DNA Structure and Replication - 1 The genetic molecule is ... · DNA Structure and Replication - 1 The genetic molecule is DNA. DNA (along with special proteins) is found in chromosomes

DNA Structure and Replication - 1

The genetic molecule is DNA. DNA (along with special proteins) is found inchromosomes. All cell activities are ultimately controlled by DNA. We also knowthat the DNA in each of our cells is identical since DNA molecules duplicate prior tocell division ensuring that the new cells formed are genetically identical to theoriginal cell.

We also know that the metabolic activities of a cell are all catalyzed by enzymes,specific proteins, and that the instructions for the synthesis of proteins are foundin the structure of DNA.

To continue with our knowledge, a gene is a functional region of DNA that specifiesa certain inheritable characteristic or trait. This region of DNA stores theinformation that specifies the sequence of amino acids that form a specificpolypeptide. The genes we inherit from our parents determine the polypeptides wesynthesize in our cells, which determine the structure and functioning of our cellsand tissues.

What DNA is and how DNA works is the subject of this section. We will look at thestructure and functions of DNA, how the information stored in DNA is used todirect cell activities and how cells regulate the activity of their genetic molecules.We shall also see how DNA duplicates itself prior to cell division.

The search for the molecule of inheritance spanned a century from the mid-1850'sto 1953, when Francis Crick and James Watson announced they had determinedthe three dimensional structure of DNA.

DNA was first isolated by Meischer in the mid-1800's. He identified a phosphoruscontaining acid found in the nuclei of cells. About the same time Feulgen developeda stain that was selective for this material of the nucleus. Fuelgen noted that thevolume of the nuclear material was the same for all body (somatic) cells, butgametes had half as much of this material. He also noted that cells that wereabout to divide had twice as much nuclear material. No one knew how to interpretthis information and nothing much happened in molecular genetics for the nextfifty years.

Although genes, chromosomes and the transmission of genetic information werestudied extensively in the first half of the 20th century, the molecular structure ofa gene was not known, nor had anyone actually shown that genetic material waseven in the nucleus.


Genetic Material is in the NucleusIn the 1930's Hammerling did a set of experiments using an alga, Acetabularia,that demonstrated that the information needed to express genetic traits waslocated in the nucleus. Acetabularia was a good choice since it is a single-celledorganism that is large (5cm) and has three morphologically distinct regions – a cap,stalk and base. The nucleus is in the base. In addition, different species ofAcetabularia are found. Hammerling transplanted stalks from one species to asecond, and the new caps regenerated were dictated by the base, not by the stalk.

Hammerling's work was confirmed in 1952 with frog nucleus transplantexperiments conducted to determine how long a cell remained totipotent (fullgenetic competence) during development. Although few frogs developed fromthese transplant experiments (the same process we use today to clone animalsand will be revisited later), those that did develop contained nuclear components ofthe transplanted nucleus, not the nuclear components of the donated enucleatedegg.

Also in the 1950's, FC Steward at Cornell University successfully cultured carrotsfrom single cells taken from the root. This was the first laboratory "clone".Although the plant cells used were differentiated root tissue cells, they weretotipotent – no genetic material had been permanently lost or "turned off".Steward demonstrated that a whole organism can be developed from a singlesomatic cell.


Discovering the Genetic MoleculeThe search for the genetic molecule was focused on the nucleus by the early1900's. It was already known that the nucleus contained concentrations of twomacromolecules: proteins and nucleic acids. In the early 20th century mostscientists believed that the genetic molecule had to be protein –because ofprotein's diversity of structures and specificity of functions. In contrast, DNA iscomposed of some fairly simple molecules: phosphates, a five-carbon sugar, andfour different nucleotides, so the means by which it could serve as the geneticmolecule was perplexing.

Evidence #1In 1928, Fred Griffith was trying to find a vaccine to protect against a pneumonia-causing bacterium, Streptococcus pneumoniae. He isolated two strains of thebacterium. One had a polysaccharide capsule that gave it a smooth (S) appearancein culture. The other form appeared rough (R) in culture. The S form is a virulentform of the bacterium, since the capsule protects it from harmful things in itsenvironment, which in this case is the immune system of the host.

Griffith injected bacteria into mice, and observed what happened. Mice injectedwith S forms died. Mice injected wit R forms lived. Mice injected with heat-killed Sforms lived. But: Mice injected with a mixture of heat-killed S forms and live Rforms died, and when necropsied, contained live S form bacteria.

What did this mean?1 . The production of a capsule is an inheritable trait that distinguishes the R

form from the S form of the bacterium.2 . Somehow, the heat which killed the S cells did not damage the material that

had genetic instructions so that this material (instructions on how to make acapsule) could be incorporated into the living R cells (The R cells could pick upthis material from the environment) transforming these R cells intovirulent S forms.

3 . Griffith called this the Transformation Principle.

Today, transformation is defined as the process by which external DNA isassimilated into a cell changing its genotype and phenotype. Transformation is oneof the processes used in DNA technologies.


Evidence #2Starting in the 1930's, a group of microbiologists, headed by Oswald Avery,repeated Griffith's experiments adding a series of enzymes (from the pancreas)that selectively destroyed DNA, RNA or protein.

They performed the following experiments:

1 . Mice + Protein-digesting enzyme + heat-killed S + R --> Dead Mice2 . Mice + RNA-digesting enzyme + heat-killed S + R ----> Dead Mice3 . Mice + DNA-digesting enzyme + heat-killed S + R ----> Live Mice

In 1944, Avery concluded that DNA was the genetic molecule.Transformation was prevented only when DNA was destroyed. Many scientists stilldisputed this conclusion, since the structure of DNA was not known, and Averycould not say how DNA might work.

Evidence # 3Bacteriophages (viruses that invade bacteria and convert the bacteria into virusmaking machines) proved to be the means by which the question was finallyanswered. In 1952, Hershey and Chase (and others) confirmed that DNA was thegenetic molecule. Viruses have just DNA (or sometimes just RNA) and a proteincoat. Proteins contain sulfur, but not phosphorus and DNA contains phosphorus,but not sulfur.

Hershey and Chase used radioactive Sulfur and Phosphorus to "label" T2 phages.They then tracked the invasion of phages into host bacteria (a strain of E coli) andwhat part of the new generation phages became radioactive. Since only the DNA ofthe new generation of phages was radioactive, Hershey and Chase were ableto confirm that DNA was the genetic molecule.


Still, the structure of DNA was unknown, so no one had an explanation for how DNAcould do its job. The search continued.

Structural Evidences supporting DNA as the Genetic MoleculeDemonstrating that DNA was the genetic molecule was one significant part of thesolution. To know how DNA works also required knowledge of the threedimensional structure of the molecule.

By the early 1950's the following was known about the DNA molecule:1 . DNA was composed of nucleotides. Each nucleotide contained:

• Phosphate (P)• The 5-carbon sugar, deoxyribose• One of four different nitrogen-containing bases

Two were double ring purinesAdenine Guanine

Two were single ring pyrimidinesThymine Cytosine


The sugar phosphate formed a backbone with one of the four bases attached tothe side of the sugar. The phosphate attaches to the number 5 carbon and thenitrogen base attaches to the number 1 carbon of the sugar.

Long chains of nucleotides are formed linking the number 3 carbon of one sugar tothe phosphate of a second sugar with a phosphodiester bond. The sugar-phosphate chains form the backbone of the nucleic acid with the nitrogen basesattached to the side of each sugar molecule (S-P-S-P-S-P-S-P, etc.). Thisdescription, regrettably, can be misleading. When DNA molecules models areassembled, one wants to make the backbone chain first, and then add the nitrogenbases. We need to remember that DNA molecules are assembled nucleotide bynucleotide.

Again, to form a nucleotide, the phosphate bonds to the 5' carbon ofthe sugar molecule, leaving the 3' carbon of the sugar to attachto the next nucleotide's phosphate. This little detail is important to thestructure of DNA. Deoxyribose is a 5-carbon sugar. Who bonds to whatcarbon is critical to DNA's structure. And the polarity of the DNA moleculeis determined by this precise bonding.


2 . Mirsky restated Feulgen's work from the 1850's that determined therelationship of the volume of DNA in the nucleus for normal body cells, cellsjust prior to division and "germ" cells or gametes. This provided evidencethat DNA was the genetic molecule because it corresponded with thebehavior of chromosomes in mitosis and meiosis.

3 . Erwin Chargaff's work in 1947:• The four nitrogen bases were not present in equal amounts• The amounts differed in different species• But

• The amount of Adenine was always the same as Thymine• The amount of guanine was always the same as Cytosine

This information is known as Chargaff's rules

4 . X-ray diffraction (best done by Rosalind Franklin at King's College in London)showed that DNA:• was long and thin• had a uniform diameter of 2 nanometers• had a highly repetitive structure with .34 nm between nitrogen bases in

the stack• was probably helical in shape, like a circular stairway

Watson and CrickFrom this information, Watson and Crick determined the structure of DNA in 1953and published their work in Nature. They surmised (and confirmed):• DNA was probably a double strand (because of the 2 nm diameter)

This was interesting because Linus Pauling, who was also working on thestructure of DNA, had just written a proposal for DNA having a triple strandedstructure.

• To maintain the uniform 2 nm diameter, a double-ring base probably would pairwith a single-ring base along the length of the molecule

Adenine can hydrogen bond to thymine at 2 places.Guanine can hydrogen bond to cytosine at 3 places

• This explained Chargaff's findings that the amounts of adenine and thyminewere the same, and the amounts of guanine and cytosine were the same for aspecies.


• Two strands of nucleotides, with their bases hydrogen-bonded to each otherwould form a ladder if, and when, the sugar phosphate backbones ran inopposite directions to each other, or anti-parallel to each other, andtwisted to form a double helix. (This is where that 5' and 3' bonding justmentioned gets important). Moreover, the base-pairs lie flat and stack at3.4nm apart because of the hydrophobic interactions. The coiling also results inalternating major and minor grooves in the DNA structure, something that isimportant for gene transcription and regulation.

The constancy of the complementary base-pairing is critical to the structureof DNA. DNA of different species and of different genes shows variation in thesequence of base pairs in the DNA chain (which base pair follows the next).

Once the structure of DNA was determined, active research could take place inhow DNA can do replication prior to cell division and in how DNA stores geneticinformation. We shall look at both.


Replication of DNA• DNA is a double stranded molecule. The two chains (or strands) are

attached by hydrogen bonds between the nitrogen bases.• The two strands are anti-parallel to each other (run in opposite

directions). That is, the 3' carbon of the sugar (the free sugar end)starts one strand and the 5' carbon sugar end (the free phosphate end)starts the other. This is necessary for the base-pairs to hydrogen bondcorrectly.

• Adenine must bond to thymine, and guanine must bond to cytosine.

Because of the complementary base pairing, if one side of the doublestranded molecule is known, we automatically know what the other half is.This model for DNA replication is known as the semi-conservative modelfor DNA replication. It was first proposed by Watson and Crick, but was notproven for several years until Stahl and Meselson devised a method usingheavy nitrogen (15N).

The process of DNA Replication (or Duplication)There are three basic steps to DNA replication:

• The two DNA strands of the parental chromosome must unwind andseparate.

• Each strand of the parent chromosome serves as a template for thesynthesis of a daughter strand. DNA is always synthesized in the 5' 3"direction from the 3' 5" template.

• The newly synthesized double helix of each parent-daughter combinationrewinds to form the DNA chains of a replicated chromosome. Each new DNAmolecule is composed of one-half of the parent chromosome and one-halfnewly synthesized DNA.

DNA Structure and Replication - 1 0

A few details about DNA Replication:1. Prior to cell division, the double-strand of DNA is unwound at places called

origins, forming replication bubbles in the DNA molecule. Initiatorproteins bind to the origin to start the process. The enzyme, DNAhelicase, facilitates the unwinding of the DNA molecule by binding to andmoving along one strand of the DNA while "pushing aside" the second DNAstrand. A set of single-strand binding proteins line up on the strandsafter DNA helicase unwinds DNA to keep the strands separated.

2. Unwinding and replication occurs in both directions, as the DNA molecule"forks" from the origins (where the unwinding starts). These regions arecalled replication forks and new DNA is replicated behind the fork as itprogresses along the DNA molecule.

3. Replication occurs rapidly, as much as 1000 nucleotides per second. SinceDNA is a helix, the uncoiling (rotation caused by helicase) occurs at 100revolutions per second (DNA coils every 10 nucleotides) – causing the DNAto twist and kink, something that is really called torque. (If you have evertried to coil a hose around your arm, you've noticed the kinks and twists thathappen to the portion of the hose not yet coiled. Just try uncoiling one ofthose coiled hoses!) Enzymes called gyrases (or properly topisomerases)solve the twisting (torque) problem for DNA by cutting one strand of theDNA and allowing it to freely swivel around the un-cut strand. The strand isthen reconnected after it has been untwisted.

4. In eukaryotic organisms, there are many, many replication units involved inthe replication of DNA on each chromosome. Each replication unit forms areplication fork at its "origin". As DNA replication progresses, replicationunits join other units when they meet an adjacent replication forks. Bacteriausually have one origin, which is a specific nucleotide sequence in itschromosome.

Origins and Replication Forks


5. Each of the two strands of the DNA molecule in the fork serves as atemplate for the attachment of its complement nucleotides (A-T, C-G, G-Cand T-A). This takes place on both sides of the replication forkssimultaneously, but in opposite directions. (This is seldom illustrated. Mostdiagrams show only one section of a replication fork, to try and simplifythings a bit.)

6. The enzyme, DNA polymerase III, promotes the synthesis of the new DNAstrands, by recognizing the appropriate complementary base needed and bybonding appropriate phosphorylated nucleotides to the growing DNA molecule.DNA polymerase III is actually a dimmer complex of 10 proteins, and is usedto simultaneously replicate both sides of the DNA molecule.

7.

DNA Polymerase III β2 subunits "sliding clamp" DNA moving through DNA Polymerase

Unfortunately, DNA polymerase has two problems: it can only work in onedirection, and it cannot get started on a single chain DNA strand.

DNA polymerase can only attach a nucleotide to the exposed -OH group onthe 3' end of the leading strand template. DNA is always synthesized inthe 5' 3" direction from the 3' 5" template. This is fine for oneof the two DNA strands of the original molecule, but not for the secondstrand, which is running in the opposite direction.

The upper 3' end of the original DNA molecule is called the leading strandof the template because replication starts at that point. The oppositestrand is called the lagging strand.


8. In addition, DNA polymerase cannot get started by itself; it needs to add to apre-existing chain. This is solved by starting replication with a RNA primer(of about 10 nucleotides), activated by an enzyme called primase. Primasecatalyzes a short RNA molecule that is used to start the synthesis of a DNAprimer at the leading strand. DNA polymerase eventually replaces the RNAnucleotides with DNA nucleotides.

9. Once the DNA strand has been "primed" by primase adding the RNA primer,DNA polymerase can go to work adding nucleotides to the 3' open end of thenewly forming molecule. The energy for this process is provided by thehydrolysis of the nucleotides. The free nucleotides have a triphosphatestructure. Two of the phosphates are removed to provide energy to bondthe nucleotides onto the growing DNA polymer.


10. Remember: DNA replication always takes place (grows) in the 5' to 3'direction from the 3' to 5' template. This sounds stranger than itreally is. Since the chains of DNA are antiparallel, or opposite, the newnucleotides attached will be in a 5' to 3' direction, while it is adding newnucleotides at the 3' template end.

11. DNA replication is continuous along one side (the leading strand) of theoriginal template DNA molecule, because the newly synthesized daughternucleotides can follow the path of DNA helicase.

12. The other template strand of DNA is unzipped in the 5' to 3" directionhowever, so new DNA synthesis is discontinuous. This new DNA strand iscalled the lagging strand, because its rate of synthesis lags behind thatof the leading strand. Additional DNA polymerase enzymes must beattached at unzipped regions, yet the synthesis direction of the laggingstrand is opposite the direction of the unzipping DNA helicase enzyme.

Since DNA polymerase must do both sides, the lagging strand has to befolded back on itself to "face" the correct 5' to 3' direction to fit into theDNA polymerase sliding rings.


New DNA polymerase enzyme molecules have to attach and work on smallportions of the unzipped DNA molecule. The enzyme, DNA ligase, links theshort pieces, called Okazaki fragments, of the lagging side together.Note: Each Okazaki fragment must be initiated by a RNA primer.

13. After the DNA is replicated along its entire length, two DNA molecules havebeen formed, each half the original, half new nucleotides. Both DNAmolecules are identical to each other and to the original.

Generalized Diagram of DNA Replication


Proofreading the DNADNA errors occur about 1 in 1 billion nucleotides in the final DNA, but pairing errorsdo occur during the process. As you might expect, DNA is proofread by DNApolymerase as it is being replicated. If there is an error, it deletes the mistake,and replaces it with the correct nucleotide. This is called mismatch repair.

Damage to DNA molecules occurs daily by exposure to routine molecules in theenvironment. DNA repairing enzymes are always at work. A set of nucleaseenzymes can cut out the damaged DNA and DNA polymerase and ligase fill in thegap with correct nucleotides, assuming there is an undamaged DNA strand to serveas the template. This is called excise repair because the damaged DNA is"excised". Earlier we mentioned the role of p53 in catching damaged DNA. p53activates damage repair nucleases.

Problem of the 5' End of the DNA strand and the Telomere solutionDNA is always synthesized in the 5' 3" direction from the 3' 5" template. The5' end of the lagging strand has no place for a primer on the last Okazaki fragmentso DNA polymerase cannot "finish" the 5' ends of the "daughter" DNA strands.After each replication, the DNA molecule gets shorter.

This is not a problem with prokaryotes that have a circular DNA molecule, but it isa problem for each of the chromosomes of an Eukaryotic organism. To solve this"problem" the ends of eukaryotic chromosomes have special non-gene repeatingnucleotide sequences, such as TTAGGG, called telomeres. The telomere regiongets shorter with each DNA replication. The number of telomere repeats variesfrom about 100 to 1000, and most cells can divide about 30 times before they runout of telomeres and can no longer replicate DNA without losing codable genes.


Telomeres also protect the DNA from cell molecules that detect damaged DNA(from the truncated replicated molecule) and so inhibit the cell pathways thatwould destroy damaged cells.

Some tissues have telomerases, special enzymes that contain an RNA sequencethat catalyzes telomeres using the RNA template at the 3' ends of chromosomes.The 5' end can then be expanded using DNA polymerase and DNA ligase.

In humans, telomerases are normally found only in tissues that produce gametes,but that ensures that gamete chromosomes have long telomeres. Shorteningtelomeres in tissues may be a factor in aging and lifespan. Regrettablytelomerases seem to be abundant in some cancer cells, so that rapid reproductiondoes not result in shortening of telomeres and cell death from lack of neededgenes.

Mouse Telemeres (Orange Tips)

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DNA Structure and Replication - 1 The genetic molecule is ... · DNA Structure and Replication - 1 The genetic molecule is DNA. DNA (along with special proteins) is found in chromosomes