The Replication of DNA

Nivedita
Ph.D. (JNU-Life Sciences)
CSIR NET-JRF, GATE
nivedita229@gmail.com
Q. In order to study the role of telomeres in DNA replication, genetically engineered
mice were prepared, where the gene for telomerase RNA was knocked out. When
cells from these knockout mice were taken and cultured in vitro, they proliferated
even after 100 cell divisions which is quite unlikely in the case of human cells. Which
of the following is the correct reason?

a. Human and mice are fundamentally different with respect to their requirements for
telomerase enzyme in the context of DNA replication.

b. In vitro, mice DNA becomes circular due to end to end chromosome fusion and doesn
not require telomerase for DNA end replication.

c. Mice have very long stretch of telomere DNA sequence compared to that of human.

d. In vitro, mice DNA replication does not require the removal of RNA primers.

Ans c
Q. Following statements were made about Human mitochondrial genome:

A. The replication of both H and L strands is unidirectional and begins at specific

regions.
B. Majority of the mitochondrial genes code for protein product
C. Though the mitochondrial genome is extremely compact, the genes never show
any sequence overlap.
D. The CR/D loop region of mitochodrial genome exhibits triple stranded structure.
E. Transcription of mtDNA initiates bidirectionally from a common promoter region
in the CR/D loop region and continuous around the circle.

Which one of the following options contains a combination of all correct statements?

1. A,B,D
2. A,D,E
3. B,D,E
4. B,C,D

Ans 2
•Strand displacement model:
•unidirectional replication
•one replication fork for each strand.
•two origins (OH and OL)
•One strand is called the light strand (L) and the other the heavy strand (H)-
different buoyant densities in denaturing CsCl density gradients.

Models for mitochondrial DNA replication

Strand coupled model:
•bidirectional mode of DNA replication.
•coupled leading (H strand) and lagging strand (L strand) synthesis.
•semidiscontinuous mode
• involving the synthesis of Okazaki fragments

Models for mitochondrial DNA replication

DNA polymerase γ is used exclusively for mtDNA replication and proofreading.

 The RNA primers are cleaved by the multifunction endoribonuclease RNase

MRP.
Rolling circle replication
Rolling circle replication
Mutation, DNA Damage and Repair
 Replication errors
Permanent change in
DNA,
Preventing
replication/transcription

Chemical damage
Mutation
Passed in progeny,
promote diseases, cell
death

Transposition
Types of nucleotide or base change substitutions-

Transition mutation:
•Pyrimidine → pyrimidine (T → C or C → T)
•Purine → purine (A → G or G → A)

Transversion mutation:
•Pyrimidine → purine (T → A, T → G, C → A, or C → G)
•Purine → pyrimidine (A → T, A → C, G → T, or G → C)

Transition Transversion
Point mutation: Mutations that alter a single nucleotide are called point mutations

A point mutation can be permanently incorporated by DNA replication.

Transitions and transversions can lead to silent, missense, or nonsense mutations

Silent mutations : Mutations that change the nucleotide sequence without

changing the amino acid sequence.

Missense mutations : Nucleotide substitutions in protein-coding regions that do

result in changed amino acids. (e.g. sickle cell anemia; AT → TA transversion
causing the normal glutamic acid codon in the β-globin chain of hemoglobin to be
replaced with valine).

Nonsense mutations : A nucleotide substitution that creates a new stop codon.

cause premature chain termination during protein synthesis.
Insertions or deletions mutation:

Insertions or deletions can cause frameshift mutations

Frameshift mutation: the addition or deletion of one or more base pairs

results in a shift in the reading frame of the resulting mRNA, and leads to
production of a nonfunctional protein.

e.g. The deletion of three base pairs in the nucleotide sequence of the cystic fibrosis
transmembrane conductance regulator (CFTR) gene results in the loss of the codon
for phenylalanine.
Types of point mutations in protein-coding sequences
Mismatch repair pathway in E. coli
Dam methylation in E. coli
Diectionality in mismatch repair, exonuclease removal of mismatched DNA
Mismatch repair pathway in mammalian cells.
Mismatch repair pathway in mammalian cells.
Mismatch repair pathway in mammalian cells.
A mutagen is any chemical agent that causes an increase in the rate of
mutation above the spontaneous background.

DNA damage:
single base changes,
structural distortion, and
DNA backbone damage.
 Deamination

Depurination

DNA damage Alkylation

Oxidation

Ionizing
radiation, UV, X-
ray, gamma rays
Deamination is the most frequent and important kind of hydrolytic damage.

Converts C to U
Converts A to hypoxanthine that base pair with C
Converts G to xanthine that base pair with C.

Depurination caused by spontaneous hydrolysis of the N-glycosyl linkage that

produces abasic site on DNA. Cause DNA backbone damage.
 Depurination

Types of DNA damage

Alkylating agents such as nitrosamines lead to the formation of O6-
methylguanine that often mispairs with thymine, resulting in the change of
a GC base pair into an AT base pair when the damaged DNA is replicated.

Another alkylating agent is N-methyl-N¹-nitro-N-nitrosoguanidine.

Methylated cytosines are hotspots for spontaneous mutations in vertebrate DNA
because deamination of 5-methylcytosine generates thymine.

This results in the change of a GC base pair into an AT when damaged DNA is
replicated.
•Potent oxidizing agents are generated by ionizing radiation and by chemical
agents that generate free radicals.

•These reactive oxygen species (for example O2-, H2O2, and OH[·]) can
generate 8-oxoguanine (oxoG), a damaged guanine base containing an extra
oxygen atom.

• OxoG is highly mutagenic because it can form a Hoogsteen base pair with
adenine.

•This gives rise to a GC → TA transversion, which is one of the most

common mutations found in human cancers.
G modification
Thymine dimer caused by UV
Structural distortion.
Impede replication/transcription

Types of DNA damage

•Gamma rays and x-rays are hazardous because they can create double-
strand break in DNA.

•Can directly attacks the deoxyribose on DNA.

•Used as therapeutic to kill rapidly proliferating cancerous cells.

•Anticancer drug such as bleomycin can cause double strand break and
called as clastogenic.
Base analog
2-Aminopurine, a purine analog of guanine and adenine, is a fluorescent
molecular marker used in nucleic acid research

2-Aminopurine
•Flat, planar,polycyclic
rings.
•cause insertion or
deletion

Intercalating agents
Q. Some errors occur during DNA replication that are not corrected by proof reading
activity of DNA polymerase. These are corrected by specialized repair pathways. Defect in
the activities of some of the following enzymes impair this process.

A. DNA polymerase III and DNA ligase

B. AP endonuclease and DNA glycosidase
C. Mut S and Mut L
D. Rec A and Rec F
Defect in which of the above enzymes impair the process?

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

Ans 4
Q. The mismatch repair activity of E. coli repairs misincorporated bases which is
not removed by the proofreading activity of DNA polymerase. However, while
doing so, it has to decide which strand of the DNA is newly synthesized and which
one is parental. Mismatch repair system does it by which one of the following
ways?

1. It recognizes nearby GATC sequence.

2. It recognizes any nearby palindromic sequence.
3. It recognizes a specific repetitive sequence.
4. It recognizes the hemi-methylated GATC sequence nearby.

Ans 4
Q. Which one of the following chemicals is a DNA intercalator?

1. 5-Bromouracil
2. Ethyl methane sulfonate
3. Acridine orange
4. UV

Ans 3

Q. 5-Bromouracil is a base analog that can cause mutation when incorporated into
DNA. Which of the following is the most likely change that 5-Bromouracil induces:

1. T:A to C:G
2. T:A to A:T .
3. G:C to T:A
4. C:G to A:T

Ans 1
Q. 2- Aminopurine induces mutation by

(1) Base pair change.

(2) Frameshift
(3) Duplication
(4) Deletion

Ans 1

Q. The following is the amino acid sequence of a part of a protein encoded by gene
'X'. …..Phe Leu Val Pro Ser Tyr Cys….. A mutant for gene 'X' is isolated following
treatment with a mutagen. The amino acid sequence of the same region encoded by
the mutant gene is as follows: …..Phe Leu Phe Arg Arg lle….. Which of the
following mutagens is most likely to have been used?

1. 5-bromouracil
2. 2-amino-purine
3. Ethyl methanesulfonate
4. Acridine orange

Ans 4
Q. The base analog 2-aminopurine pairs with thymine, and can occasionally
pair with cytosine. The type of mutation induced by 2-aminopurine is

(1) transversion
(2) Transition.
(3) deletion
(4) nonsense.

Ans 2
 Identification of
chemical agents that act
as a mutagen

Mutant (S. typhimurium)

has defective operon
involved in His
biosynthesis.

Liver enzymes can

convert chemicals into
potent mutagen.

The Ames test

Q. Ames test is used to evaluate mutagens in the environment. Which of the
following statements, about Ames test are true?
A. The mutagenic effect of a compound tested using an auxotrophic strain of
Salmonella typhimurium
B. The mutagenic effect of a compound is tested using His strain of Escherichia
coli.
C. Using appropriate strains, compounds causing base substitutions and frame
shift
D. Liver enzymes are important as they are activated by test compound to evaluate
its mutagenicity potential.
E. Many compounds may have to be converted to bioactive metabolites, which is
carried out by the enzymes from the liver

(1) A, C and D
(2) A, B and D
(3) A, C and E
(4) A and E only

Ans 3
Q. Three met– E. coli mutant strains were isolated. To study the nature of mutation these
mutant strains were treated with mutagens EMS or proflavins and scored for revertants. The
results obtained are summarized below:
( + stands for revertants of the original mutants and – stands for no revertants obtained)
Based on the above and the typical mutagenic effects of EMS and proflavin, what was the
nature of the original mutation in each strain?

Mutant strain Mutagen treatment

EMS Proflavin

A - +
B + -
C - -

1.A-Transversion
B- Insertion or deletion of a single base
C- Deletion of multiple bases
2. A-Transition
B- Transversion
C- Insertion or deletion of a single base
3. A-Insertion or deletion of a single base
B- Transition
C- Deletion of multiple bases
4. A- Transition
B- Insertion or deletion of multiple bases
C- Transversion Ans 3
Reconstitution of human
mismatch repair in an in
vitro system
Q. A 6.4 Kb plasmid DNA has two restriction endonuclease sites, HindIII and EcoRI.
Complete double digestion of the plasmid with both the enzymes yields two fragments
of 3.1 and 3.3 Kb. In order to study DNA repair process, a G:T mismatch was
introduced in one strand of HindIII site and the damaged plasmid was incubated in a
reconstituted repair system containing all the factors and enzymes required for repair. If
the efficiency of the repair system is 50%, which one of the following band patterns on
agarose gel will be obtained after treating the repaired plasmid with both HindIII and
EcoRI?

Ans 4
Hereditary nonpolyposis colorectal cancer: a defect in mismatch repair

HNPCC is an autosomal dominant condition resulting from mutations in one of

several DNA mismatch repair genes.
Mutations in either the MSH2 (MutSa) or MLH1 (MutLa) genes account for more than
90% of mutations in HNPCC families.

•Germline mutation in one allele of the mismatch repair genes

•Somatic loss of the wild-type allele
•Defective mismatch repair mechanism
•Accumulation of mistakes in DNA replication
•Microsatellite instability
DNA repair systems.
Type Damage Repair proteins

Damage bypass

Translesion DNA Pyrimidine dimer or DNA polymerases IV and V in E. coli

synthesis apurinic site Pol ζ, pol η, pol ι, pol κ, and pol λ in humans

Damage reversal

Photoreactivation Pyrimidine dimers DNA photolyase*

Removal of methyl groups O6-methylguanine Methyltransferase

Damage removal

Base excision repair Damaged base DNA glycosylases

MutS, MutL, and MutH in E. coli

Mismatch repair Replication errors
MutSα, MutLα, and EXO1 in humans
Pyrimidine dimer UvrA, UvrB, UvrC, and UvrD in E. coli
Nucleotide excision repair
Bulky adduct on base XPA, XPB, XPC, XPD, ERCC1/XPF, and XPG in humans
RecA and RecBCD in E. coli
MRN complex, Rad51, Rad 52, BRCA1, BRCA2,
Double-strand break repair Double-strand break XRCC3, etc. in humans for homologous recombination
Ku proteins, Artemis/DNA-PKCS, XRCC4 in humans for
nonhomologous end-joining
 DNA photolyase uses energy from near UV to
blue light to break the covalent bonds holding
the two adjacent pyrimidines together

DNA repair by photoreactivation (Direct reversal of DNA damage)

 A sulfhydryl group of a cysteine residue of the
methyltransferase accepts the methyl group
from guanine.

DNA repair by methyl group removal (Direct reversal of DNA damage).

OxoG:A repair- DNA damage recognition by 8-oxoguanine DNA glycosylase 1
(hOGG1)
Base excision repair pathway in mammalian cells.
Base excision repair pathway in mammalian cells.
Base excision repair pathway: the uracil glycosylase reaction