals are obtained, the protein structure can be elucidated by x-ray diffraction and…

 Biochemistry

Replication in Prokaryotes

JoVE 10788

DNA replication has three main steps: initiation, elongation, and termination. Replication in prokaryotes begins when initiator proteins bind to the single origin of replication (ori) on the cell’s circular chromosome. Replication then proceeds around the entire circle of the chromosome in each direction from two replication forks, resulting in two DNA molecules.

Replication is coordinated and carried out by a host of specialized proteins. Topoisomerase breaks one side of the double-stranded DNA phosphate-sugar backbone, allowing the DNA helix to unwind more rapidly, while helicase breaks the bonds between base pairs at the fork, separating the DNA into two template strands. Proteins that bind single-stranded DNA molecules stabilize the strands as the replication fork travels along the chromosome. DNA can only be synthesized in the 5’ to 3’ direction, so one strand of the template—the leading strand—is elongated continuously, while the other strand—the lagging strand—is synthesized in shorter pieces of 1000-2000 base pairs called Okazaki fragments. Much of the research to understand prokaryotic DNA replication has been performed in the bacterium Escherichia coli, a commonly-used model organism. E. coli has 5 DNA polymerases: Pol I, II, III, IV, and V. Pol III is responsible for the majority of DN

 Core: DNA Structure and Function

Transcription

JoVE 10794

Transcription is the process of synthesizing RNA from a DNA sequence by RNA polymerase. It is the first step in producing a protein from a gene sequence. Additionally, many other proteins and regulatory sequences are involved in the proper synthesis of messenger RNA (mRNA). Regulation of transcription is responsible for the differentiation of all the different types of cells and often for the proper cellular response to environmental signals. In eukaryotes, the DNA is first transcribed into a primary RNA, or pre-mRNA, that can be further processed into a mature mRNA to serve as a template for the synthesis of proteins. In prokaryotes such as bacteria, however, translation of RNA into polypeptides can begin while the transcription is still ongoing, as RNA can be quickly degraded. Transcription can also produce different kinds RNA molecules that do not code for protein, such as microRNAs, transfer RNA (tRNA), and ribosomal RNA (rRNA)—all of which contribute to protein synthesis. With few exceptions, all of the cells in the human body have the same genetic information in them, from neurons in the brain to muscle cells in the heart. So how do cells assume such diverse forms and functions? To a large extent, the answer lies in the regulation of transcription during development of the organism. Specifically, transcriptional regulation plays a central ro

 Core: DNA Structure and Function

Combining Single-molecule Manipulation and Imaging for the Study of Protein-DNA Interactions

1LENS - European Laboratory for Non-linear Spectroscopy, University of Florence, 2Chemistry Research Laboratory, University of Oxford, 3Department of Biology, University of Florence, 4Department of Physics and Astronomy, University of Florence, 5National Institute of Optics-National Research Council, Italy, 6International Center of Computational Neurophotonics

JoVE 51446

 Biology

Screening for Functional Non-coding Genetic Variants Using Electrophoretic Mobility Shift Assay (EMSA) and DNA-affinity Precipitation Assay (DAPA)

1Center for Autoimmune Genomics and Etiology, Cincinnati Children's Hospital, 2Medical Scientist Training Program, University of Cincinnati, 3Immunology Graduate Program, University of Cincinnati, 4Divisions of Biomedical Informatics and Developmental Biology, Cincinnati Children's Hospital

JoVE 54093

 Biology

Performing Data Mining And Integrative Analysis Of Biomarker in Breast Cancer Using Multiple Publicly Accessible Databases

1Department of Thyroid and Breast Surgery, First Affiliated Hospital of Shantou University Medical College, 2Department of Medical Oncology, Cancer Hospital of Shantou University Medical College, 3Shantou University Medical College

JoVE 59238

 Cancer Research

A Protein Preparation Method for the High-throughput Identification of Proteins Interacting with a Nuclear Cofactor Using LC-MS/MS Analysis

1Graduate School of Frontier Biosciences, Osaka University, 2Department of Biotechnology and Genetic Engineering, Jahangirnagar University, 3Division of Cell Regeneration and Transplantation, School of Medicine, Nihon University, 4Institute of Genetics and Animal Breeding of the Polish Academy of Sciences, 5Graduate School of Life and Medical Sciences, Doshisha University

JoVE 55077

 Biochemistry

Genome-wide Mapping of Drug-DNA Interactions in Cells with COSMIC (Crosslinking of Small Molecules to Isolate Chromatin)

1Department of Biochemistry, University of Wisconsin–Madison, 2Department of Electrical and Computer Engineering, University of Wisconsin–Madison, 3Graduate Program in Cellular and Molecular Biology, University of Wisconsin–Madison, 4The Genome Center, University of Wisconsin–Madison

JoVE 53510

 Biology
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