DNA Synthesis Guided Discussion

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DNA Synthesis Guided Discussion Introduction The goal of this discussion is for the students to lead a description of DNA synthesis. This discussion can later be extended to describe the process of sequencing by synthesis within the MiSeq DNA sequencer. Visual aids (see images included below) may be helpful in describing the process of DNA synthesis and sequencing by synthesis. (Images for the template strand and primers are currently not included in this document.) DNA synthesis First review the DNA synthesis reaction: One strand of DNA is copied to make a second strand. The sequence of the new strand will be the “reverse complement” of the original sequence. Ask the Students: What components are required for DNA synthesis? What does DNA polymerase need to make a new strand of DNA? Answers should include the following components. Component Role DNA polymerase Enzyme that synthesizes DNA Template DNA that will be copied in the DNA synthesis reaction Short piece of single-stranded DNA that is complementary to the template Primer strand Nucleotides Building blocks of DNA Enzyme that unwinds double-stranded DNA to form single-stranded DNA Helicase, etc. (Helicase and other accessory proteins involved in DNA replication may be (optional) considered optional for the purpose of this discussion. Among the enzymes in replication, DNA polymerase is the most important player.) Walk through the DNA synthesis: 1. Primer binds to complementary region of template DNA 2. DNA polymerase binds to primer 3. Nucleotides randomly float into the active site of the DNA polymerase 4. When complementary nucleotide moves into position, DNA polymerase attaches it to the growing strand 5. Once the correct base has been added, DNA polymerase moves to the next position Additional points: Polymerase can’t begin synthesis until it is bound to primer Polymerase can only add one nucleotide at a time. Polymerase only adds the complementary base (mistakes are possible but rare) This process happens FAST – consider how quickly your cells need to divide. (There are 3 billion bases in the human genome, so even the division of one single cell involves quite a bit of DNA synthesis!) A T C G Laser http://cliparts.co/clipart/2387676 Laser http://cliparts.co/clipart/2387676 DNA Polymerase doi: 10.2210/rcsb_pdb/mom_2000_3 DNA Polymerase doi: 10.2210/rcsb_pdb/mom_2000_3 Camera http://cliparts.co/clipart/14746 Camera http://cliparts.co/clipart/14746 x x .

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Glossary - Cellbiology
1 Glossary - Cellbiology Blotting: (Blot Analysis) Widely used biochemical technique for detecting the presence of specific macromolecules (proteins, mRNAs, or DNA sequences) in a mixture. A sample first is separated on an agarose or polyacrylamide gel usually under denaturing conditions; the separated components are transferred (blotting) to a nitrocellulose sheet, which is exposed to a radiolabeled molecule that specifically binds to the macromolecule of interest, and then subjected to autoradiography. Northern B.: mRNAs are detected with a complementary DNA; Southern B.: DNA restriction fragments are detected with complementary nucleotide sequences; Western B.: Proteins are detected by specific antibodies. Cell: The fundamental unit of living organisms. Cells are bounded by a lipid-containing plasma membrane, containing the central nucleus, and the cytoplasm. Cells are generally capable of independent reproduction. More complex cells like Eukaryotes have various compartments (organelles) where special tasks essential for the survival of the cell take place. Cytoplasm: Viscous contents of a cell that are contained within the plasma membrane but, in eukaryotic cells, outside the nucleus. The part of the cytoplasm not contained in any organelle is called the Cytosol. Cytoskeleton: (Gk. ) Three dimensional network of fibrous elements, allowing precisely regulated movements of cell parts, transport organelles, and help to maintain a cell’s shape. • Actin filament: (Microfilaments) Ubiquitous eukaryotic cytoskeletal proteins (one end is attached to the cell-cortex) of two “twisted“ actin monomers; are important in the structural support and movement of cells. Each actin filament (F-actin) consists of two strands of globular subunits (G-Actin) wrapped around each other to form a polarized unit (high ionic cytoplasm lead to the formation of AF, whereas low ion-concentration disassembles AF).
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Paul Modrich Howard Hughes Medical Institute and Department of Biochemistry, Duke University Medical Center, Durham, North Carolina, USA
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Chromosome Replication Duringmeiosis
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Arthur Kornberg Discovered (The First) DNA Polymerase Four
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Control of Eukaryotic DNA Replication Initiation—Mechanisms to Ensure Smooth Transitions
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De Novo DNA Synthesis Using Polymerase- Nucleotide Conjugates
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DNA Replication
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De Novo DNA Synthesis Using Polymerase
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Helicase-DNA polymerase interaction is critical to initiate leading-strand DNA synthesis Huidong Zhang1, Seung-Joo Lee1, Bin Zhu, Ngoc Q. Tran, Stanley Tabor, and Charles C. Richardson2 Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA 02115 Contributed by Charles C. Richardson, April 27, 2011 (sent for review March 3, 2011) Interactions between gene 4 helicase and gene 5 DNA polymerase (gp5) are crucial for leading-strand DNA synthesis mediated by the replisome of bacteriophage T7. Interactions between the two pro- teins that assure high processivity are known but the interactions essential to initiate the leading-strand DNA synthesis remain uni- dentified. Replacement of solution-exposed basic residues (K587, K589, R590, and R591) located on the front surface of gp5 with neu- tral asparagines abolishes the ability of gp5 and the helicase to mediate strand-displacement synthesis. This front basic patch in gp5 contributes to physical interactions with the acidic C-terminal tail of the helicase. Nonetheless, the altered polymerase is able to replace gp5 and continue ongoing strand-displacement synthesis. The results suggest that the interaction between the C-terminal tail of the helicase and the basic patch of gp5 is critical for initiation of strand-displacement synthesis. Multiple interactions of T7 DNA polymerase and helicase coordinate replisome movement. DNA polymerase-helicase interaction ∣ strand-displacement DNA synthesis ∣ T7 bacteriophage ∣ T7 replisome acteriophage T7 has a simple and efficient DNA replication Bsystem whose basic reactions mimic those of more complex replication systems (1). The T7 replisome consists of gene 5 DNA polymerase (gp5), the processivity factor, Escherichia coli Fig.
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Part 2 - Modeling DNA Function: Replication Questions to Jumpstart Your Thinking 1
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