Quiz Me!	DNA Structure and Replication
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Last revised: Friday, October 15, 1999
Reading: Ch. 16 in text

Note: These notes are provided as a guide to topics the instructor hopes to cover during lecture. Actual coverage will always differ somewhat from what is printed here. These notes are not a substitute for the actual lecture!

Copyright 1999. Thomas M. Terry

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Types of Nucleic Acids

• Nucleic acids function primarily as informational molecules, for the storage and retrieval of information regarding the primary sequence of polypeptides.
• There are two types of nucleic acids:
  1. Deoxyribonucleic acid (DNA), which serves as a cellular database by storing an immense amount of information regarding all possible polypeptides a cell can make.
  2. Ribonucleic acid (RNA), which occurs in several different forms (messenger RNA, ribosomal RNA, transfer RNA) and is needed to convert DNA information into polypeptide sequences. In some viruses, RNA serves as the primary database with no DNA involvement. Certain RNAs have catalytic ability similar to that of protein enzymes; these are called ribozymes.

Nucleotide anatomy

• Nucleic acids are built from subunits called nucleotides.
• Each nucleotide includes three components:
  1. a ring-shaped molecule belonging to the class of purine or pyrimidine bases
  2. a 5-carbon, or pentose, sugar
  3. one or more phosphate groups

View anatomy of a nucleotide

Purines & Pyrimidines

• Every nucleotide contains a nitrogenous base. These bases are classified as purines (two ring-shaped molecules joined together, one with 6 and one with 5 atoms) and pyrimidines (a single ring made from 6 atoms).
• In DNA, there are four different bases: Adenine (A) and Guanine (G) are the larger purines. Cytosine (C) and Thymine (T) are the smaller pyrimidines. These are frequently symbolized by their single letter abbreviations.
• RNA also contains four different bases. Three of these are the same as in DNA: Adenine, Guanine, and Cytosine. RNA contains Uracil (U) instead of Thymine (T).

View purines and pyrimidines

How did Purines and Pyrimidines evolve? A possible origin for Adenine.

• At first glance, molecules such as adenine look very complex. How did they evolve to become part of nucleic acids? We can only speculate about such questions, but there are reasons for thinking that adenine is not all that complex a molecule.
• We know that the primitive Earth evolved around 4.5 billion years ago, and that initially there was no free oxygen. Compounds such as water (H₂O), ammonia (NH₃) and methane (CH₄) were abundant, as was energy in the forms of heat, UV radiation, lightning, radioactivity, etc.
• Scientists have tried simulating such environments, boiling mixtures of gases and water with electric spark discharges. In some such experiments, purine and pyrimidine bases have been formed! A possible mechanism is shown in the accompanying diagram. This is not the way adenine is synthesized in cells today, but it suggests why such molecules may have been available in the primitive earth when life first evolved.

View Possible Mechanism for Adenine evolution

Ribose & Deoxyribose

• The sugars found in nucleic acids are pentose sugars, with five Carbon atoms.
• Ribose, found in Ribonucleic acid (RNA), is a "normal" sugar, with one oxygen atom attached to each carbon atom.
• Deoxyribose, found in Deoxyribonucleic acid (DNA), is a modified sugar, lacking one oxygen atom (hence the name "de-oxy"). This difference of one oxygen atom is an important one for the enzymes which recognize DNA and RNA, allowing these two molecules to be easily distinguished inside organisms.

View Ribose and Deoxyribose

Phosphate

• Phosphate groups can be joined together to form phosphodiester bonds.
• Nucleotides typically have one, two, or three phosphate groups, and are named monophosphate, diphosphate, or triphosphate accordingly.
• When phosphate groups are joined together, they have a strong tendency to repel each other, because of the high concentration of negative charge in the very polar and usually ionized oxygen atoms. As a result, molecules with two or three phosphate groups are good energy donors, readily releasing energy along with the transfer of phosphate groups. Nucleotides such as ATP and GTP are used not just for RNA or DNA synthesis, but also as energy donors for many cellular reactions.

View Phosphate groups

ATP, ADP, AMP

• Let's examine a set of nucleotides built with the purine base Adenine, the sugar deoxyribose, and one, two, and three phosphate groups.
• A combination of a base and a sugar is called a nucleoside.
• When the base Adenine is added to the sugar deoxyribose, the resulting nucleoside is deoxyadenosine. When one or more phosphates are added to this nucleoside, we have a nucleotide.
• We can name nucleotides by combining the nucleoside name (Deoxyadenosine) with the number of phosphates (mono-, di-, or tri-phosphate), as shown in the figure.
  1. Adenosine monophosphate, or AMP
  2. Adenosine diphosphate, or ADP
  3. Adenosine triphosphate, or ATP

View nucleotides

The following tables gives the names of bases and their corresponding nucleosides and nucleotides

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Nucleotides involved in DNA

Base	Deoxyribonucleoside	Deoxyribonucleotides
Adenine	Adenosine	dAMP, dADP, dATP
Cytosine	Cytidine	dCMP, dCDP, dCTP
Guanine	Guanosine	dGMP, dGDP, dGTP
Thymine	Thymidine	dTMP, dTDP, dTTP

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Nucleotides involved in RNA

Base	Ribonucleoside	Ribonucleotides
Adenine	Adenosine	AMP, ADP, ATP
Cytosine	Cytidine	CMP, CDP, CTP
Guanine	Guanosine	GMP, GDP, GTP
Uracil	Uridine	UMP, UDP, UTP

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DNA and RNA

• Nucleotides can serve as monomers for the assembly of polymeric nucleic acids. When the nucleotides contain deoxyribonucleotides, the polymer is deoxyribonucleic acid, or DNA. DNA is normally double-stranded (although some viruses contain single-stranded DNA).
• Each strand of DNA consists of a "backbone" of alternating units of phosphate and deoxyribose. Purine or pyrimidine bases are attached to the 5-C deoxyribose sugar, and form base pairs with purine or pyrimidine bases from the opposite strand. The only effective pairs are Adenine with Thymine (A-T pairs) and Guanine with Cytosine (G-C pairs).

  View DNA model
  View interactive PDB file of DNA (if Chime plug-in is installed)

• When ribonucleotides serve as monomers, the resulting polymer is ribonucleic acid, or RNA. RNA is normally single-stranded (although some viruses contain double-stranded RNA). Many bases in RNA molecules such as ribosomal RNA and transfer RNA are chemically modified after polymerization, a process which makes these molecules more stable.
• An RNA consists of a "backbone" of alternating units of phosphate and deoxyribose. Purine or pyrimidine bases are attached to the 5-C ribose sugar.

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  Evidence for DNA as the genetic material

  • Early in this century, little known about DNA; regarded as uninteresting junk
  • Proteins were thought to be the only truly complex molecules in cells, and therefore must be responsible for heredity
  • 1928: Frederick Griffith discovered phenomenon of transformation in bacteria
    1. Used organism Streptococcus pneumoniae.
    2. Strep bacillus has two forms:
       1. slimy colonies (S strain) forms mucous capsules, survives attack by macrophages in lung, kills mice
       2. rough colonies (R strain) lacks capsules, quickly killed by macrophage, no disease
    3. When Griffith mixed heat-killed S-strain with live R-strain, resulting organisms killed mice, and lungs were filled with S-strain.
    4. View diagram of Griffith experiment
    5. Conclusion: some chemical is surviving heat treatment, retains genetic information, is able to transmit that information to some R-strain bacteria, convert them to S. Griffith didn't know what was responsible.
  • 1944: Avery, McCarty and MacLeod demonstrate that DNA is responsible for transformation in bacteria
    1. Fractionated different chemicals in S-strain bacteria, tested each separately to see what would cause transformation.
    2. Isolated DNA could transform, but no other isolated fraction could (RNA, protein, lipids, polysaccharides). Conclusion: DNA is transforming principle
    3. But critics attacked slight (<1%) presence of protein in the DNA extracts used, claimed this might be responsible for transformation
  • 1952: Hershey & Chase prove that only DNA is responsible for bacterial virus infection of host cells.
    1. Viruses (called phage if host cells are bacteria) are much simpler than cells, contain only DNA & protein.
    2. H&C were able to use different radioactive isotopes to distinguish DNA from protein: for DNA, used P-32 (lots of P in DNA, but none in protein); for protein, used S-35 (proteins contain S in certain amino acids, but DNA lacks S).
    3. H&C grew phage in hosts with either P or S radioisotope. Then infected different bacteria for short time, vortexed in blendor to separate phage coats from cells, and separated phage (very small) from cells (larger) by centrifugation.
    4. Result: only P-32 isotope found in cells. All S-35 could be knocked loose by blending, but cells were still infected and produced new phage. Therefore only DNA, not protein, was responsible for inheritance.
    5. View diagrams of H&C experiment (scroll down the page)

  Structure of DNA

  • DNA known to contain purine & pyrimidine bases, deoxyribose, and phosphate -- but how are they arranged?
  • 1947: Chargaff published data showing that % of A, T, C, and G showed certain regularities
    1. % of bases varies from organism to organism
    2. % A = % T, and % C = % G. This is called Chargaff's rule. What did it mean?
  • 1952: X-ray pictures of DNA taken by Rosalind Franklin in Wilkins lab in London showed some kind of helix.
  • 1953: Watson & Crick published a model built from Franklin data = double helix. Suggested that Chargaff's rule was due to base-pairing of A with T, C with G.
  • Interact with DNA as Chime graphic
  • In linear molecule, one strand has free 3'-end, where the other (complementary) strand has 5'-end.
  • View 3' and 5' ends of DNA (from The Biology Place)
  • Two chains of DNA face in opposite directions, called antiparallel (defined by which way 3' and 5' sides of sugar molecule are facing). In linear molecule, one strand has free 3'-end, where the other (complementary) strand has 5'-end.
    

    5'-CAGCTAGAGTCATCG-3'
    3'-GTCGATCTCAGTAGC-5'
    

  • W&C also suggested simple model for replication: if double stranded DNA uncoiled, each strand could serve as template for replication of new DNA. This was an exciting experimental prediction, and many labs set out to try to prove it.

  Replication of DNA

  • First enzyme isolated by Kornberg (----> Nobel prize): DNA polymerase.
    1. Reaction: [dATP, dCTP, dGTP, dTTP] -------(DNA polymerase, Mg⁺⁺, template DNA)--------> new DNA + P~P (pyrophosphate)
    2. Note 1: P~P is immediately split into 2 P_i (inorganic phosphate ions).
    3. Note 2: energy for forming new sugar-phosphate bond comes from splitting a high-energy phosphate bond as P~P is removed. This always occurs at free 3'-OH group on deoxyribose (and on ribose in RNA synthesis). All nucleic acids grown by addition at 3'-end, not at 5'-end. Often referred to as 5' -----> 3' synthesis.
    4. View animation of DNA synthesis (from The Biology Place)
  • Eventually discovered that cells have a variety of DNA polymerase enzymes; some serve for DNA repair rather than for new synthesis.
  • Other enzymes and proteins involved:
    1. DNA helicase: unwinds DNA in front of opening replication fork (otherwise DNA would quickly tangle). Uses ATP, makes single-stranded cut, allows one strand to swivel freely around the other.
    2. RNA primase: DNA polymerase III cannot start a growing chain from scratch; needs a short primer (a few nucleotides) to add to. This is carried out by DNA-dependent RNA primase, makes very short piece of RNA by base-pairing RNA nucleotides with template DNA.
    3. DNA polymerase : adds new nucleotides at free 3'ends of growing chain, uses base-pairing rules to insert complementary nucleotides (A opposite T, G opposite C, etc.) Can keep on adding indefinitely for millions of nucleotides if not blockage. Also removes RNA primers, fills in gaps by base pairing, inserts new DNA nucleotides to replace RNA primer. (several types of this enzyme)
    4. DNA ligase: seals any gaps where adjacent nucleotides on one strand have not been covalently joined.
  • Note: many gaps result on lagging strand (see below), so lots of need for enzymes (5) and (6).

  Leading and Lagging strands

  • Since two strands in DNA are antiparallel, new DNA must be synthesized in opposite directions on the two template strands.
  • But overall, DNA must unwind in one direction (at replication fork), overall DNA synthesis has one direction.
  • No problem for the strand growing in same direction as unwinding = leading strand. Can make one long, continuous piece of DNA
  • Big problem for strand growing in opposite direction to unwinding = lagging strand; must grow away from unwinding. As new template is opened up by DNA unwinding, will have to start a new copy.
  • In fact, just this situation was discovered experimentally by Okazaki; found many short DNA fragments newly synthesized from lagging strand = Okazaki fragments. Must be joined together by DNA ligase to make continuous DNA strand.
  • View animation of DNA synthesis on both template strands of DNA helix (from The Biology Place).

  DNA repair

  • Any damage to DNA would be lethal. Cells often spend much more energy repairing DNA than synthesizing it.
    1. Correcting damage due to enviromental effects
       • Example: UV light --> thymine dimers. Energy in UV links thymine where it occurs side-by-side on one strand of DNA, screws up the ability of this bit of DNA to serve as template for replication or for correct reading of proteins.
       • One good 4-hour day at beach ---> 10 UV-induced errors in DNA of every skin cell
       • Your skin cells spend lots of energy patrolling DNA, detecting such errors, cutting them out, and using the remaining good strand as a template for repair synthesis.
    2. Correcting errors during replication (proofreading)
       • When new DNA is synthesized, occasional errors in base pairing occur with frequency ~ 1 in 10,000 base pairs
       • If not corrected, could lead to mutations, loss of functions, loss of competitiveness, evolutionary weeding out.
       • Proofreading carried out by DNA polymerases enzymes; if base mismatch spotted, cut out new bases (keep track of which is template strand and which is new strand during replication), resynthesize copy strand from that neighborhood of template.

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