DNA REPLICATION 2.7 (part) & HL Topic 7Understandings, Applications and Skills

Statement / GuidanceWORKBOOK
7.1.U1 / Nucleosomes help to supercoil the DNA. / Activity 208 p. 288
2.7.U1 / The replication of DNA is semi-conservative and depends on complementary base pairing. / Activity 56 p. 75
2.7.U2 / Helicase unwinds the double helix and separates the two strands by breaking hydrogen bonds. / Activity 56 p. 75
2.7.U3 / DNA polymerase links nucleotides together to form a new strand, using the pre-existing strand as a template. / The different types of DNA polymerase do not need to be distinguished.Activity 56
7.1.U2 / DNA structure suggested a mechanism for DNA replication.
7.1.U3 / DNA polymerases can only add nucleotides to the 3’ end of a primer.
7.1.U4 / DNA replication is continuous on the leading strand and discontinuous on the lagging strand. / Details of DNA replication differ between prokaryotes and eukaryotes. Only the prokaryotic system is expected.
7.1.U5 / DNA replication is carried out by a complex system of enzymes. / Activity 211 p. 292
The proteins and enzymes involved in DNA replication should include helicase, DNA gyrase, single strand binding proteins, DNA primase and DNA polymerases I and III.
2.7 S2 / Analysis of Meselson and Stahl’s results to obtain support for the theory of semi-conservative replication of DNA. / Activity 57 p. 77
DBQ p. 113-114 in Text Book
2.7 A1 / Use of Taq DNA polymerase to produce multiple copies of DNA rapidly by the polymerase chain reaction (PCR).
7.1.A1 / Rosalind Franklin’s and Maurice Wilkins’ investigation of DNA structure by X-ray diffraction. / Activity 209 p. 288
7.1.A2 / Use of nucleotides containing dideoxyribonucleic acid to stop DNA replication in preparation of samples for base sequencing. / Activity 211 p. 292

Pre Unit Connections

7.1 U1 - Nucleosomes help to supercoil the DNA.

•The DNA is complexed with ______(an octamer) to form a complex called a______

•Nucleosomes are linked by an additional histone protein (H1 histone) to form a string of______

•These then coil to form a______(~6 chromatosomes per turn) which is condensed to form a______

•These fibres then form loops, which are compressed and folded around a protein scaffold to form______

•Chromatin will then supercoil during cell division to form______that are visible (when stained) under microscope

Why does Eukaryotic DNA need to be supercoiled?

  • essential to ______into the nucleus
  • to ______to allow cell division to occur (most DNA supercoiling occurs at this time)
  • to ______- supercoiled DNA cannot be transcribed
  • allow ______by permanently supercoiling DNA (heterochromatin)
  • transcription of active chromatin (Euchromatin)can be ______by the associated histones

7.1.S2Utilization of molecular visualization software to analyse the association between protein and DNA within a nucleosome.

  1. Use the RCSB Protein Bank to read about nucleosomes and examine Jmol images of them.

Article on nucleosomes:

Jmol visualisation of a nucleosome:

  1. Identify the two copies of each histone protein. This can be done by locating the ‘tail of each protein’. The tails of the proteins are involved in regulating gene expression.
  1. Suggest how the positive charges help to form the nucleosome with the negatively charged DNA molecule.

2.7 U1 - The replication of DNA is semi-conservative and depends on complementary base pairing.

•All DNA must be copied before cell division (Mitosis or Meiosis) so each daughter cell can ______

•DNA replication occurs during the ______, before chromatin condenses into chromosomes.

•Each of the nitrogenous bases can only pair with its partner (A=T and G=C) this is called ______.

•Each new strand contains one original and one new strand, therefore DNA Replication is said to be a ______. Process.

(2.7 S2 examines the evidence for this)

There are 3 Main Steps in DNA Replication

•Step 1 – Helicase unzips the DNA strand by breaking the hydrogen bonds between base pairs; creates two new “template” strands

•Step 2 – DNA polymerase inserts new complementary bases (and builds P/S backbone)

•Step 3 –DNA polymerase proofreads the sequence; fixes errors

2.7 U2 - Helicase unwinds the double helix and separates the two strands by breaking hydrogen bonds.

Helicase

•The ‘ase’ ending indicates it is an ______

•Unwinds the DNA Helix

•Separates the two polynucleotide strands by ______between complementary base pairs

•______is needed by helicase to both move along the DNA molecule and to break the hydrogen bonds

•The two separated strands become parent/template strands for the replication process

2.7 U3 - DNA polymerase links nucleotides together to form a new strand, using the pre-existing strand as a template.

DNA Polymerase

•This protein family consists of multiple polypeptides sub-units

•The polymerisation reaction is a ______reaction

Recall that the DNA strands are antiparallel to one another

•DNA polymerase always moves in a 5’ to 3’ direction

•DNA polymerase catalyses the ______between sugars and phosphate groups

•Free nucleotides are deoxynucleosidetriphosphates

•The extra phosphate groups carry energy which is used for formation of covalent bonds

•DNA Polymerase______. Consequently mistakes are very infrequent occurring approx. once in every billion bases pairs

Core Review From 2.6. Make notes on anything that you did not recall from the last lesson

7.1 U2 - DNA structure suggested a mechanism for DNA replication.

DNA replication and mechanisms by which it can happen are implied by complementary base pairing. Outline the evidence that supports complementary base pairing:

•(X-ray diffraction showed that) the DNA helix is both tightly packed and regular* therefore ______

•______of adenine and thymine are compatible (and opposite) allowing ______to form between them.

•The pairing of cytosine with guanine allows for ______to form between them.

* This evidence also showed that opposite bases need to be “upside down” in relation each other - the helix is anti-parallel however this is not important for complementary base pairing.

7. 1 U3 - DNA polymerases can only add nucleotides to the 3’ end of a primer.

  • DNA Replication is initiated at many points in eukaryotes
  • This makes DNA replication faster and more efficient

*In this topic prokaryote DNA replication is examined; prokaryotes have a single replication fork and a simpler mechanism of replication

These points are known as ______and will have ______

base sequences

Proteins called ______will bind here and then ______will be able to attach, to begin replication.

RNA primers provide an attachment and initiation point for DNA polymerase III.

7.1 U4 - DNA replication is continuous on the leading strand and discontinuous on the lagging strand.

.______provide an ______point for DNA polymerase III.

RNA primers consists of a ______of ______

DNA polymerase III adds new nucleotides to the C3 hydroxyl group on the ribose/deoxyribose sugar such that the strand grows from the 3' end therefore DNA polymerase III moves along the new strand in a 5' - 3' direction (3' - 5' direction on the template strand)

The primers are replaced with DNA bases and the fragments joined together by a combination of DNA pol I and DNA ligase

Leading versus Lagging Strands

Because double-stranded DNA is ______, DNA polymerase must move in opposite directions on the two strands

•On theleading strand, DNA polymerase is moving______ and so can copy______

•On thelagging strand, DNA polymerase is moving______, meaning copying is______

  • As DNA polymerase is moving away from helicase, it must constantly return to copy newly separated stretches of DNA
  • This means the lagging strand is copied as a series of short fragments (______), each preceded by a primer

7.1 U5 - DNA replication is carried out by a complex system of enzymes.

Helicase

Helicase unwinds and separates the ______by breaking the ______between base pairs

This occurs at specific regions (origins of replication), creating a ______

DNA Gyrase

DNA gyrase reduces the ______created by the unwinding of DNA byhelicase

Single Stranded Binding (SSB) Proteins

SSB proteins bind to the DNA strands after they have been separated and ______These proteins also help to prevent the single stranded DNA from being digested

DNA Primase

DNA primase generates a ______(~10–15 nucleotides) on each of the template strands

The RNA primer provides ______for DNA polymerase III, which can extend a nucleotide chain but not start one

DNA Polymerase III

DNA pol III attaches to the ______and covalently joins the free nucleotides together in a ______direction

As DNA strands are antiparallel, DNA pol III moves in opposite directions on the two strands

On the______, DNA pol III is moving towards the replication fork and can synthesise continuously

On the______, DNA pol III is moving away from the replication fork and synthesises in pieces (Okazaki fragments)

DNA Polymerase I

As the lagging strand is synthesised in a series of short fragments, it has multiple RNA primers along its length. DNA pol I ______from the lagging strand and replaces them with DNA nucleotides

DNA Ligase

DNA ligase joins the ______together to form a continuous strand

It does this by covalently joining the sugar-phosphate backbones together with a ______

2.7 S2 - Analysis of Meselson and Stahl’s results to obtain support for the theory of semi-conservative replication of DNA.

Prior to this experiment, three hypotheses had been proposed for the method of replication of DNA:

•______– An entirely new molecule is synthesised from a DNA template

•______– Each new molecule consists of one newly synthesised strand and one template strand

•______– New molecules are made of segments of new and old DNA

•Meselson and Stahl were able to experimentally test the validity of these three models using radioactive isotopes of nitrogen

•Nitrogen is a key component of DNA and can exist as a heavier15N or a lighter14N

•DNA molecules were prepared using the heavier15N then replicated in the presence of the lighter14N

•DNA samples were then separated via centrifugation to determine the composition of DNA in the replicated molecules

•The results after two divisions supported the ______of DNA replication

•After one division, DNA molecules were found to contain a mix of15N and14N, disproving the ______

•After two divisions, some molecules of DNA were found to consist solely of14N, disproving the ______

Conservative
DNA Density / Semiconservative
DNA Density / Dispersive
DNA Density
After 1 rep
After 2 rep

2.7 A1 - Use of Taq DNA polymerase to produce multiple copies of DNA rapidly by the polymerase chain reaction (PCR).

Taq polymeraseis an enzyme isolated from the thermophilic bacteriumThermusaquaticus

As this enzyme’s optimal temperature is ~75ºC, it is able to function at the high temperatures used in PCR without denaturing

PCR is a way of producing large quantities of a specific target sequence of DNA. It is useful when only a small amount of DNA is available for testing e.g. crime scene samples of blood, semen, tissue, hair, etc.

PCR occurs in a thermal cycler and involves a repeat procedure of 3 steps:

1. ______ DNA sample is heated to separate it into two strands

2. ______ DNA primers attach to opposite ends of the target sequence

3. ______ A heat-tolerant DNA polymerase (Taq) copies the strands

•One cycle of PCR yields two identical copies of the DNA sequence

•A standard reaction of 30 cycles would yield 1,073,741,826 copies of DNA (230)

Take Notes while you watch the video’s (and watch more videos if you need to understand)

7. 1 A1 - Rosalind Franklin’s and Maurice Wilkins’ investigation of DNA structure by X-ray diffraction.

7.1.A1Rosalind Franklin’s and Maurice Wilkins’ investigation of DNA structure by X-ray diffraction.

When X-rays are directed at a material some is scattered by the material. This scattering is known as diffraction. For X-ray diffraction to work well the material ideally should be crystallised so that the repeating pattern causes diffraction to occur in a regular way. DNA cannot be crystallised but the molecules were arranged regularly enough for the technique to work.


  1. Use the animation ( to understand how to interpret the Rosalind Franklin’s and Maurice Wilkins’ X-ray diffraction photographs of DNA. To the right is an example of a X-ray diffraction photograph of DNA.

Your answers to the questions below may need diagrams.

  1. What can be deduced from the X-shaped pattern?
  1. What deduction can be made about the regular nature of the pattern?
  1. The vertical distance between the horizontal bars is a measure of what feature of the DNA helix
  1. The distance from the middle of the image to the top measure what feature of the DNA molecule?
  1. What can be deduced given the answers to c and d?
  1. What can be deduced from the angle between the horizontal axis and the arms of X-shaped pattern?
  1. From their images what deduction did Franklin make about the positions of molecular units within the helical structure?

Go to the Bio-Ninja Website and play around with the interactive model there. Also Watch the video linked on the power point.

7.1 A2 - Use of nucleotides containing dideoxyribonucleic acid to stop DNA replication in preparation of samples for base sequencing.

State how dideoxyribonucleicacid affect DNA replication.

Outline how do the answers to the above two questions enable scientists to identify the base sequence of DNA.

Review questions

  1. State during which phase of the cell cycle DNA replication occurs.
  1. State the function of DNA replication.
  1. Which of the following is the end product of DNA replication in a human somatic cell?
  2. 23 chromosomes
  3. 46 chromosomes
  4. 23 pairs of chromosomes
  5. 23 pairs of sister chromatids
  1. Explain why the free nucleotides used in DNA replication are deoxynucleoside triphosphates not deoxynucleoside(mono) phosphates.
  1. State the direction in which DNA polymerase catalyses the formation of the new strand.
  1. Explain the importance of complementary base pairing in conserving the base-sequence during DNA Replication.
  1. Polymerase chain reaction (PCR) is a key technique in DNA manipulation and analysis.
  2. State the main uses of PCR.
  1. State the name of the specialised apparatus needed to support this process.
  1. Complete outline of the three keys steps in the process of PCR.
  • Denaturation:
  • Annealing:
  • Elongation:
  1. If one cycle of PCR yields two identical copies of the DNA sequence. Calculate how many copies 20 cycles would yield.

The image to the right details the three possible methods of DNA replication.


Review your understanding of Meselson and Stahl’s experiments by using using the presentation, theMcGraw and Hill animation (clear and accessible) or the Scitable article (Nature education) (more detailed).

At the start of a Meselson and Stahl experiment (generation 0) a single band of DNA with a density of 1.730 g cm-3 was found. After 4 generations two bands were found, but the main band had a density of 1.700 g cm-3.

  1. Explain why the density of the main band changed over four generations. (2)
  1. After one generation only one DNA band appeared, but the density had changed.
  2. Estimate the density of the band. (1)
  1. Which (if any) mechanisms of DNA replication are falsified by this result? (1)
  1. Explain why the identified mechanism(s) are falsified. (1)
  1. Describe the results after two generations and which mechanisms and explain the identified mechanism(s) (if any) are falsified as a consequence. (3)
  1. Mechanisms for DNA replication are implied by the presence of complementary base pairing in DNA. Explain why it is only possible for cytosine to pair with guanine and adenine to pair with thymine.
  1. Explain why Prokaryotic DNA is described as being ‘naked’.
  1. In the space below, draw and label the structure of a simplified nucleosome, including the H1 linker and octamer (which consists of two copies of four different types of histone proteins).
  1. Nucleosomes both protect DNA andallow it to be packaged, this in turn allows DNA to be supercoiled.
  2. Review 1.6.U2 and briefly outline why it is essential to supercoil chromosomes.
  1. Outline how nucleosomes help regulate transcription.
  1. State the part of the cell cycle in which the most DNA would be supercoiled.

7.1.U3DNA polymerases can only add nucleotides to the 3’ end of a primer.

  1. Outline what a primer is and the role it has in DNA Replication.
  1. In which direction does DNA polymerase move along the template strand? What implication does this have for the addition of bases on the growing strand?

7.1.U4DNA replication is continuous on the leading strand and discontinuous on the lagging strand.

7.1.U5DNA replication is carried out by a complex system of enzymes.

  1. Explain the process of DNA Replication (focusing on prokaryotes):
  2. Distinguish between the lead strand and the lagging strand.
  1. Explain the process of DNA replication on the lagging strand, with reference to DNA primase, RNA primers, DNA gyrase, single strand binding proteins,DNA polymerase III, Okazaki fragments, DNA polymerase I and DNA ligase.

Some biochemists are making a mixture of enzymes for DNA replication in the lab. In each of these cases, something was missing from the mixture. For each situation, deduce which one enzyme was missing, with a reason:

  1. The DNA produced came out as lots of short sections of DNA, a few hundred base-pairs long, rather than one continuous strand.
  1. Only the lead strand was replicated.
  1. No DNA was replicated. The original DNA remained untouched.

7.1.U6Some regions of DNA do not code for proteins but have other important functions.

  1. Distinguish between coding and non-coding regions of DNA.
  1. Outline how non-coding regions can be involved in gene expression.
  1. Most of the eukaryotic genome is non-coding.There are two types of repetitive sequences: moderately repetitive sequences and highly repetitive sequences otherwise known as satellite DNA.Give an example of a region of DNA that contains highly repetitive sequences and outline the function of that region.

7.1.A3Tandem repeats are used in DNA profiling.

  1. State the two different sources of DNA used in paternal and maternal profiling.
  1. Suggest a reason why non-coding regions are more useful than coding regions in DNA profiling.
  1. Describe what is meant by the term tandem repeat sequence.
  1. Describe why and how andem repeats are useful in DNA profiling.

Citations: