??????????(Microbial Genetics and Biotechnology)

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??????????(Microbial Genetics and Biotechnology)

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Bacterial DNA structure and replication

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I.Structures of nucleic acids (DNA and RNA)
1. Composition (1) Base
Purines A (adenine), G (guanine)
Pyrimidines in DNA C (cytosine), T
(thymine) in RNA C (cytosine), U
(uracil) (2) nucleoside A base attaches to
the 1 end of a pentose (a sugar (
DNA deoxyribose RNA ribose) in DNA
deoxyadenosine, deoxyguanosine, deoxycytidine,
deoxythymidine in RNA
adenosine, guanosine, cytidine, uridine (3)
nucleotide phosphate(s) attaches to 3 end of
the pentose one phosphate group
in DNA dNMP - deoxyadenosine monophosphate
(dAMP), deoxyguanosine monophosphate
(dGMP), deoxycytidine monophosphate
(dCMP), deoxythymidine monophosphate
(dTMP)

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I.Structures of nucleic acids (DNA and RNA)
in RNA NMP - adenosine monophosphate
(AMP), guanosine monophosphate (GMP),
cytidine monophosphate (CMP), uridine
monophosphate (UMP) two phosphate groups
dNDP NDP (D di-) dADP, dGDP, dCDP, dTDP
ADP, GDP, CDP, UDP three phosphate groups
dNTP NTP (T tri-) dATP, dGTP, dCTP, dTTP
ATP, GTP, CTP, UTP

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I.Structures of nucleic acids (DNA and RNA)

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I.Structures of nucleic acids (DNA and RNA)

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I.Structures of nucleic acids (DNA and RNA)

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I.Structures of nucleic acids (DNA and RNA)

• Structure - Double helix(???)
• a. H bond(??) AT vs. GC
• b. Chargaffs rule in natural DNAs, the
  total amount of A
• is always equal to the total amount of T
  The total
• amount of G is always equal to the total
  amount of C.
• c. Antiparallel(?????) 5 to 3 direction
• d. Complementarity(???)
• e. Hydrophobic (???) vs hydrophilic (???)
• f. covalent bond (???) vs non-covalent bomd
  (????)

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I.Structures of nucleic acids (DNA and RNA)

• 2. The double helix In the early 1950s, the
  X-ray diffraction
• studies of Franklin and Wilkins showed that
  DNA is double helix.
• (1). The DNA chain phosphodiester bonds join
  each
• deoxynucleotide-
• The phosphate attached to the 5
  position of the carbon of
• the deoxyribose of one nucleotide is
  attached to the
• hydroxyl group at the 3 position of
  the carbon of the
• deoxyribose of the next nucleotide to
  form one strand of
• nucleotides, to form a 5 to 3, 5 to
  3 backbone.
• (2). Base pairing
• i. Chargaff rule the concentration of
  G always equals the
• concentration of C and the
  concentration of A always
• equals the concentration of T no
  matter the source of the
• DNA.

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I.Structures of nucleic acids (DNA and RNA)

• ii. Complementary base pair Watson and Crick
  proposed that
• the two strands of the DNA are held
  together by specific
• hydrogen bonding between A and T, and G
  and C in opposite
• strands.
• iii. Antiparallel construction The two chains
  of DNA run in
• opposite directions causing the 5
  phosphate end of one
• strand and the 3 hydroxyl end of the
  other to be on the same
• end of double-stranded DNA molecule.
• iv. The major and minor grooves Because the
  two strands of the
• DNA are wrapped around each other to form
  a double helix,
• the helix has two grooves between the two
  strands major
• groove (the wide one) and minor groove
  (the narrow one).

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I.Structures of nucleic acids (DNA and RNA)

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I.Structures of nucleic acids (DNA and RNA)
5? end
3? end
Each strand of the double helix is oriented in
the opposite direction
3? end
5? end
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I.Structures of nucleic acids (DNA and RNA)

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I.Structures of nucleic acids (DNA and RNA)

• High humidity (92) DNA is called the B-form. It
  is probably close to the conformation of the most
  DNA in cells.
• Lower humidity from cellular conditions to about
  75 and DNA takes on the A-form
• Plane of base pairs in A-form is no longer
  perpendicular to the helical axis
• A-form seen when hybridize one DNA with one RNA
  strand in solution
• A- and B-form are right-handed helix.

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II. DNA replication

• A. Semiconservative(?????)- 14N vs 15N
• B. template(??)- leading strand vs lagging strand
• a. Okazaki fragments .
• D. Base pairing(????)
• E. Enzymes(???)
• a. substrates(??)- dNTPs
• b. DNA primerase(?????) RNA primer(??)
• c. Helicase(????)- unwinding(??)
• d. SSB (single strand binding proteins)
• e. DNA polymerase III(DNA???III)-
  Polymerization(?
• ???)and proofreading(??)
• f. Ligase(???)
• g. Topoisomerase II(???II)

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II. DNA replication

• The basic process of replication involves
  polymerizing, or
• linking the precursors (nucleotides) into
  long chains or strands,
• using the sequence on the other strand as a
  guide.
• 2. DNA replication involves many enzymes.
• The DNA polymerase attaches the first phosphate
  (called a) of
• one deoxyribonucleoside triphosphate to the
  OH of 3 carbon
• of sugar of another deoxyribonucleoside
  triphosphate, and
• release the last two phosphates (called ß
  and ? ) of the first
• deoxyribonucleoside triphosphate to produce
  energy for the
• reaction. The same reaction occurs over and
  over until a long
• chain is synthesized.
• DNA polymerase can not start the synthesis of a
  new strand of
• DNA without a primer (a preexisting 3 OH
  of a
• deoxyribonucleotide).
• The short RNA is used as primer to initiate the
  synthesis of
• new DNA strand either by RNA polymerase or
  a special
• enzyme, primase. During DNA replication,
  special enzymes
• recognize and remove the RNA primer.

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II. DNA replication

• 6. DNA polymerases also need a template strand to
  direct the
• nucleotide to be inserted at each step of
  polymerization
• reaction by complementary base-pairing. For
  example, the DNA
• polymerase will insert a T into the new
  strand when there is an
• A in the template strand.
• 7. Some proteins travel with DNA polymerase as
  part of a DNA
• replication complex, replicosome. The
  functions of these DNA
• polymerase accessory proteins are listed in
  table 1.
• 8. DNA replication is a semiconservative process
  each of the new
• molecules will consist of one old conserved
  strand and one
• newly synthesized strand. (The Meselson-Stahl
  experiment).
• 9. Okazaki fragments and the replication fork
• (1) DNA polymerase can move only in the 3-
  to 5- direction on
• the template strand and synthesize the
  new DNA strand in
• the 5- to 3- direction.

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II. DNA replication
Base pairing provides the mechanism for
replicating DNA

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II. DNA replication

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II. DNA replication

Meselson-Stahl experiment
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II. DNA replication

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II. DNA replication

• (2) Because DNA molecule is an antiparallel
  structure, the DNA
• polymerase on one of the two strands
  would have to move in the
• wrong direction overall at the
  replication fork.
• (3) There are three types of DNA polymerase, I
  II, and III in E. coli.
• (4) On one template strand, DNA polymerase
  III initiates synthesis from
• an RNA primer and moves along the
  template DNA in the 3-to 5
• direction. The newly synthesized DNA
  strand is referred as the
• leading strand. On the other strand,
  DNA polymerase also moves in
• the 3-to 5 direction, but works in
  opposition to the movement of
• the replication fork as a whole. In
  order to synthesize the second
• new strand (called lagging strand), DNA
  polymerase III makes short
• pieces of DNA fragments (called Okazaki
  fragments) from a new
• RNA primer about 10 to 12 nucleotides
  long synthesized by DnaG
• primase.
• (5) DnaG primase produces a new primer about
  once every 2 kilobases,
• recognizing the sequence 3-GTC-5.
• (6) DNA polymerase I removes the RNA primer
  by its 5 exonuclease
• and replaces the RNA primer with DNA by
  its polymerization activity.

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II. DNA replication

• (7) The Okazaki fragments are joined
  together by DNA ligase before the
• replication fork moves on.
• 10. Replication errors
• (1) To maintain the stability of a species,
  replication of DNA must be
• almost free of error. However, the
  wrong base is sometimes
• inserted into the growing DNA chain.
  Mismatches can occur when
• the bases take on forms called
  tautomers, which pair differently
• from the normal form of the base.
• (2) The cell can reduce mistakes during
  replication through one of the
• following ways
• i. Editing function - Editing
  function can be performed by either
• DNA polymerase or other proteins.
  The 3 exonuclease of DNA
• polymerase can remove the last
  incorrectly inserted base. The
• editing proteins probably
  recognize a mismatch because the
• mispairing will cause a minor
  distortion in the structure of double
• helix of the DNA.
• ii. Methyl-directed mismatch repair
  The state of methylation of the
• DNA strands allows the mismatch
  repair system of E. coli to
• distinguish the new strand from
  the old strand after replication.

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Tautomerization
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II. DNA replication

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II. DNA replication

Looping models for replication in eukaryotes
allow DNA polymerases to move in the same
direction on the leading and lagging strands. In
prokaryotic replication, the core DNA polymerase
III is composed of two a-subunits. Additional
proteins, PCNA and RF-C are involved in the
eukaryotic replication complex.
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II. DNA replication

• 11. Replication of bacterial chromosome and cell
  division
• (1) Most bacteria only have one circular
  chromosome that means there
• is only one unique DNA molecule per
  cell. In some bacteria,
• chromosome replicates but the cell for
  some reason does not
• divide.
• (2) The DNA replication initiates at a
  unique site called origin of
• chromosome replication (oriC).
• i. A primosome (consisting of DnaA,
  B, C and other proteins)
• may help the DnaG primase or other
  RNA polymerase to
• synthesize an RNA to start
  replication at oriC.
• (3) In E. coli, chromosome replication
  usually terminates in
• certain region but not at
  well-defined unique site.
• i. A termination region, ter,
  contains cluster of sites called
• ter sequences, which are only 22
  bp long.
• ii. Cluster terA and terB bracker the
  termination region.
• iii. Replication fork can pass terA in
  the clockwise direction but not
• in counterclockwise direction.
• iv. Replication fork can pass terB in
  the counterclockwise direction
• but not in clockwise direction.

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II. DNA replication

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II. DNA replication

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II. DNA replication

• 12. According to the Watson-Crick structure,
  the two strands are
• wrapped around each other about once
  every 10.5 base pairs
• to form the double helix. If two strands
  are wrapped around
• each other more than once every 10.5
  base pairs, the DNA is
• said to be positively supercoiled, and
  to be negatively
• supercoiled if less than 10.5 base
  pairs.
• 13. The supercoiling of DNA in the cell is
  modulated by enzymes
• called topoisomerases which bind to DNA,
  break one or both
• of the strands and pass the DNA strands
  through the break
• before resealing it, and will either
  introduce or remove
• supercoils from DNA.

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II. DNA replication

• 14. There are two types of topoisomerases
  TopI cuts one strand
• and pass the other strand through the
  break before resealing
• and will change DNA one supercoil at a
  time. TopII cuts both
• strands and pass two other strands from
  somewhere else in
• the DNA through the break before
  resealing and will change
• DNA two supercoils at a time. The major
  bacterial TopI
• removes negative supercoils from DNA.
  Bacteria have more
• than one type of TopII. TopII can
  remove negative supercoils
• from DNA. The major bacterial TopII is
  called gyrase and can
• add supercoils.
• 15. Gyrase acts by first wrapping the DNA
  around itself and then
• cutting the two strands before passing
  another part of the
• DNA through the cuts, thereby
  introducing two negative
• supercoils. Adding negative supercoils
  increases the stress
• in the DNA and so requires energy,
  here is ATP.

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II. DNA replication
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III. Chromosome replication is coordinated
with cell division

• 1. The bacterial nucleoid
• (1) The bacterial chromosome is not
  enclosed in the nuclear
• membrane as eukaryotic chromosomes.
• (2) DNA is about 1000 times longer than the
  bacterium itself
• and must be condensed to fit in the
  cell but also must be
• folded in such a way that it is
  available for its functions.
• (3) The area that condensed chromosome
  located is called
• nucleoid.
• (4) The nucleoid is composed of 30 to 50
  loops of DNA
• emerging from a more condensed
  region, or core. Most of
• the DNA loops are twisted up on
  themselves, called
• supercoiling. Whether core
  attachment sites on DNA are
• unique or random is not clear.
  However, the repeated
• sequences called rep that are almost
  identical in all bacteria
• have been implicated as sites at
  which the DNA might
• attach to core.