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Department of Biological and Environmental Sciences

Cell & Molecular Biology
Dr. David A. Johnson
Biol 405    4 Credits   Spring 2017  MWF 11:45-12:50 AM   PH

DNA Replication 

DNA must be able to carry out its basis role as the genetic material. What must the genetic material be able to do? 1) carry information, 2) copy that information (replication), 3) give meaning to that information (determine traits). The information is carried in the nucleotide sequence. We will now look at the second of these roles.
  • The Basic Model of DNA Replication: Watson and Crick proposed the basic model for DNA replication at the time they proposed the double helix: the template DNA specifies the nascent DNA sequence by base pairing.
  • DNA Polymerase: In 1956, Kornberg discovered the first DNA polymerase: E. coli's DNA Polymerase I, not the main replication enzyme (it works primarily in DNA repair). DNA replication occurs at the replication fork. This enzyme needs the 4 deoxynucleoside 5'-triphosphates, primer DNA, and template DNA and directs the synthesis of a DNA molecule following the sequence of the template strand. When the nucleotide is added, nucleophilic attack on the 3'-OH of the primer strand's last deoxyribose cleaves off a pyrophosphate and a phosphodiester bond is formed between the 3' O of the primer and the 5' O of the newly added nucleotide. The enzyme undergoes a 3D conformational change when correct base pairing occurs, and then catalyzes the formation of the phosphodiester bond. The enzyme pyrophosphatase cleaves the pyrophosphate into two inorganic phosphates, so the reaction cannot be reversed. NOTE: DNA synthesis is a one way process. The new DNA molecule grows in the 5' to 3' direction. That is, it grow by primer extension at the 3' end. (Nucleotide nomenclature.)

  • Origin of Replication: DNA replication begins at a sequence specific site. In bacteria like E. coli, there is one origin and replication proceeds bidirectionally from that fork (autoradiography photographs). In higher organisms, there are numerous replication origins along each chromosome and replication is bidirectional. Mammalian replication origins are spaced 50 - 300 kb apart (kb=kilobase, 1000 bases). In various tissues and developmental stages, the rate of replication varies. The rate of replication is apparently determined by how many replication forks are activiated, not the speed of fork movement.

  • Semiconservative DNA Replication (pp. 110-111): Watson and Crick's model of DNA replication can be called a semiconservative model, since the newly made molecule has one old strand and one newly made strand. Meselson and Stahl (original paper) proved that DNA replication is semiconservative in E. coli in an experiment using DNA labeled with a heavy isotope of nitrogen (15N versus the normal 14N). Bacteria were grown in growth medium with heavy nitrogen. When these cells were transferred to medium with light nitrogen and allowed to go through one round of replication, their DNA was shown by CsCl density gradient centrifugation to be hybrid in density (between the density of all heavy and all light DNA). These results were consistent with the semiconservative (but not the conservative) model of DNA replication. Further experiments also showed replication was not dispersive. Later, Taylor, Woods, and Hughes showed DNA was semiconservative in eukaryotes (plants) using autoradiography.

  • Continuous versus Discontinuous DNA Replication (Leading Strand versus Lagging Strand Replication), An Overview: The problem of how replication can occur on both strands since the process only goes in one direction was solved by Okazaki with the discovery of Okazaki fragments. In E. coli, these are 1 - 3 kb long fragments synthesized on the lagging strand. In eukaryotes they are only 100 - 200 bp long. Leading strand replication is continuous and does not involve Okazaki fragments. Lagging strand replication is discontinuous and involves Okazaki fragments. Each Okazaki fragment must starts with the synthesis of short 3-10 nt RNA primer by action of the RNA polymerase called primase. These RNA primers will be removed by exonuclease activity and replaced by DNA. Finally, when the growing 3' end of one Okazaki fragment reaches the 5' end of the previous fragment, the resulting nick is sealed by the enzyme DNA ligase. (DNA replication is sometimes said to be semidiscontinuous.)

  • Unraveling the DNA: The action of topoisomerases induce breaks in the DNA ahead of the replication fork to allow for unwinding (swiveling). Type I topoisomerases make single-strand breaks and type II make double-strand breaks. Type II enzymes, like the bacterial gyrase, are needed in replication-related unraveling. These enzymes are also important in untangling DNA molecules during DNA replication and cell division and during crossing over. (Topoisomerase info.)(Topoisomerase I and II video)

  • The Enzymes of Replication: Many enzymes besides DNA polymerase are required to replicate a DNA molecule in vivo. (Video)(more)
    • The DNA Polymerases: This class of enzymes synthesizes the new DNA, adding a nucleotide to the 3' OH of a primer.
      • Prokaryotic DNA Polymerases: There are three DNA polymerases in E. coli: DNA polymerases I, II, and III. DNA polymerase III is the main enzyme of DNA replication, although I is also involved (see below). The main job of DNA polymerases I and II in the process of DNA repair.
      • Eukaryotic DNA Polymerases: Eukaryotes have three polymerases involved in nuclear DNA replication: DNA polymerases α, δ, and ε. A fourth polymerase, γ, is the DNA polymerase that replicates mitochondrial DNA. DNA polymerase δ was formerly thought to be the main replication enzyme, while α is involved with primase. α appears to have DNA-dependent RNA polymerase activity (primase-like enzyme: see Primase below). Some new evidence indicates that ε is the primary DNA polymerase working on the leading stand while δ is the primary enzyme on the lagging strand. A more recent article suggests that while δ is the enzyme responsible for lagging strand replication, it may also be involved with ε in leading strand replication. (So the picture still is not clear.)
    • Primase: This is a DNA-dependent RNA polymerase, like the ones we will see later that make mRNA. Primase synthesizes the short RNA primer and does not need a primer itself, so it can start a new Okazaki fragment. In humans, DNA polymerase α is composed of 4 subunits, two of which begin the primer by synthesizing RNA complementary to the template DNA. However, DNA polymerase α also has DNA polymerase activity and extends the RNA primer by adding DNA nucleotides. Then, it is thought that DNA polymerase δ or ε takes over to synthesize most of the Okazaki fragment. (DNA polymerase α does not have the 3' to 5' exonuclease activity needed for proofreading.)
    • 5' ---> 3' Exonuclease Activity: An enzyme must remove the RNA primer. This is accomplished by some enzyme that has 5' ---> 3' exonuclease* activity. The exonucleases involved in removing the RNA primer remove one nucleotide at at time from the 5' end.
      • Prokaryotes: In E. coli, DNA polymerase I (which has both 5' ---> 3' and 3' ---> 5' exonuclease activity in addition to its polymerase activity) removes the RNA primer and simultaneously synthesizes new DNA to replace it.
      • Eukaryotes: In eukaryotes, a special exonuclease called RNase H appears to work along with other exonucleases to remove the RNA primer in the 5' to 3' directions. DNA polymerase δ then returns to synthesize DNA where the RNA primer was.
    • DNA Ligase: The nick left between two Okazaki fragments must be sealed. DNA ligase joins the two Okazaki fragments. Before ligation, there is a phosphate on the 5' end of one fragment and a hydroxyl on the 3' end of the other. DNA ligase first reacts with ATP and AMP is covalently bond to ligase (releasing pyrophosphatase). The AMP is then transferred to the 5' phosphate of one fragment (adenosine--phosphate--phosphate--then rest of the primer 5' to 3'). Then, nucleophilic attack of the 3' OH releases the AMP, ligating the two Okazaki fragments. (Know the difference between a nick and a gap and a double-strand break.)
    • Single-Stranded DNA Binding Protein: These bind to and prevent single-stranded DNA from collapsing on itself.
    • Helicases: This enzyme breaks the hydrogen bonds holding the two strands together and unwind the two strands at the replication fork.

    • Clamp Protein: Clamp-related proteins recruit and hold DNA polymerase to the helix at the site of polymerization. (Slides down the molecule)
    • Topoisomerases: Topoisomerases are a class of enzymes that induce double-strand breaks or nicks in the DNA backbone to allow for untangling. Type I toposisomerases produce single-strand nicks while type II topoisomerases (like DNA gyrase of E. coli) make double-strand break. Type II are important in the swiveling needed for prokaryote and eukaryote untangling and unwinding during replication. Type II can actually allow two helices to pass through one another.
    • A Video (from your text's web site)
    • DNA replication summary video
  • The Replication of Telomeres and Telomerase: The ends of chromosomes are called telomeres. The end of DNA molecules tend to react with other molecules and cause problems. The loop structure of the telomere helps get around this. Also, DNA replication at the telomere occurs by way of the enzyme telomerase and thereby gets around the 5' ---> 3' problem. Telomerase is a reverse transcriptase that carries its own template RNA (called TERC: telomerase RNA component). TERC in humans is transcribed from an RNA gene on the long arm of chromosome 3 (from a site other than the telomere).
  • Aging and telomeres: Most human somatic cells lack much telomerase activity. The length of the telomere correlates with the number of  divisions remaining before cell death. Cancer cells have telomerase activity. (Some cervical cancers have extra copies of TERC.)
  • 2009 Nobel Prize (Medicine) to Telomere Researchers.
  • Research News: Telomeres, telomerase, and cancer.

*Nucleases are enzymes that digest nucleic acids. Some are specific for DNA and are called DNases. Some DNases digest only double stranded DNA and some digest only single stranded DNA. Some nucleases are specific for RNA and are called RNases. Some are non-specific and digest either DNA or RNA. Nucleases are also classified according to whether they break the nucleic acid backbone internally (endonucleases) or cleave off one nucleotide at a time from one end (exonucleases).

Nucleotide Nomenclature

Nitrogen Base Nucleoside (N Base+Sugar) Nucleotide (N Base+Sugar+PO4) Nucleotide used in RNA synthesis
adenine (A) adenosine adenylic acid
(adenosine monophosphate: AMP)
adenosine triphosphate (ATP)
guanine (G) guanosine guanylic acid
(guanosine monophosphate: GMP)
guanosine triphosphate (GTP)
cytosine (C) cytidine cytidylic acid
(cytidine monophosphate: CMP)
cytidine triphosphate (CTP)
uracil (U) uridine uridylic acid
(uridine monophosphate: UMP)
uridine triphosphate (UTP)
Nitrogen Base Nucleoside (N Base + Sugar) Nucleotide (N Base + Sugar + PO4) Nucleotide used in DNA synthesis
adenine (A) deoxyadenosine deoxyadenylic acid
(deoxyadenosine monophosphate: dAMP)
deoxyadenosine triphosphate (dATP)
guanine (G) deoxyguanosine deoxyguanylic acid
deoxyguanosine monophosphate: dGMP)
deoxyguanosine triphosphate (dGTP)
cytosine (C) deoxycytidine deoxycytidylic acid
deoxycytidine monophosphate: dCMP)
deoxycytidine triphosphate (dCTP)
thymine (T) deoxythymidine deoxythymidylic acid
deoxythymidine monophosphate: dTMP)
deoxythymidine triphosphate (dTTP)

Quote of the Day: Who was the first person to look at a cow and say, "I think I'll squeeze these dangly things and drink whatever comes out?"