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D.
A replication fork contains a:
1.
Leading strand
that is synthesized continuously by
DNA polymerase
(delta).
2.
Lagging strand
that is synthesized discontinuously by
DNA polymerase
(alpha). DNA pri-
mase
synthesizes short RNA primers along the lagging strand. DNA polymerase
uses the
RNA primer to synthesize DNA fragments called
Okazaki fragments.
Okazaki fragments
end when they run into a downstream RNA primer. To form a continuous DNA strand
from the Okazaki fragments, a
DNA repair enzyme
erases the RNA primers and replaces it
with DNA.
DNA ligase
subsequently joins the all the DNA fragments together.
E.
The anti-neoplastic drugs
camptothecins (e.g., irinotecan, topotecan)
;
anthracyclines (e.g.,
doxorubicin); epipodophyllotoxins (e.g., etoposide VP-16, teniposide VM-26);
and
amsacrine
are
topoisomerase inhibitors.
F.
The anti-microbial drugs
quinolones (e.g., ciprofloxacin, ofloxacin, levofloxacin, fluoro-
quinolones)
are also topoisomerase inhibitors.
A.
The human telomere is a 3-20 kb repeating nucleotide sequence (
TTAGGG
) located at the end
of a chromosome. The 3-20 kb (TTAGGG)
n
array is preceded by 100-300 kb of telomere—
associated repeats before any unique sequence is found.
B.
The telomere allows replication of linear DNA to its full length. Because 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 leading 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 of lagging strand shortening is solved by a special
RNA-directed DNA poly-
merase or reverse transcriptase
called
telomerase
(which has a RNA and protein component).
The RNA component of telomerase carries a
CCCUAA
sequence (antisense sequence of the
TTAGGG telomere) that recognizes the TTAGGG sequence on the leading strand and adds
many repeats of TTAGGG to the leading strand.
D.
After the repeats of TTAGGG are added to the leading strand,
DNA polymerase
uses the
TTAGGG repeats as a template to synthesize the complementary repeats on the lagging
strand. Thus, the lagging strand is lengthened.
DNA ligase
joins the repeats to the lagging
strand and a
nuclease
cleaves the ends to form double helix DNA with flush ends.
E.
Telomerase is NOT utilized by a majority of
normal somatic cells,
so that chromosomes nor-
mally get successively shorter after each replication; this contributes to the finite lifespan of
the cell.
F.
Telomerase is utilized by
stems cells
and
neoplastic cells
so that chromosomes remain per-
petually long. Telomerase may play a clinical role in
aging
and
cancer
.
IV. TYPES OF DNA DAMAGE AND DNA REPAIR
A.
Chromosomal breakage refers to breaks in chromosomes due to sunlight (or ultraviolet) irra-
diation, ionizing irradiation, DNA cross-linking agents, or DNA damaging agents. These
insults may cause
depurination of DNA
,
deamination of cytosine to uracil
,
or
pyrimidine dimer-
ization,
which must be repaired by DNA repair enzymes.
20
BRS Genetics
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B.
DNA repair involves
DNA excision
of the damaged site,
DNA synthesis
of the correct
sequence, and
DNA ligation
.
Some types of DNA repair use enzymes that do not require DNA
excision.
C.
The normal response to DNA damage is to stall the cell in the
G
1
phase
of the cell cycle until
the damage is repaired.
D.
The system that detects and signals DNA damage is a multiprotein complex called
BASC
(BRCA1-associated genome surveillance complex).
Some the components of BASC include:
ATM (ataxia telangiectasia mutated) protein, nibrin, BRCA1 protein,
and
BRCA2 protein
.
E.
The clinical importance of DNA repair enzymes is illustrated by some rare inherited diseases
that involve genetic defects in DNA repair enzymes such as xeroderma pigmentosa (XP),
ataxia-telangiectasia, Fanconi anemia, Bloom syndrome, and hereditary nonpolyposis col-
orectal cancer.
F.
Types of DNA damage include:
1. Depurination.
About 5,000 purines (A’s or G’s) per day are lost from DNA of each human
cell when the N-glycosyl bond between the purine and deoxyribose sugar-phosphate is
broken. This is the most frequent type of lesion and leaves the deoxyribose sugar-phos-
phate with a missing purine base.
2. Deamination of cytosine to uracil.
About 100 cytosines (C) per day are spontaneously
deaminate to uracil (U).If the U is not corrected back to a C, then upon replication instead
of the occurrence of a correct C-G base pairing and U-A base pairing will occur instead.
3. Pyrimidine dimerization.
Sunlight (UV radiation) can cause covalent linkage of adjacent
pyrimidines forming for example,
thymine dimers
.
V. SUMMARY TABLE OF DNA MACHINERY
Chapter 3
Chromosome Replication
21
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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
FIGURE 3-1. Replication fork. (A)
A diagram of double helix DNA (Chromosome 1) at a replication origin (RO) site. DNA heli-
case (H) will bind at the RO and unwind the double helix into two DNA strands. This site is called a replication bubble (RB).
At both ends of a replication bubble a replication fork (RF) forms. DNA synthesis occurs in a bidirectional manner from
each RF (arrows). (B) Enlarged view of a RF at one end of the replication bubble. The leading strand serves as a template
for continuous DNA synthesis in the 5
→
3
direction using DNA polymerase
(P
). The lagging strand serves as a tem-
plate for discontinuous DNA synthesis in the 5
→
3
direction using DNA polymerase
(P
). Note that DNA synthesis on
the leading and lagging strands is in the 5
→
3
direction but physically are running in opposite directions. (C) DNA syn-
thesis on the lagging strand proceeds differently than on 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 it with DNA. Finally, DNA ligase joins all the Okazaki fragments together.
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Chapter 3
Chromosome Replication
23
t a b l e
3-1
Summary of DNA Replication Machinery
Component Function
Topoisomerase
Nicks (or breaks) a single strand of DNA which causes DNA unwinding
DNA helicase
Recognizes the replication fork and opens up the double helix
High Fidelity DNA-Directed DNA Polymerases
DNA polymerase
Synthesizes the lagging strand; 3
→
5
exonuclease absent*
DNA polymerase
Repairs DNA by base excision; 3
→
5
exonuclease absent
DNA polymerase
Synthesizes mitochondrial DNA; 3
→
5
exonuclease present
DNA polymerase
Synthesizes the leading strand; 3
→
5
exonuclease present; repairs DNA by nucleotide
and base excision
DNA polymerase
Repairs DNA by nucleotide and base excision; 3
→
5
exonuclease present
Low Fidelity DNA-Directed DNA Polymerases
DNA polymerase
ζ
Involved in hypermutation in B and T lymphocytes
DNA polymerase
η
Involved in hypermutation in B and T lymphocytes
DNA polymerase
ι
Involved in hypermutation in B and T lymphocytes
DNA polymerase
Involved in hypermutation in B and T lymphocytes
RNA-Directed DNA Polymerase (Reverse Transcriptase)
Telomerase
Lengthens the end of the lagging strand
LINE 1/endogenous retrovirus
Converts RNA into cDNA, which can integrate elsewhere in the genome
reverse transcriptase
Primase
Synthesizes short RNA primers
Ligase
Catalyzes the formation of the 3
,5-phosphodiester bond; joins DNA fragments
Single-stranded binding proteins
Maintain the stability of the replication fork
High fidelity
DNA sequence faithfully copied; low fidelity DNA sequence not faithfully copied (error prone)
* 3
→
5
exonuclease serves as proofreading activity
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24
1.
Human cells have a finite lifespan and this
contributes to the aging process. Stem cells
and neoplastic cells have indefinite life
spans. The reason for these observations is
that chromosomes in a cell get progressively
shorter with each cell division because the
telomere sequences at the ends of the chro-
mosomes get shorter with each cell division.
The chromosomes in stem cells and neo-
plastic cells do not generally shorten with
each cell division. The enzyme utilized by
stem cells and neoplastic cells to lengthen
the telomeres is which of the following?
(A)
DNA polymerase delta
(B)
DNA polymerase alpha
(C)
DNA ligase
(D)
topoisomerase
(E)
telomerase
2.
Some antineoplastic drugs act by inhibit-
ing which of the following?
(A)
DNA helicase
(B)
topoisomerase
(C)
telomerase
(D)
DNA polymerase delta
(E)
DNA polymerase alpha
3.
Which one of the following is an accurate
statement regarding chromosome replication?
(A)
It is semiconservative.
(B)
It occurs during G1 in the cell cycle.
(C)
Inactive genes are replicated first.
(D)
It starts with the synthesis of Okazaki
fragments.
4.
The leading strand of DNA in the replica-
tion fork is synthesized by which one of the
following mechanisms?
(A)
continuously by DNA polymerase alpha.
(B)
discontinuously by DNA polymerase delta.
(C)
continuously by DNA polymerase delta.
(D)
discontinuously by DNA polymerase alpha.
5.
The autosomal recessive disease Fanconi
anemia is characterized by chromosome
breakage and rearrangements and most indi-
viduals with the disease will develop some
kind of cancer. Which one of the following is
defective in individuals with Fanconi anemia?
(A)
DNA polymerase delta
(B)
DNA repair enzyme
(C)
DNA ligase
(D)
DNA primase
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