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DNA Replication - Amazon S3s3. Semiconservative replication. Conservative replication. Dispersive replication. • In semiconservative replication, the parental DNA strands separate

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  • DNA Replication DNA Strands are templates for DNA synthesis:

     Watson and Crick suggested that the existing strands of DNA served as a template for the producing of new strands, with bases being added to the new strand’s according to complementary base pairing

    Biologists then proposed three alternative hypotheses for how the old and new DNA strands interacted during replication:

     Semiconservative replication.

     Conservative replication.

     Dispersive replication.

    • In semiconservative replication, the parental DNA strands separate and each is used as a template for the synthesis of a new strand. Daughter molecules each consist of one old and one new strand.

    • In conservative replication, the parental molecule serves as a template for the synthesis of an entirely new molecule.

    • In dispersive replication, the parental molecule is cut into sections such that the daughter molecules contain old DNA interspersed with newly synthesized DNA.

    • Meselson and Stahl showed that each parental DNA strand is copied in its entirety, but did not illustrate a mechanism for this process.

    • The discovery of DNA polymerase, the enzyme that catalyzes DNA synthesis, cleared the way for understanding DNA replication reactions.

  • • Arthur Kornberg (et al.) were credited with isolating the first DNA polymerase and in 1959 won the Nobel prize (Physiology/Medicine).

    • A critical characteristic of DNA polymerases is that they

    can only work in one direction.

     DNA polymerases can add deoxyribonucleotides to only the 3′ end of a growing DNA chain. As a result, DNA synthesis always proceeds in the 5′ → 3′ direction.

    • DNA polymerization is exergonic because the monomers that act as substrates in the reaction are deoxyribonucleoside triphosphates (dNTPs), which have high potential energy because of their three closely packed phosphate groups.

    Assume that the following single strand of DNA was

    synthesized using DNA polymerase and standard ATP, GTP, and CTP along with a form of TTP that had each of the three

    phosphates labeled with 32P. 5'—GGTTGAACATGG—3'

    To determine how nucleotides are linked together, an investigator cleaved the newly formed strand with the

    enzyme spleen diesterase, which cleaves DNA at the covalent bond connecting the 5' carbon of the sugar to the phosphate. Which of the resulting nucleotide(s) would you expect to now

    carry the 32P? a. A only b. T only c. T, A, G d. C, T, G, A How does replication get started?

     A replication bubble forms in a chromosome that is actively being replicated

  • o Replicated bubbles grow as DNA replication proceeds, because synthesis is bidirectictional

     In bacterial chromosomes, the replication process begins at a single location, the origin of replication

     Eukaryotes also have bidirectional replication but they have multiple origins of replication and thus have multiple replication bubbles

     A replication fork is the Y-shaped region where the DNA is split into two separate strands for copying

    How is the helix opened and stabilized?

     Several proteins are responsible for opening and stabilizing the double helix o The enzyme helicase catalyzes the breaking of

    hydrogen bonds between the two DNA strands to separate them

    o Then single stranded DNA binding protein (SSBPs) attach to the separated strands to prevent them from closing

     Unwinding the DNA helix creates tension farther down the helix o The enzyme topoisomerase cuts down and rejoins

    the DNA downstream of the replication fork, relieving the tension

    How is the leading strand synthesized?

     DNA polymerase (III) requires a primer – which consists of a few nucleotides binded to the template strand – because it provides a free 3’ hydroxyl group that can combine with an incoming dNTP to form a phosphodiester bond

     Primase, a type of RNA polymerase, synthesizes a short RNA segment that serves as a primer for DNA synthesizes that is on the order of 1-60 nucleotides long (species dependent)

  •  The enzymes product s called the leading strand, or continuous strand, because it leads into the replication fork and is synthesizes continuously in the 5’-3’ direction

    What about the other strand of DNA? (The Lagging strand) how is it replicated?

    - the other DNA strand is called the lagging strand because it is synthesize discontinuously in the direction away from the replication fork and so lags behind the fork - as with the leading strand, synthesis of the lagging strand starts when primase synthesizes a short stretch of RNA that acts a primer. DNA polymerase III then adds bases to the 3’ end of the primer

  • - DNA polymerase III moves away from the replication fork, even though helicase continues to open The discontinuous replication hypothesis

    - states that once primase synthesizes an RNA primer on the lagging strand, DNA polymerase will synthesize short fragments of DNA along the lagging strand, and that these fragments would later be linked together to form a continuous whole

    - This hypothesis was tested by Okazaki and his colleagues, hence these short fragments came to be known as ―Okazaki fragments‖. The Discovery of Okazaki Fragments

    - the lagging strand is synthesized as short discontinuous fragments called Okazaki fragments (1000- 2000 bases long)

    - DNA polymerase I removes the RNA primer at the beginning of each Okazaki fragment and fills in the gap

    - The enzyme DNA ligase then joins the Okazaki fragments to form a continuous DNA strand

    - Because Okazaki fragments are synthesized independently and joined together later, the lagging strand is also called the discontinuous strand

  • DNA synthesizes enzymes are well organized

    - most of the enzymes responsible for DNA synthesis around the replication fork are joined into one large multi- enzyme machine called the replisome

  • What about the replication of the ends of linear chromosomes…leading and lagging strands? Are there differences in somatic and gametic cells? - the region at the end of a linear chromosome is called a telomere. Telomeres consist of short segments of repeating base sequences…in humans this sequence is ―TTAGGG‖ These repeating sequences can be 10K bases long.. - replication of telomeres can be problematic – while leading-strand synthesize results in a normal copy of the DNA molecule, the telomere on the lagging strand shortens during DNA replication Replicating the ends of linear chromosomes from somatic cells… - as the replication fork reaches the end of a linear chromosome, there is no way to replace the RNA primer from the lagging strand with DNA, because there is no available prime for DNA synthesis - this leaves a section of single-stranded DNA on the lagging strand at the end of each new chromosome - this remaining single-stranded DNA is eventually degraded, resulting in shortening of the telomere (and chromosome)

  • Repeating the ends of linear gametic chromosomes - telomeres do not contain genes, but consist of short

    repeating stretches of bases - the enzyme telomerase adds more repeating base sequences to the end of the lagging strand, catalyzing the synthesis of DNA from an RNA template that it carries with it - primase then makes an RNA primer which DNA polymerase uses to synthesize the lagging strand, the RNA primer is removed, and ligase connects the new sequence together - this prevents the lagging strand from getting shorter with each replication

  • Replication in somatic cells - somatic cells normally lack telomerase. The chromosomes of somatic cells progressively shorten as the individual ages - this has led biologists to hypothesize that telomere shortening has a role in limiting the amount of time ells remain in an actively growing state DNA replication at the ends of linear chromosomes requires the special enzyme telomerase because _____. a. DNA polymerase cannot copy all the way to the end of the leading strand b. removal of the last RNA primer on the lagging strand leaves a recessed end that the DNA polymerase cannot fill c. the ends contain repeated DNA sequences that DNA polymerase cannot copy d. the ends contain special histone proteins that inhibit DNA polymerase Repairing mistakes and damages - DNA replication is very accurate, with an average error rate of less than one mistake per billion bases - DNA polymerase is highly selective in matching complementary bases correctly. As a result, DNA polymerase

  • inserts the incorrect base only about once every 100,000 bases added

    - If mistakes remain after synthesis is complete or if DNA is damaged, repair enzymes can remove the defective bases and repair them. How does DNA polymerase proofread? - DNA polymerase can proofread its work – it checks the match between paired bases, and can correct mismatched bases when they do occur - if the enzyme finds a mismatch, it pauses and removes the mismatched bases that was just added. DNA polymerase III can do this because its epsilon subunit acts as an exonuclease that removes mismatched deoxyribonucleotides from newly synthesized DNA - if – in spite of its proofreading ability – DNA polymerase leaves a mismatched pair behind in the