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8
CHAPTER 1
2.
Chemical environment
a.
DNA in the presence of monovalent cations (e.g., Na
ions) will have a high
T
M
because monovalent cations stabilize double-stranded DNA (c Na
c
T
M
).
b.
DNA in the presence of alkaline pH will have a low T
M
because alkaline pH
disrupts the hydrogen bonds (c pH
T T
M
).
c.
DNA in the presence of urea will have a low T
M
because urea disrupts the
hydrogen bonds (c urea
T T
M
).
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9
A. Chromosome replication occurs during S phase of the cell cycle and involves both DNA
synthesis and histone synthesis to form chromatin.
B. An inactive gene packaged as heterochromatin is replicated late in S phase. An active
gene packaged as euchromatin is replicated early in S phase.
C. DNA-directed DNA polymerases have the following properties:
1.
DNA polymerases require a 3
-OH end of a primer strand as a substrate for strand
extension. Therefore, an RNA primer (synthesized by a DNA primase) is required
to provide the free 3
-OH group needed to start DNA synthesis.
2.
DNA polymerases copy a DNA template in the 3
S 5 direction, which produces
new DNA strand in the 5
S 3 direction.
3.
DNA polymerases have proofreading ability which depends on 3
S 5 proof-
reading exonuclease activity that is associated with the DNA polymerase complex.
These are called high-fidelity DNA polymerases.
D. DEOXYRIBONUCLEOSIDE 5
-TRIPHOSPHATES (dATP, dTTP, dGTP, dCTP) pair
with the corresponding base (A-T, G-C) on the template strand and form a 3
,5-phos-
phodiester bond with the 3
-OH group on the deoxyribose sugar which releases a
pyrophosphate.
E.
Chromosome replication is semiconservative, which means that double-helix DNA
contains one intact parental DNA strand and one newly synthesized DNA strand.
F.
Chromosome replication is bidirectional, which means that replication begins at a
replication origin and simultaneously moves out in both directions from the replica-
tion origin.
The Chromosome Replication Process (Figure 2-1)
A. Chromosome replication begins at specific nucleotide sequences located throughout
the chromosome called replication origins. Eukaryotic DNA contains multiple repli-
cation origins to ensure rapid DNA synthesis.
B. The enzyme DNA helicase recognizes the replication origin and opens up the dou-
ble helix at that site forming a replication bubble with a replication fork at each end.
The stability of the replication fork is maintained by single-stranded binding proteins.
The replisome refers to a complex molecular machine that carries out DNA replication.
C. As the replication fork moves along the double-stranded DNA, the DNA ahead of the
replication fork becomes overwound or positively supercoiled, whereas the DNA
behind the replication fork becomes underwound or negatively supercoiled. DNA
topoisomerases solve this problem by altering DNA supercoiling.
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10
CHAPTER 2
A
B
C
5
5
5
5
5
5
3
3
3
3
3
5
5
5
5
5
5
5
5
3
3
3
3
3
5
5
3
3
5
5
3
3
Parental
strand
Leading
strand
Lagging
strand
Parental
strand
Dna2 helicase/nuclease
FEN1
RNase H
DNA ligase
I
● Figure 2-1 Replication Fork.
(A) Double-helix DNA (chromo-
some 1) at a replication origin
(RO) site. DNA helicase (H) will
bind at the RO and unwind the
double helix into two DNA
strands. This site is called a repli-
cation bubble (RB). At both ends
of a replication bubble a replica-
tion fork (RF) forms. DNA synthe-
sis occurs in a bidirectional man-
ner from each RF (arrows). (B)
Enlarged view of an RF at one end
of the replication bubble. A
parental strand serves as a tem-
plate for continuous DNA synthe-
sis in the 5
S 3 direction using
DNA polymerase
(P). The other parental strand serves as a template for discontinuous DNA synthesis in
the 5
S 3 direction using DNA polymerase (P). Note that DNA synthesis of the leading and lagging
strands is in the 5
S 3 direction but physically running in opposite directions. (C) DNA synthesis of the
lagging strand proceeds differently from that of the leading strand. DNA primase synthesizes RNA primers.
DNA polymerase
uses these RNA primers to synthesize DNA fragments called Okazaki fragments (OF).
Okazaki fragments end when they run into a downstream RNA primer. Subsequently, DNA repair enzymes
remove the RNA primers and replace them with DNA. Finally, DNA ligase joins all the Okazaki fragments
together. FEN1
flap endonuclease 1.
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11
CHROMOSOME REPLICATION
D. A replication fork contains a:
1.
Leading strand
that is synthesized from the parental strand continuously by DNA
polymerase
(delta).
2.
Lagging strand
that is synthesized from the parental strand discontinuously by
DNA polymerase
(alpha).
a.
DNA primase synthesizes RNA primers along the lagging strand.
b.
DNA polymerase
uses the RNA primers to synthesize DNA fragments called
Okazaki fragments. Okazaki fragments end when they run into a downstream
RNA primer.
c.
Okazaki fragments contain an RNA/DNA primer that must be removed before
the Okazaki fragments can be joined. The RNA/DNA primer is raised into a flap.
d.
Dna2 helicase/nuclease and flap endonuclease 1 (FEN 1) remove the flap.
e.
RNase H degrades the RNA.
f.
DNA ligase I subsequently joins all the DNA fragments together.
E.
PROKARYOTIC DNA REPLICATION. Prokaryotic (bacterial) DNA replication has
some important distinctions compared with eukaryotic DNA replication as follows:
1.
DNA polymerase III
replicates DNA on both the leading strand and the lagging
strand.
2.
DNA polymerase I
removes RNA primers and replaces them with DNA to form a
continuous DNA strand with the Okazaki fragments on the lagging strand.
3.
Dna A protein
recognizes the replication origin and opens up the double helix at
that site forming a replication bubble.
4.
Okazaki fragments are processed by DNA polymerase I, RNase, and DNA ligase H.
DNA Topoisomerases (Figure 2-2).
DNA topoisomerases are enzymes that alter the
supercoiling of double-stranded DNA. Supercoiling of DNA is a mathematical property rep-
resenting the sum of the DNA twists (the number of helical turns in the DNA) plus the
DNA writhe (number of times the DNA loops over itself). Positive supercoiling refers to
overwound DNA. Negative supercoiling refers to underwound DNA.
A. TYPE I TOPOISOMERASE
1.
Type I topoisomerase produces a transient
single-strand nick in the phosphodiester
backbone which allows the DNA on either
side of the nick to rotate freely using the
phosphodiester bond in the unnicked
strand as a swivel point and then the nick
is resealed. ATP hydrolysis (energy) is not
required for this process.
● Figure 2-2 Supercoiling of DNA.
Writhe
Writhe
Twists
2.
Type I topoisomerase introduces negative supercoiling (or relaxes positive su-
percoiling). That is to say, Type I topoisomerase unwinds DNA.
3.
The chemotherapy drugs irinotecan and topotecan inhibit Type I topoisomerase
in cancer cells.
B. TYPE II TOPOISOMERASE
1.
Type II topoisomerase produces a transient double-strand nick in the phosphodi-
ester backbone at a site where the DNA loops over itself. Type II topoisomerase
breaks one double helix of the loop so that a DNA gate is formed, the DNA gate
allows the other nearby double helix of the loop to pass through, and then the nick
is resealed. ATP hydrolysis is required for this process.
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CHAPTER 2
2.
Type II topoisomerase introduces negative supercoiling (or relaxes positive
supercoiling). That is to say, Type II topoisomerase unwinds DNA.
3.
The chemotherapy drugs etoposide, teniposide, and HU-331 (a quinolone syn-
thesized from cannabidiol) inhibit Type II topoisomerase in cancer cells.
C. DNA GYRASE
1.
DNA gyrase is a variant of Type II topoisomerase.
2.
DNA gyrase introduces negative supercoiling (or relaxes positive supercoiling).
That is to say, DNA gyrase unwinds DNA.
3.
DNA gyrase is found in bacteria and is inhibited by two classes of antibiotics: the
aminocoumarins (e.g., novobiocin) and the quinolones (e.g., ciprofloxacin and
nalidixic acid).
D. REVERSE GYRASE
1.
Reverse gyrase is a variant of Type I topoisomerase found in hyperthermophilic
bacteria.
2.
Reverse gyrase introduces positive supercoiling. That is to say, reverse gyrase
winds DNA.
A. The human telomere is a 3–20 kb repeating nucleotide sequence (TTAGGG) located
at the end of a chromosome.
B. The telomere allows replication of linear DNA to its full length. Since DNA polymerases
CANNOT synthesize in the 3
S 5 direction or start synthesis de novo, removal of
the RNA primers will always leave the 5
end of the lagging strand shorter than the
parental strand. If the 5
end of the lagging strand is not lengthened, a chromosome
would get progressively shorter as the cell goes through a number of cell divisions.
C. This problem is solved by a special RNA-directed DNA polymerase or reverse tran-
scriptase called telomerase (which has an RNA and protein component).
D. The RNA component of telomerase carries an AAUCCC sequence (antisense sequence
of the TTAGGG telomere) that recognizes the TTAGGG sequence on the parental strand
and adds many repeats of TTAGGG to the parental strand.
E.
After the repeats of TTAGGG are added to the parental strand, DNA polymerase
uses
the TTAGGG repeats as a template to synthesize the complementary repeats on the lag-
ging strand. Thus, the lagging strand is lengthened.
F.
DNA LIGASE joins the repeats to the lagging strand and a nuclease cleaves the ends
to form double-helix DNA with flush ends.
G. Telomerase is present in human germline cells (i.e., spermatogonia, oogonia) and stem
cells (e.g., in skin, bone marrow, and gut), but is absent from most other somatic cells.
A. GENERAL FEATURES
1.
Chromosomal breakage refers to breaks in chromosomes due to sunlight (or ultra-
violet) irradiation, ionizing irradiation, DNA cross-linking agents, or DNA-damaging
agents.
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