The Replication of DNA

Nivedita
Ph.D. (JNU-Life Sciences)
CSIR NET-JRF, GATE
nivedita229@gmail.com
Genetic identification of the
replicators
Mapping eukaryotic DNA
replication origins.
Mapping eukaryotic DNA replication origins
ORC as a molecular machine that stimulates pre-replication complex
assembly
The associated proteins were analyzed by immunoblotting with antibodies specific for
the following components of the prereplication complex: Mcm2-7, Orc2, and Cdc6.
Q. In eukaryotic cells, replication initiation from a replication origin occurs only
once per cell cycle and S-phase CDKs play avital role in the regulation of DNA
replication. In budding yeast, a protein complex known as a origin recognition
complex (ORC) is associated with DNA replication origin in G1; however, origin fire
only once at the beginning of S-phase. DNA replication does not start in G1 because:

A. MCM helicases are inactive in G1

B. Spindle checkpoint is active in G1
C. DNA polymerase is not recruited in G1
D. ORC and initiation factors cdc6 and cdt1do not recruit MCM helicase to the site of
replication initiation in G1

Which of the above statements are correct?

1. A and B
2. A and C
3. B and C
4. B and D

Ans 2
The following statements are made on DNA replication:
A. The replication fork is a branch point in replication “eye” or “bubble”.
B. A replication bubble contains two replication fork.
C. DNA replication is continuous according to the interpretation made by
Okazaki.
D. Multiple priming events are required for both leading and lagging strands to
initiate DNA synthesis.

Which one of the following is a correct combination:

1. A and B
2. B and C
3. C and D
4. A and C

Ans 1
Q. Plasmid copy number achieved by plasmid encoded control elements that
regulate the initiation of the replication step. For example in stringent plasmid
protein Rep A dimerize and binds to origin of replication and do not allow
replication more than once. What mutation may convert this stringent mode of
replication in plasmid into relaxed one?

a. Over expression in repA protein

b. Mutation in repA gene in dimerization domain
c. Mutation in repA other than dimerization domain
d. Gain of function in recognition domain of repA

Ans b
Q. Each Origin of replication is activated only once. This is achieved because:

a. Pre-replicative complex can only form in G1 and replication can only be initiated
when pre-replicative complex is dissembled at the beginning of S-phase.
b. replication can only be initiated when pre-replicative complex is intact.
c. replication can only be initiated when unphosphorylated Rb is present.
d. Pre-replicative complex can only form in S phase.

Ans a
Termination of DNA replication in circular DNA

Topoisomerase II catalyzes the decatenation

of replication products
 Termination of Replication in eukaryotic chromosome

End replication problem : When the final primer is removed from the lagging
strand at the end of a chromosome, this 8–12 nt region is left unreplicated.
Protein priming is a solution to end replication problem
 Telomeres
•The ends of the eukaryotic chromosomes are called telomeres.

•Telomeres were identified in 1938 by Barbara McClintock working with

maize (corn).

•Telomeres are comprised of tandem repeats of a TG-rich sequence.

•Telomeres seal the ends of chromosomes and confer stability by keeping

the chromosomes from ligating together.

•Loss of telomeres leads to end-to-end chromosome fusions, facilitates

increased genetic recombination, and triggers cell death through apoptosis.

•The 3’ end of each chromosome extends beyond the 5’end as ssDNA.

 Telomerase

•Telomerase is a ribonucleoprotein (RNP) complex enzyme that has an RNA

subunit and a protein subunit that are both essential for activity.

•The protein component of telomerase characterized as a reverse

transcriptase – a polymerase that synthesizes DNA using an RNA template
and called as telomerase reverse transcriptase (TERT).
•Telomerase extends 3’end of the DNA like other DNA polymerase.

•Telomerase does not need an exogenous DNA template to direct DNA

synthesis.

•Instead, RNA component of the telomerase serves as a template.

Replication of telomere
by telomerase
Replication of telomere
by telomerase
Extension of 3’ end of the
telomere by telomerase
solves the end problem
 Regulation of telomerase activity

Telomere length control by POT1, TRF1, and TRF2:

•The proteins POT1 (protection of telomeres), TRF1 (TTAGGG repeat binding

factor 1), and TRF2 (TTAGGG repeat binding factor 2) may prevent telomerase
access to telomeres by forming a folded chromatin structure.

•When the telomere is long enough, the levels of POT1 on the overhang are
high, and telomerase is inhibited.

•When the telomere is too short, little or no POT1 is transferred to the end and
telomerase is no longer inhibited, allowing it to add DNA back to the telomere.
Model for telomere length control.
t-loop formation
•
In a wide range of eukaryotes, telomeric DNA has been shown to form a
unique t-loop structure.

•In this structure, the 3′ single-stranded DNA tail invades the double-stranded
telomeric DNA to form the loop in which the 3′ overhang is base paired to the
C strand sequence.
Telomerase activity :
 Telomerase, aging, and cancer

•The point at which cells stop dividing (limited capacity for replication)
was called the Hayflick limit (Leonard Hayflick, 1962).

•After the Hayflick limit, telomere shortening triggers an irreversible state

of cellular aging or senescence, a state characterized by continued cell
viability without further cell division.
Telomere shortening is a “molecular clock” that triggers aging.

One of the hallmark features of cancer cells is that they become

immortalized and can grow uncontrollably.

In most human cancer cells, telomerase has been reactivated.

Telomerase may thus be a more attractive target for anticancer therapy

rather than anti-aging therapy.
Effect of experimental activation of telomerase on normal human somatic cells

Telomerase activity increases the lifespan

Gene therapy for liver
cirrhosis
 Dyskeratosis congenita: loss of telomerase activity

Dyskeratosis congenita is a rare inherited disease in which patients have problems

in tissues where cells multiply rapidly and where telomerase is normally
expressed .
Symptoms of dyskeratosis congenita
Reduced telomerase activity, abnormally short telomeres, age-
General dependent
increase in chromosomal rearrangements
Hair Hair loss (including eyelashes and eyebrows), premature graying
Mouth Precancerous oral lesions (leukoplasia), tooth loss, and cavities
Skin Abnormal pigmentation, skin cancer
Finger and toenails Nail dystrophy
Lung Fibrosis
Liver Cirrhosis
Intestine Gut disorders, cancer
Testes Hypogonadism (defects in sperm formation)
Poor wound healing, frequent infections, failure to produce blood
Bone marrow
cells
•There are two forms of this disease:
X-linked recessive dyskeratosis congenita : results from mutations in the gene coding
for a protein called dyskerin.

•Dyskerin is a pseudouridine synthase that binds to many small nucleolar RNAs

(snoRNAs) and is proposed to play a role in ribosomal RNA processing.

• Patients with dyskerin mutations have five-fold less telomerase RNA than unaffected
siblings.

• Autosomal dominant dyskeratosis congenita : results from mutations in the

telomerase RNA gene.

•The disease results from partial loss of function of telomerase RNA, either through
deletions or through one or two single base changes.

•As in the X-linked recessive form of the disease, patients have abnormally short
telomeres in dividing cells types where telomerase is normally expressed.
Q. Telomerase, a RNA-protein complex which completes the replication of telomeres
during DNA synthesis, is a specialized

1. RNA dependent DNA polymerase

2. DNA dependent DNA polymerase
3. DNA dependent RNA polymerase
4. RNA dependent RNA polymerase

Ans 1

Q. Telomerase, a protein-RNA complex, has a special reverse transcriptase activity

that completes replication of telomeres during DNA synthesis. Although it has many
properties similar to DNA polymerase, some of them are also different. Which one of
the following properties of telomerase is different from that of
DNA polymerase?

1. Telomerase requires a template to direct the addition of nucleotides

2. Telomerase can only extend a 3 -OH end of DNA
3. Telomerase does not carry out lagging strand synthesis
4. Telomerase acts in a processive manner

Ans 3
Q. Which statement is correct in relation of activity of telomerase?

a. Increase with age

b. Observed in all cancers and responsible for immortality
c. Responsible for apoptosis but not for ageing
d. Re‐synthesize telomeres

Ans b

Q. Ciprofloxacin is a synthetic chemotherapeutic antibiotic of the fluoroquinolone

drug class. The target of antibiotic ciprofloxacin is…

a. Replication
b. Protein synthesis
c. Cell wall synthesis
d. Membrane structure

Ans a
Explanation: Ciprofloxacin is a broad-spectrum antibiotic of the fluoroquinolone class. It is active
against some Gram-positive and many Gram-negative bacteria. It functions by inhibiting a type II
topoisomerase (DNA gyrase) and topoisomerase IV, necessary to separate bacterial DNA
(replication), thereby inhibiting cell division.