Title: ??????????(Microbial Genetics and Biotechnology)
1??????????(Microbial Genetics and Biotechnology)
2Bacterial DNA structure and replication
3I.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)
4I.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
5I.Structures of nucleic acids (DNA and RNA)
6I.Structures of nucleic acids (DNA and RNA)
7I.Structures of nucleic acids (DNA and RNA)
8I.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
(????)
9I.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.
10I.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).
11I.Structures of nucleic acids (DNA and RNA)
12I.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
13I.Structures of nucleic acids (DNA and RNA)
14I.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.
15II. 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)
16II. 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.
17II. 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.
18II. DNA replication
Base pairing provides the mechanism for
replicating DNA
19II. DNA replication
20II. DNA replication
Meselson-Stahl experiment
21II. DNA replication
22II. 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.
23II. 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.
24Tautomerization
25II. DNA replication
26II. 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.
27II. 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.
28II. DNA replication
29II. DNA replication
30II. 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.
31II. 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.
32II. DNA replication
33III. 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.