Chapter 16THE MOLECULAR BASIS OF INHERITANCE

Summary of Chapter 16, BIOLOGY, 10TH ED Campbell, by J.B. Reece et al. 2014.

CONCEPT I. DNA IS THE GENETIC MATERIAL

1. THE SEARCH FOR GENETIC MATERIAL

A. Evidence that DNA can transform bacteria

Genes carry information

Early geneticists thought that genes were made of proteins.

Archibald Garrod (1908) introduced the idea that genes and enzymes are related.

  • Discussed the genetic disease alkaptoneuria.
  • Homogentisic acid (HA), an intermediate of the breakdown of phenylalanine and tyrosine, is excreted in the urine.
  • Garrod theorized that an enzyme that oxidizes HA was lacking and that this was due to a mutation of the gene.

James Sumner (1926) showed that enzymes were proteins.

Frederick Griffith (1928) converted an avirulent pneumococcus to the virulent strain.

  • Experiment with mice injected with avirulent live cells and heat-killed virulent cells.
  • Avirulent cells were converted to virulent cells.
  • Transformation due to a transforming principle

O.T. Avery, C.M. McLeod and M. McCarty (1944) identified the transforming principle to be DNA.

George Beadle and Edward Tatum (1940s) suggested that a single gene specifies each protein.

  • They worked with fungus Neurospora crassa.
  • Neurospora is a haploid organism.
  • One gene, one protein hypothesis.

Alfred Hershey and Martha Chase (1952) conducted experiments on the reproduction of bacteriophages.

  • They showed that DNA enters the cell.
  • DNA is required for synthesis of new protein coats and DNA.

B. Additional Evidence That DNA is the Genetic Material

Erwin Chargaff (1950) determined the composition of DNA: ratios of adenine-thymine and guanine-cytosine were very close to 1.

Rosalind Franklin and M.H. Wilkins conducted experiments on the x-ray diffraction of the DNA molecule in the early 50’s.

James Watson and Francis Crick (1953) proposed a model for the structure of the DNA molecule based on the work done by Franklin and Wilkins. They proposed the double helix structure.

2. BUILDING A STRUCTURAL MODEL OF DEOXYRIBONUCLEIC ACID - DNA

  1. DNA, a polymer, is made of two polynucleotide chains intertwined to form a double helix.
  1. Each nucleotide monomer contains a nitrogenous base which may be one of the
  • Purines: adenine or guanine

or

  • Pyrimidine: thymine or cytosine.
  1. Each base is covalently linked to deoxyribose, a 5C sugar.
  1. Deoxyribose is covalently bonded to a phosphate.
  1. The backbone of each single DNA chain is formed by alternating deoxyribose and phosphate groups joined by phosphodiester linkages.
  1. Each phosphate group is linked to the 5’ carbon of one deoxyribose and to the 3’ carbon of the other deoxyribose.
  1. Hydrogen bonds form between adenine and thymine (two bonds), and between guanine and cytosine (three bonds). The sequence of bases is complementary but not identical. This allows to predict the sequence bases in one strand if one knows the sequence of bases in the other strand.
  1. Each pair base is 0.34 nm from the adjacent pair bases.
  1. There are ten base pairs in each turn of the helix making each turn 3.4 nm high.
  1. The double helix is 2 nm wide.
  1. The chains run in an opposite direction and are said to be antiparallel to each other. At the end of each DNA molecule there is an exposed 5’ carbon on one strand and an exposed 3’ carbon on the other strand.
  1. Complementary base paring of adenine and thymine and guanine and cytosine are the basis of Chargaff ‘s rule, which is A = T and C = T in DNA.

CONCEPT II. DNA REPLICATION AND REPAIR

Many proteins work together in DNA replication and repair.

1. THE BASIC PRINCIPLE: BASE PAIRING TO A TEMPLATE STRAND.

A process called replication can precisely copy DNA.

The essential features of DNA replication are universal but there are some differences between prokaryotes and eukaryotes due to the difference in DNA organization.

In prokaryotes, DNA consists of a circular double-stranded molecule, while in eukaryotes it is made of a linear double-stranded molecule associated with great deal of proteins.

The two strands of the double helix unwind. Each strands serves as a template for the formation of a new complementary strand.

DNA replication is semiconservative: each daughter double helix contains one strand from the parent DNA and one newly synthesized strand.

More than a dozen enzymes and proteins are involved in DNA replication.

2. DNA REPLICATION: A CLOSER LOOK

A. Getting started: the mechanism

  1. DNA begins at specific sites in the molecule named origins of replication and forms the replication bubble. Here at each end of the replication bubble, DNA helicase creates a replication fork.
  • A 6-subunit DNA binding complex is required to initiate replication and the attachment of helicase.
  • This group of proteins known collectively as thepre-replication complex(pre-RC).
  • ATP is required.
  1. The position of the replication fork is constantly moving as replication proceeds.
  1. The enzyme DNA helicase travels along the helix opening it as they move.
  1. Single-strand binding proteins bind to the single DNA strands preventing reformation of the double helix.
  1. Topoisomerases break and rejoin sections of the DNA to relieve strain and prevent knots during replication.
  1. DNA synthesis always proceeds in a 5’ 3’ direction: 5' phosphate at one end and 3' hydroxyl at another end.
  1. The two DNA strands are antiparallel, that is, their sugar phosphate backbones run in opposite directions.

B. Synthesizing a New DNA Strand

  1. DNA polymerases catalyze the linking together of the nucleotide subunits. There are at least eleven DNA polymerases involved in eukaryote replication.
  1. Nucleotides with three phosphate groups are used as substrates for the polymerization reaction. Two of the phosphates are removed and the nucleotide is added to the 3’ end of the growing strand.
  1. These reactions are exergonic and do not require ATP.
  1. DNA polymerase cannot initiate the synthesis of polynucleotide; they can only add nucleotides to the 3’ end of an already existing chain that is base-paired with the template strand.
  1. DNA synthesis requires an RNA primer to initiate the synthesis reaction. The RNA primer is made of about ten nucleotide long in eukaryotes.
  1. The RNA primer is synthesized by a protein complex known as a primosome, which includes an enzyme, primase, that is able to start a new strand of DNA opposite a DNA strand.

C. Antiparallel Elongation

  1. DNA replication is continuous in one strand and discontinuous in the other.
  1. DNA polymerase adds nucleotides to the 3’ of the new strand that is always growing toward the replication fork. This strand is called the leading strand.
  1. DNA polymerase adds nucleotides to the 3’ of the new strand that is growing away from the replication fork. This strand is called the lagging strand. The rate of elongation is the addition of about 500 nucleotides per second in bacteria and 50 in humans.
  1. Primase synthesizes a short RNA primer, which is extended by DNA polymerase to form an Okazaki fragment.
  1. The lagging strand is synthesized in short pieces called Okazaki fragments, which are made of 100 to 1000 nucleotides. The fragments were discovered by Reijii Okazaki.
  1. These fragments grow in a direction away from the replication fork.
  1. Each Okazaki fragment begins with an RNA primer.
  1. After it has been elongated by DNA polymerase III, the RNA primer is degraded by DNA polymerase I and the gaps are filled with DNA. The adjoining fragments are linked together by DNA ligase.
  1. DNA ligase links the 3’ end of one fragment with the 5’ end of the adjoining fragment.

DNA replication is bidirectional starting at the origin of replication and proceeding in both directions.

An eukaryotic chromosome may have several origins of replication and may be replicating at several points at any one time.

D. The DNA Replication Complex.

Two DNA polymerase III molecules work together in a complex with helicase and other proteins.

By interacting with other proteins at the fork, primase apparently acts as a molecular brake, slowing progress of the replication fork and coordinating the placement of primers and the rates of replication on the leading and lagging strands.

One DNA polymerase acts on each template strand.

DNA moves through the complex during the replication process.

The DNA polymerase molecules, one on each template strand, reel in the parental DNA and extrude the newly made daughter DNA molecules.

3. PROFREADING AND REPAIRING DNA.

DNA polymerase proofreads each nucleotide against its template as soon as it is added. If there is an error, the nucleotide is removed and the correct one is added in its place.

Errors that arise after replication are also corrected.

Nucleotide excision repair.

  • The mismatch pair of nucleotide distorts the DNA molecule;
  • A nuclease enzyme cuts the damaged DNA strand at two points;
  • The DNA is repaired by DNA polymerase by filling the gap with the correct nucleotides;
  • DNA ligase attaches the new sequence to the rest of the molecule.

4. EVOLUTIONARY SIGNIFICANCE OF ALTERED DNA NUCLEOTIDES

The error rate after proofreading and repair is extremely low, but mistakes do slip through.

Once the mismatched pair is replicated, the change is permanent in the daughter molecule and subsequent copies.

This is called a mutation, a permanent change in the DNA base arrangement.

Mutations can change the phenotype of an organism.

The vast majority of these changes has no effect or is harmful; a small percentage is beneficial. This is the origin of variation within the species.

Natural selection acts on variation.

The selection of beneficial mutations is ultimately responsible for the appearance of new species.

5. REPLICATING THE ENDS OF DNA MOLECULES

The usual replication machinery cannot complete the 5' ends of daughter DNA strands.

DNA polymerase can add nucleotides only to the 3' end of a preexisting nucleotide.

The 3' end of the lagging strand is provided by the primer. When the primer is removed at the end of strand, no more nucleotides can be added. This results in a shorter strand.

Repeated replication results in shorter DNA molecules with uneven ends.

The presence of telomeres postpones the erosion of genes at the end of the lagging strand.

Eukaryotic chromosomal DNA molecules have special nucleotide sequences called telomeres at their ends.

These end caps of repetitive DNA are called telomeres.

Telomeres do not contain genes. They are made of multiple repetitions of a nucleotide sequence, e. g. TTAGGG is the repetitive unit in humans.

Recent research supports the idea that the repetitive DNA at the end of the chromosome has a protective function.

The number or repetitions in a telomere varies from 100 to 1000.

A small amount of telomeric DNA fails to replicate each time the DNA replicates. No essential genetic information is lost.

Telomeric DNA can be lengthened by a DNA replicating enzyme called telomerase.

Telomerase molecules have a small RNA molecule together with the protein.

Cells that produce telomerase continue to divide indefinitely beyond the point at which cell division would normally cease.

Active telomerase is found in germ cells that give rise to sperm and eggs in animals, but it is absent in somatic cells.

The absence of telomerase activity in animal cells may be the cause of cellular aging.

It is possible that telomeres are a limiting factor in the life span of certain tissues.

CONCEPT III. A CHROMOSOME CONSISTS OF DNA AND PROTEINS

A chromosome consists of a DNA molecule packed together with proteins.

Chromatin consists of DNA and histones. Chromatin is 10 nm thick.

Proteins called histones are responsible for the first level of DNA packing.

Most of the histone amino acids are positively charged (lysine or arginine) and bind tightly to the negatively charge DNA.

DNA winds twice around the histones and form a nucleosome. Nucleosomes resemble beads in a string. The DNA between nucleosomes is called the linker.

The amino end of each histone extends outward from the nucleosome. This is the N-terminus.

The histone tails of one nucleosome and the linker DNA and nucleosomes on either side interact and cause the DNA to coil forming a chromatin fiber 30 nm thick.

The 30-nm fiber, in turn, forms loops called loop domains. Loop domains are 300 nm thick.

The loops are attached to a DNA scaffold made of proteins.

In a mitotic chromosome, the loop domains coil and fold further compacting the chromatin to produce the mitotic DNA found in the metaphase of cell division.

Metaphase chromosomes are about 700 nm thick.

Heterochromatin is a tightly packed form of DNA. Because it is tightly packed, this DNA is inaccessible to polymerases and it cannot be transcribed.

Euchromatin is less packed and therefore, accessible to polymerases. The genes here can be expressed.

Progressive levels of DNA coiling and folding:

1. DNA, a double helix (chromatin)

2. Histones (chromatin)

3. Nucleosome or “beads on a string”.

4. 30-nm fiber.

5. Looped domains (300-nm fiber).

6. Chromosome.

SUMMARY

You must know the function of the following enzymes:

DNA helicase

Single-strand binding proteins

Topoisomerases

DNA polymerases

Primase

Nuclease

Ligase

Telomerase

Also the structure, function and meaning of the following:

Primer3’ end of chromosome (phosphate)

Leading strand5’ end of chromosome (hydroxyl)

Lagging strandantiparallel

Okazaki fragmentbidirectional

Telomeresreplicating fork

Repetitive DNAsemiconservative

Chromatinreplication

Histonesheterochromatin

Nucleosomeseuchromatin

Linkerloop domain