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Information about IntroDNACloningDNARep

Published on October 16, 2007

Author: Jolene


DNA Replication:  DNA Replication Synonyms: Doubling, Duplication, Copying, Biosynthesis and Synthesis From Monomers to Polymers:  From Monomers to Polymers Complementary surfaces Watson-Crick base pairs DNA Replication:  DNA Replication Replication of DNA occurs during the process of normal cell division cycles. The DNA of the daughter cells must be the same as the parental cell. DNA replication must possess a very high degree of fidelity. The entire process of DNA replication is complex and involves multiple enzymatic activities. DNA Synthesis:  DNA Synthesis Biochemical process of making new DNA strand from nucleotides using various enzymes and a DNA molecule as a template. All DNA replication uses DNA synthesis, but not all DNA synthesis need give complete DNA replication of DNA. Sometimes referred to a “biosynthesis” since you can make “synthetic” DNA using a synthesis machine and chemicals. Template Replication Model:  Template Replication Model The two strands could separate from one another, each still containing the complete information, and synthesize a new strand. Did the two strands unwind and each act as a template for new strands? Did the strands not unwind, but somehow generate a new double stranded DNA copy of entirely new molecules? Or was there some combination of both? A Classic Experiment:  A Classic Experiment In 1957 Messelson and Stahl added heavy nitrogen (15N) to bacteria and arrested growth at various times. Heavier 15N DNA could be separated from lighter 14N DNA in Cesium Chloride gradient centrifugation. This was a very important method used in other experiments. Slide13:  (a) is a photograph of the tube under UV light. (b) is a graph of darkness of each band Results of the Experiment:  Results of the Experiment Results show that after one generation, the double stranded DNA is 1/2 heavy (from the parent) and 1/2 light (newly synthesized). This result is predicted by semiconservative replication. Conclusion- (as predicted by Watson and Crick) DNA strands serve as templates for their own replication. Biochemical Mechanism of DNA Replication:  Biochemical Mechanism of DNA Replication DNA replication is not a passive and spontaneous process. Many enzymes are required to unwind the double helix and to synthesize a new strand of DNA. Biochemical Mechanism of DNA Replication:  Biochemical Mechanism of DNA Replication Enzymes are biochemical catalysts- They speed up a reaction without being consumed by the reaction. Enzymes break and form covalent bonds. Every chemical event in the cell depends on the action of an enzyme. Studying DNA Replication:  Studying DNA Replication In Vitro Extracts: Biochemists break open cells and “extract” their contents. They use biochemical techniques to separate the proteins based on size and charge as well as their ability to bind to DNA. “ A biochemist devoted to enzymes could, if persistent, reconstitute any metabolic event in the test tube as well as the cell does it.” Arthur Kornberg Nobel Prize for DNA Replication. DNA Polymerase I Discovery :  DNA Polymerase I Discovery Kornberg decided to study bacteria, reasoning that they replicated DNA rapidly. Whatever enzymes was required must be present in simple bacterial cells. He took rapidly growing bacteria and broke them open. The DNA replication enzymes were separated into fractions and each fraction was assayed to identify the DNA polymerase enzyme. Studying DNA Replication:  Studying DNA Replication Scientists need an assay or test to determine if their extracts are making DNA. Basic assay for DNA replication is to add various DNAs to a tube with extracts, nonradioactive dNTPs and one radioactive dTTP. Separate the small dNTPs from the bigger radioactive DNA pieces and measure radioactivity incorporated. Pulse-Chase Assays :  Pulse-Chase Assays Add the radioactively labeled molecules for a short time period (pulse). Add a large amount of nonradioactive (cold) molecules of the same sort after the short time (chase). Molecules synthesized during the pulse will be labeled and chase molecules will not be labeled. Combining In Vitro Extracts With Genetics:  Combining In Vitro Extracts With Genetics Kornberg combined biochemistry and genetics to study DNA replication. Isolated "conditional mutant bacteria" which were temperature sensitive; Temperature sensitive bacteria made DNA and replicated the bacterial chromosome at 37° C (permissive) but couldn’t at 42° C (restrictive). Two Kinds of Conditional Mutants:  Two Kinds of Conditional Mutants Quick stop mutants ­ at a shift to restrictive temperature, DNA synthesis stopped immediately along with replication of the bacterial chromosome. This was due to a malfunction in the elongation machinery itself or in the enzymes required for essential precursors Slow stop mutants ­ at a shift to restrictive temperature, the cell is able to finish replication of the bacterial chromosome but couldn’t begin another round. In Vitro Complementation:  In Vitro Complementation If normal enzyme extract is added to a template DNA, replication occurs. If an extract from conditional mutants cells is added to a template, no replication occurs. If this same mutant extract is added along with purified normal proteins, replication can take place if the new purified enzyme replaces the mutant enzyme. Kornberg’s Success:  Kornberg’s Success Kornberg was awarded the Nobel Prize in Medicine in 1959. Mutation Analysis Reveals Polymerase I Can’t Be the Only DNA Polymerase:  Mutation Analysis Reveals Polymerase I Can’t Be the Only DNA Polymerase These types of heat sensitive mutants revealed that Kornberg’s Polymerase I could be mutated. The bacterial cells were weak growers but still lived. Scientists reasoned there must be more to the situation. No, he didn’t have to give back the Nobel Prize…. The real situation is more complex:  The real situation is more complex Polymerase III is the critical DNA polymerase. Polymerase I is an important repair enzyme Slide30:  New DNA strands are always synthesized in the 5' to 3' direction. The 5' triphosphate can only be added to a free 3'OH of deoxyribose. Why Does DNA Polymerase Only Add Nucleotides to 3‘ OH? :  Why Does DNA Polymerase Only Add Nucleotides to 3‘ OH? A triphosphate is required to provide energy for the bond between a newly attached nucleotide and the growing DNA strand. However, this triphosphate is very unstable and can easily break into a monophosphate and an inorganic pyrophosphate, which floats away into cell. Why Does DNA Polymerase Only Add Nucleotides to 3'? :  Why Does DNA Polymerase Only Add Nucleotides to 3'? At the 5' end of the DNA, this triphosphate can easily break. If a strand has been sitting in the cell for a while, it would not be able to attach new nucleotides to the 5' end once the phosphate had broken off. The 3' end only has a hydroxyl group, so as long as new nucleotide triphosphate are always brought by DNA polymerase, synthesis of a new strand can continue no matter how long the 3' end has remained free. This Presents a Problem:  This Presents a Problem One strand of the double helix is 5' to 3' and the other one is 3' to 5'. How can DNA polymerase synthesize new copies of the 5' to 3' strand, if it can only travel in one direction? DNA Synthesis Occurs at Replication Forks.:  DNA Synthesis Occurs at Replication Forks. Synthesis is bidirectional from a replication origin (or origins). Replication forks are highly organized structures in cells (DNA synthesis "machines"). What is going on in the fork? Several models were proposed. Slide37:  The two antiparallel strands are replicated simultaneously in both directions and semi-discontinuously. RNA primers are used to initiate a new strand. DNA polymerase uses the RNA primers as starting points to synthesize progeny DNA strands. Both strands are replicated 5’ to 3’ direction. Slide39:  Pulse Chase Experiments reveal DNA is made in short pieces that become longer when combined. Adding Other Enzymes to In Vitro Extracts:  Adding Other Enzymes to In Vitro Extracts RNases degrade RNA. DNases degrade DNA. They can be single or double-stranded DNase. Make an In Vitro extract and add these enzymes. This showed Kornberg and others that there could be no replication if there was not RNA made. Other Proof RNA Is Involved:  Other Proof RNA Is Involved Radioactive UTP is added to the labeled fragments and over time in a pulse chase assay, removed. Helicase Function:  Helicase Function Helicase unwinds duplex DNA at the replication fork. DnaB helicase is a 5' to 3' helicase that travels along the lagging single strand as it unwinds upstream duplex DNA. This creates two antiparallel DNA single strands. The leading DNA strand is the template for DNA polymerase III holoenzyme, which synthesizes a continuous strand. Primase Makes the RNA Primers:  Primase Makes the RNA Primers The function of primase is to initiate, or prime, DNA synthesis. Primase is required because DNA polymerases cannot initiate polymer synthesis; DNA polymerases can only elongate polymers that have already been initiated. RNA polymerases can initiate polymer synthesis in addition to being able to elongate. There are two RNA polymerases in E. coli and the one involved in DNA replication is called primase. Electron Micrographs and Pulse Chase:  Electron Micrographs and Pulse Chase Label the molecules with short burst of radioactivity. Take electron micrograph pictures of DNA molecules and look at where the radioactivity is. Use short circles of DNA called plasmids. How Does the Enzyme Complex Know Where to Start Replication?:  How Does the Enzyme Complex Know Where to Start Replication? Origin of Replication:  Origin of Replication Strand separation occurs at what is termed an "origin of replication". Bacteria typically have a single origin of replication. Eukaryotes have many origins of replication in each chromosome. Viruses have an origin of replication. Abbreviation is ORI. OBR= Origin or Bidirectional Replication = ORI:  OBR= Origin or Bidirectional Replication = ORI Genetically defined origins of replication (ori) are DNA sequences that are necessary and sufficient for initiation of replication In most cases, the molecularly defined OBR's and the genetically defined oris coincide. A few cases exist, however, where an ori causes bidirectional replication to initiate in an adjacent region. Slide57:  Bidirectional Circular DNA Replication ORIs:  ORIs DnaB can’t find the origin of replication by itself; it needs some help DnaA binds to the specific sequences that define the origin (oriC) and separates the strands yielding an open complex DnaC delivers DnaB to this template One DnaB hexamer clamps around each single strand forming the pre-priming complex Terminators:  Terminators Terminators function unidirectionally and stop replication as the fork passes through those sequences. The second sequence of the pair is thought to act as a back-up, in case the first fails. When two forks collide, either between the two pairs of termination signals, or at a termination signal, the forks are annihilated. Concatenated circles should result. Electron Micrograph Information:  Electron Micrograph Information The replication bubble enlarges as replication proceeds. The double-stranded DNA template is not completely denatured before DNA synthesis begins. Unwinding and synthesis are coupled. This is indicated by the uniformly equal thicknesses of the bubble walls. Electron Micrograph Information:  Electron Micrograph Information The DNA template at the replication fork must be unwound to allow access by the DNA polymerase and DNA synthesis. An enzyme activity is needed to relieve superhelical stress that results from the unwinding process. Otherwise, the newly replicated bubble would be extensively interwound. DNA Replication: Important Points:  DNA Replication: Important Points DNA polymerases cannot melt or unwind duplex DNA- they need help from helicases and gyrases in vivo or heating in vitro. DNA polymerases cannot initiate chains,but can only extend a pre-existing DNA or RNA strand. DNA replication can only start at an origin of replication. All DNA strands grow in a 5’ to 3’ direction during replication. Slide67:  The central dogma of molecular biology: DNA makes RNA makes Protein is also violated by some viruses that use RNA to make DNA. Slide68:  In 1975 Howard Temin was given Nobel Prize for discovery of the enzyme reverse transcriptase which used RNA as a template to make DNA in certain viruses. Slide69:  Retroviruses have a single stranded RNA genome that is converted to double stranded DNA in three steps: RNA is the template for RT-catalyzed synthesis of an RNA-DNA duplex. The RNA is degraded by the RNaseH activity of the viral RT. The DNA is copied (by host cell enzymes) to produce double stranded DNA. In this form, the retroviral genome can be inserted into the cellular chromosomes. Slide70:  Host-cell RNA polymerase is used to make virus-related RNA. These RNA strands serve as templates for making new copies of the viral chromosomal RNA and serve also as mRNA. Slide71:  Reverse Transcriptase lacks a proofreading 3' --> 5' exonuclease. “Error-prone replication" leads to mutations in all HIV-1 genes. The rapidly changing genome that results, is a difficult moving target to hit with therapeutic drugs

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