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DNA Replication

DNA Replication. Synonyms: Doubling, Duplication, Copying, Biosynthesis and Synthesis. From Monomers to Polymers. Complementary surfaces Watson-Crick base pairs. DNA Replication. Replication of DNA occurs during the process of normal cell division cycles.

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DNA Replication

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  1. DNA Replication Synonyms: Doubling, Duplication, Copying, Biosynthesis and Synthesis

  2. From Monomers to Polymers Complementary surfaces Watson-Crick base pairs

  3. 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.

  4. 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.

  5. 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?

  6. 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.

  7. (a) is a photograph of the tube under UV light. (b) is a graph of darkness of each band

  8. 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.

  9. Biochemical Mechanism of DNA Replication • DNA replication is not a passive and spontaneous process. • Many enzymesare required to unwind the double helix and to synthesize a new strand of DNA.

  10. 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.

  11. 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.

  12. 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.

  13. 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.

  14. 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.

  15. 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).

  16. 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.

  17. 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.

  18. Kornberg’s Success • Kornberg was awarded the Nobel Prize in Medicine in 1959. • http://www.nobel.se/medicine/laureates/1959/

  19. 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….

  20. The real situation is more complex • Polymerase III is the critical DNA polymerase. • Polymerase I is an important repair enzyme

  21. 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.

  22. 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.

  23. 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.

  24. 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?

  25. 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.

  26. 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.

  27. Pulse Chase Experiments reveal DNA is made in short pieces that become longer when combined.

  28. 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.

  29. Other Proof RNA Is Involved • Radioactive UTP is added to the labeled fragments and over time in a pulse chase assay, removed.

  30. 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.

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