GEN 272 Introductory Molecular Genetics

1
GEN 272Introductory Molecular Genetics

• Lecture Three

2
REMINDER!

• First test 27 February 2008 (during lecture
  period)

3
Characteristics of the Genetic Material

• Replication exactly the same information as in
  parent cells must be reproduced in daughter cells
• This is achieved via the processes of mitosis and
  meiosis that form part of the general phenomenon
  of cellular reproduction
• Storage of information entails being able to
  serve as a repository of genetic information,
  whether the information is expressed or not
• Most cells contain a full complement of DNA, but
  only a subset of genes is expressed at a given
  time, e.g. skin cells may display active melanin
  genes, but never their hemoglobin genes or
  digestive cells activate many genes specific to
  their function, but do not activate melanin genes
  
• Expression of information the basis of the
  central dogma of molecular genetics/biology (Fig.
  10-1)
• Variation by mutation genetic material must
  serve as the basis for newly arising variability
  among organisms, e.g. disease resistance genes
• If a mutation occurs, the alteration will be
  reflected during transcription and translation
  and may often affect the specified protein

3
4
Central Dogma of Molecular Biology
Fig. 10-1
Klug et al. (2006). Concepts of Genetics, 8th
Edition
4
5
DNA Replication

• An essential function of the genetic material
  that ensures the genetic continuity between cells
  during cell division
• must be executed precisely if genetic continuity
  between cells is to be maintained
• for example, human haploid genome 3 x 109 base
  pairs (spread over 23 chrs). One error per 106
  bp 3 x 103 errors per replication cycle of the
  genome (too much!!!)
• nonetheless, evolution would not have occurred in
  the absence of (such) error
• The double helix model proposed by Watson and
  Crick (1953) provided the initial insight into
  DNA replication semiconservative replication
• if DNA were to unwind, each nucleotide along the
  two parent strands would have an affinity for its
  complementary strand, thus, each strand could
  serve as template for the synthesis of its
  complement
• based on complementarity, A would attract T while
  G attracts C (and vice versa in both cases)
• in short, each replicated DNA molecule would
  consist of one old strand (original template)
  and one new strand semiconservative
  replication
• two other theoretical modes of replication are
  also possible, i.e. conservative replication and
  dispersive replication (see Fig. 11-2)

5
6
Semiconservative Replication
Fig. 11-1
Klug et al. (2006). Concepts of Genetics, 8th
Edition
7
The Meselson-Stahl Experiment

• First report that provided strong evidence for
  semiconservative replication as the mode used by
  bacteria to produce new DNA molecules
• Grew E. coli in medium containing 15NH4Cl
  (ammonium chloride)
• 15N is more dense than 14N, and more stable than
  radioactive isotopes
• The presence of hybrid molecules at Generation I
  and that not all molecules at Generation 0 are of
  an intermediate density rules out the
  conservative and dispersive replication modes
  (see also Fig. 11-4)

Fig. 11-3
Density gradient centrifugation
Klug et al. (2006). Concepts of Genetics, 8th
Edition
8
The Meselson-Stahl Experiment
Generation 0
Klug et al. (2006). Concepts of Genetics, 8th
Edition
Fig. 11-4
8
9
The Taylor-Woods-Hughes Experiment
Fig. 11-5

• Grew root tips for 1 generation in the
  presence of a radioisotope (3H-thymidine)
• Remove and place in unlabelled medium allow
  cell division to continue
• End of each generation, added colchicine and
  examined chrs by radiography
• Radioisotope emits energy, the emulsion turns
  black at the approximate point of emission
• Radioactive thymidine associated with sister
  chromatids containing newly synthesized DNA
  molecules
• After 2nd replication cycle, only 1 of 2 sister
  chromatids was radioactive

Klug et al. (2006). Concepts of Genetics, 8th
Edition
10
Characteristics of Replication

• Where along the chr does replication initiate?
• Is there a single or multiple points of
  initiation?
• Is the point of origin random or specific?
• Once initiated, does replication continue in a
  single (unidirectional) or both directions
  (bidirectional)?

11
Replication in prokaryotes

• In prokaryotes
• there is a single, origin of replication (oriC)
  and
  terminates at the ter region
• replication is bidirectional
• Two other terms to remember,
• replication fork a point along the chr where
  
  replication initiates and the strands of the
  helix have
  unwound
• since replication is bidirectional, there
  will be two
  replication forks moving along the DNA
  duplex in
  opposite directions (Fig. 11-6)
• replicon the length of DNA that is replicated
  
  following one initiation event at a single
  origin
• in E. coli 4.2 Mb, which represents the
  entire single,
  circular chr of the bacterial cell

Fig. 11-6
Klug et al. (2006). Concepts of Genetics, 8th
Edition
11
12
DNA synthesis in bacteria

• Performed by an enzyme termed DNA polymerase I
  (DNA pol I), the first to be identified by A.
  Kornberg and colleagues (1957)
• able to direct DNA synthesis in a cell free
  environment (in vitro)
• however, enzyme requires (in vitro) two major
  components, i.e. all four deoxyribonucleoside
  triphosphates (dNTPs) and template DNA
• lack of these components results in little
  (or no) DNA synthesis
• importantly, DNA pol I is functionally specific,
  thus, chain elongation occurs in 5 to 3
  direction (see Fig. 11-8) and accurate (see Table
  11.1)
• Some researchers were not convinced
  that
  DNA pol I was the enzyme that
  replicated DNA
  in vivo (within
  bacterial cells)
• subsequently proven by transfecting
  
  bacterial protoplasts with phage (?X174)
  
  DNA, from which mature
  phages could be
  produced
• thus, an enzyme in bacterial protoplasts
  
  is able to synthesize
  biologically active
  DNA
  which directed phage reproduction

Klug et al. (2006). Concepts of Genetics, 8th
Edition
13
Other polymerase enzymes

• Paula DeLucia John Cairns (1969) discovered a
  mutant E. coli strain deficient in DNA pol I
  activity
• mutation designated polA1
• mutant E. coli strain able to duplicate DNA and
  reproduce successfully (in the absence of pol I).
  However, cells were highly deficient in DNA
  repair, e.g. sensitive to UV light and radiation
• at least 1 other enzyme responsible for
  replication in vivo is present in E. coli cells
• DNA pol I may serve a secondary function in
  vivo this function is believed to be critical
  to the fidelity of DNA synthesis
• Four other unique DNA pol enzymes have been
  isolated from cells lacking pol I activity
• DNA pol I, II, and III cannot initiate DNA
  synthesis but can elongate an existing DNA
  strand, termed a primer
• these enzymes are large proteins (100 000
  Daltons, Da)
• all possess 3 to 5 exonuclease activity
  (proofreading capability), but only pol I
  demonstrates 5 to 3 exonuclease activity (note
  do not confuse with 5 to 3 polymerization,
  which all three can perform)
• If there are so many pol enzymes, why did
  Koornberg isolate DNA pol I only?
• DNA pol I is more abundant than others in a cell
  (see Table 11.2) and its much more stable

14
Roles of polymerases in vivo

• Pol III is responsible for the 5 to 3
  polymerization activity that is essential to in
  vivo replication
• its 3 to 5 exonuclease activity also provides a
  proofreading function that is activated when it
  inserts an incorrect nucleotide
• when this occurs, synthesis stalls, and the pol
  enzyme reverse course, excise the incorrect
  nucleotide and then proceeds with the 5 to 3
  polymerization
• Pol I is believed to be responsible for removing
  the primer as well as filling gaps during the
  synthesis
• its exonuclease activity allows it to participate
  in the DNA repair system
• Pols II, IV, and V are involved in various
  aspects of repair of DNA that has been damaged by
  external forces such as UV light

15
Features of DNA polymerase III

• It is a very large (900 000 Da) and complex
  enzyme
• Its active form, called a holoenzyme, consists of
  10 different polypeptide subunits

Loading the enzyme onto the template at the
replication fork
Klug et al. (2006). Concepts of Genetics, 8th
Edition
16
Pertinent issues during DNA replication

• A mechanism must exist by which the helix
  undergoes unwinding and remains in this
  configuration for replication to occur
• Increased coiling creates more tension further
  down the helix, which must be reduced
• A primer must be synthesized for polymerization
  to commence. Notably, RNA (and not DNA) is used
  for this purpose
• Replication must proceed in only one direction of
  each strand
• RNA primers must be removed prior to completion
  of replication. The gaps are replaced with DNA
  complementary to the template at each location
• The newly synthesized DNA that filled the
  locations previously occupied by RNA primers must
  be joined to the adjacent DNA strands
• A proofreading mechanism must be operational
  during replication to correct any polymerization
  errors arising

17
DNA helix unwinds

• In bacteria and viruses, DNA synthesis initiates
  at the oriC region, consisting of 245 base pairs
  (bp) characterized by repeating sequences of 9
  and 13 bases (9mers and 13 mers, respectively)
• Unwinding is initiated by the protein DnaA, where
  a number of its subunits will bind to each of the
  9mers
• This binding recruits other proteins involved in
  the process of destabilizing the helix. Such
  proteins include DnaB, DnaC and Single-Stranded
  Binding Proteins (SSBPs) and they are known as
  helicases
• Coiling tension strengthens ahead of the
  replication fork to a state termed supercoiling.
• Supercoiling is relaxed by the enzyme DNA gyrase,
  a member of a larger group of enzymes called DNA
  topoisomerases

Klug et al. (2006). Concepts of Genetics, 8th
Edition
Fig. 11-9
18
Primer synthesis

• DNA pol III requires a nucleotide chain with a
  free 3-hydroxyl group in order to add more
  nucleotides and elongate the polynucleotide chain
• An RNA polymerase called primase is known to
  synthesize a primer, a short segment of RNA
  nucleotides (about 5 15 nucleotides long), in
  the absence of a free 3-end
• Note RNA priming is recognized as a universal
  phenomenon during the initiation of DNA synthesis

Fig. 11-10
Klug et al. (2006). Concepts of Genetics, 8th
Edition
19
Continuous and Discontinuous DNA synthesis

• DNA strands are antiparallel to each other
  meaning DNA pol III can only synthesize DNA in
  only one direction (5 to 3) per strand
• For this reason, only one strand can serve as
  template for continuous DNA synthesis. This newly
  synthesized DNA is referred to as the leading
  strand
• As the fork progresses, many points of initiation
  are necessary on the opposite DNA template,
  resulting in discontinuous DNA synthesis of the
  lagging strand
• The short pieces of DNA that form the lagging
  strand are known as Okazaki fragments
• These fragments will later be joined, following
  the removal of the RNA primer by DNA pol I, by
  DNA ligase

Klug et al. (2006). Concepts of Genetics, 8th
Edition
Fig. 11-11
20
Concurrent leading and lagging strand synthesis

• Evidence suggests that both strands are
  replicated simultaneously
• This involves the lagging strand forming a loop
  (lie in parallel to the leading strand) and
  nucleotide polymerization on both strands
  occurring under the direction of a dimer of the
  DNA pol III enzyme
• Upon encountering a completed Okazaki fragment on
  the lagging strand, the monomer of the enzyme
  releases from the lagging strand and a new loop
  is formed
• Key to this scenario will be the ?-subunit of the
  enzyme sliding clamp that prevents the core
  enzyme (subunits ?, ?, and ?) from falling off
  the template
• Note as polymerization proceeds, mismatched
  nucleotides inserted into the growing
  polynucleotide chain are excised and replaced
  with matching complementary nucleotides by the
  epsilon (?) subunit of the core enzyme

Fig. 11-12
Klug et al. (2006). Concepts of Genetics, 8th
Edition
21
Coherent model summarizing DNA replication

• Helicases unwind DNA
• SSBPs associate with strands to destabilize the
  configuration
• DNA gyrase diminishes the coiling tension
• Each monomer of the core enzyme subunit of
  polymerase binds to one of the template strands
  by a ?-subunit sliding clamp
• Continuous synthesis occurs on the leading strand
• Lagging forms a loop to allow simultaneous
  synthesis to occur on both strands
• DNA pol I and DNA ligase facilitate the joining
  of Okazaki fragments

Fig. 11-13
Klug et al. (2006). Concepts of Genetics, 8th
Edition
22
Take Home Message

• DNA replication is an essential process in the
  cell cycle as it ensures that genetic information
  contained in the genetic material is fairly
  passed onto the succeeding generations
• Replication occurs via a semiconservative model,
  where each newly synthesized DNA duplex consist
  of one old and one new DNA strands
• There are several DNA polymerase enzymes involved
  in DNA replication. These include DNA pol III (5
  to 3 exonuclease activity), pol I (3 to 5
  exonuclease activity), and pols II, IV, and V
  (DNA repair)
• The process of DNA replication has been
  elucidated and it involves a number of protein
  molecules with the DNA strands assuming different
  structural configurations

22