1.

Discuss the following enzymes/proteins and their respective roles in DNA replication:

a. DNA polymerase
 The one responsible for DNA chain elongation by copying the DNA templates which can read parent
nucleotide sequences in 3’-5’ direction and synthesize new DNA strands in 5’-3’ direction.
 Needs RNA primer as this will act as the acceptor of the 1st deoxyribonucleotide and will allow the
DNA polymerase to start the process of elongation by addition of nucleotides along the single
stranded template.
 Remains bound to the template strand as it moves along and does not diffuse away and rebind
before adding a nucleotide
 New daughter strand formed is antiparallel to the parent strand
 3 types of prokaryotic DNA polymerase (Pol I, II, III) while 5 types of eukaryotic DNA polymerase
(DNA alpha, beta, gamma, delta, epsilon)

b. DNA helicase
 Responsible for separating the parent DNA strands by unwinding the double helix
 ATP driven process and initiates the start of the replication
 Helicases that unwinded the DNA at the replication fork can cause supercoiling in other regions of
the DNA

c. SSBP
 Prevents the single strands of DNA by reassociating or premature reannealing of dsDNA
 Can keep the 2 strands of DNA separated in the area of the replication of origin and protects the
DNA from nucleases which degrades the ssDNA

d. DNA primase
 Initiates the synthesis of RNA primers because DNA polymerase cannot initiate the synthesis of a
complementary strand of DNA on a single stranded template without its presence
 RNA primer- short segment of RNA that is base-paired to the DNA template and produces a double-
stranded DNA-RNA hybrid. The –OH group on the 3’ end of the RNA primer acts as the acceptor of
the deoxyribonucleotide by the action of the DNA polymerase.
 Prokaryotes: dnaG
 Eukaryotes: DNA Pol alpha

e. DNA ligase
 Joins the two adjacent DNA strands that are bound to the same template via formation of a
phosphodiester bond
 Seals the single strand nick between the nascent chain and Okazaki fragments on the lagging strand
 Okazaki fragments: short segments of DNA which are synthesized in 5’-3’ direction by the DNA delta
in eukaryotes

f. Topoisomerase
 Responsible for relieving torsional strain on the parental duplex caused by unwinding of the strands
 Relieve torsional strain that results from helicase-induced unwinding of DNA
 Two types: Type I and Type II DNA topoisomerase
o Type I
 Can reversibly cleave 1 strand of the double strand of the double helix
  Have both the strand cutting and strand-resealing capabilities and does not require
the usage of ATP
 Can relax the negative supercoils in E. coli and both the positive and negative
supercoils in many prokaryotic cells (except E coli) and in eukaryotes
 + supercoil: is wounded more tightly in the direction of the DNA helix (right
handed)
 - supercoil: made less tightly wound (left-handed)
o Type II
 Can bind tightly to the double helix of the DNA and forms transient breaks in both
strands
 Can relieve both the positive and negative supercoils
 Example: DNA gyrase which can introduce negative supercoils into the circular DNA
of bacteria via ATP. This can facilitate the replication of DNA due to the negative
supercoils neutralizing the positive supercoils introduced during the opening of the
double helix.
g. Exonuclease
 3’-5’ exonuclease
o removes the error that the DNA polymerase III has detected due to its own proofreading
ability
o This ability of the DNA polymerase III ensures that the correct nucleotide is being added to
the chain and is matched to its correct complementary base
o If there is an error present, the 3’-5’ exonuclease will cleave the error and then will be
replaced with the correct nucleotide by the 5’-3’ polymerase
 5’-3’ exonuclease
o DNA polymerase I has its 5’-3’ exonuclease activity which hydrolytically remove RNA primers
o DNA pol I will search and locate the space between the 3’ end of the newly synthesized DNA
by the DNA polymerase III and the 5’ end of the adjacent RNA primer
o DNA pol I will then remove the RNA primer while moving in 5’-3’ direction. As it removes it,
the DNA pol I will replace it with deoxyribonucleotides which synthesizes DNA in 5’-3’
direction and also proofreads the new chain using its 3’-5’ exonuclease activity to remove
errors

QUESTIONS:
 What does helicase do? Unzip the double stranded DNA
 Who is primase? initiates the synthesis of RNA primers because DNA polymerase cannot initiate the
synthesis of a complementary strand of DNA on a single stranded template without its presence
 DNA polymerase, what does it do? DNA chain elongation by copying the DNA templates which can
read parent nucleotide sequences in 3’-5’ direction and synthesize new DNA strands in 5’-3’
direction
 Why not begin with DNA polymerase? RNA primers have a free 3’-OH to which the 1st
deoxynucleotide is added

2. Discuss the three properties of DNA polymerase. Discuss the different DNA polymerases in
prokaryotes and eukaryotes and function of each of this polymerase in prokaryotes and eukaryotes

There are 3 important properties a DNA polymerase has and it includes: (1) chain elongation, (2)
processivity, and (3) proofreading.
 Chain elongation
o Involves the rate at which the polymerization of the newly strand of DNA occurs
o Elongation of the DNA strand is via the presence of DNA polymerases strand via the addition
of deoxyribonucleotides one at a time to the 3’ end of the growing chain
o The nucleotides that are added into the new strand is complementary base-paired
o Usually catalyzed by DNA polymerase III which uses the 3’-OH end of the RNA primer as the
acceptor of the 1st deoxyribonucleotide
o By this, polymerase then starts to add nucleotides along the template which is specific for
the sequence of bases in the newly synthesized chain
o New strand then will grom in the 5’-3’ direction which is antiparallel to the parent strand
o For this step to occur, all four deoxyribonucleoside triphosphates (dATP, dTTP, dCTP, and
dGTP) are needed and will not occur is one of them is depleted or missing
 Processivity
o This explains that the DNA polymerase remains bound to the parent template strand while
the enzyme creates new phosphodiester bonds while they are reading the template
o The DNA polymerase will not dissociate and reassociate after each nucleotide is added to
the existing DNA strand
o This feature is based on the beta subunit present in the enzyme which forms a ring that
encircles and moves along the template strand of the DNA which serves as a sliding DNA
clamp
 Its role is to increase the Pol III-DNA stability, processivity, and rate of chain
elongation
 Proofreading
o DNA polymerase III has its own proofreading ability using the 3’-5’ exonuclease
o It functions to check the strand to make sure that the newly added nucleotide is correct and
matches the complementary base on the template
o If there is error detected, 3’-5’ exonuclease will correct the mistake
o This removes most of the base pairing errors as they occur in the chain

TYPES OF DNA POLYMERASES IN PROKARYOTES

TYPES OF DNA POLYMERASES IN EUKARYOTES
They are categorized based on their molecular weight, cellular location, sensitivity to inhibitors, and the
templates or substrates on which they can act

 Pol alpha
o Multisubunit enzyme as one subunit has primase activity
 initiates the synthesis of the new strand on the leading strand and at the beginning
of each Okazaki fragments on the lagging strand
 Pol epsilon and delta
o Pol epsilon: DNA synthesis on the leading strand
o Pol delta: elongation of the Okazaki fragments on the lagging strand
o Both uses 3’-5’ exonuclease activity in proofreading the newly synthesized DNA
 Pol beta and Pol gamma
o Pol beta: used for the gap filling of DNA repair
o Pol gamma: cause mitochondrial DNA synthesis
QUESTIONS:
 What are the 3 properties? (1) chain elongation, (2) processivity, and (3) proofreading.
 Importance of proofreading? To detect presence of errors during the elongation phase and remove
it and replace it with correct nucleotide

3. What are the 3 major stages in DNA replication? Describe the different processes that occur in each
stage

3 steps in DNA replication: initiation, elongation, and termination

 Initiation
o DNA replication initiation involves the separation of complementary DNA strands, starting at
specific origins of replication. In prokaryotes, a consensus sequence marks the origin, while
eukaryotes have multiple initiation sites. The unwinding of strands forms a replication fork
where synthesis occurs bidirectionally, creating a replication bubble. Proteins like DnaA,
helicases, and SSB maintain strand separation, and DNA topoisomerases resolve
supercoiling issues. DNA replication proceeds in the 5'-3' direction, with leading and lagging
strands synthesized differently. RNA primers, synthesized by primase, are essential for DNA
polymerases to initiate synthesis. The primosome, formed by adding primase, facilitates
leading strand synthesis and initiates Okazaki fragment formation on the lagging strand.
 Elongation
o During DNA elongation, DNA polymerase III in both prokaryotes and eukaryotes extends a
new DNA strand by sequentially adding deoxyribonucleotides to the 3'-end, utilizing the 3'-
hydroxyl group of the RNA primer. Its high processivity, facilitated by the B subunit forming
a sliding DNA clamp, ensures continuous template binding. The synthesis occurs in the 5'-3'
direction, releasing pyrophosphate with each addition. DNA polymerase III's proofreading,
through 3' 5' exonuclease activity, corrects mismatched nucleotides, maintaining replication
fidelity and minimizing errors.
 Termination
 o During DNA replication termination, DNA polymerase III synthesizes DNA on the lagging
strand until it encounters an RNA primer. DNA polymerase I, with its 5'-3' exonuclease
activity, removes the RNA primer, replacing it with deoxyribonucleotides while proofreading
using 3' 5' exonuclease activity. This continues until the RNA primer is entirely degraded,
and the gap is filled with DNA. The distinct 5'-3' exonuclease activity of DNA polymerase I
allows removal from properly base-paired regions and handling of altered nucleotide
groups. DNA ligase then connects the DNA chains synthesized by DNA polymerases III and I,
utilizing energy from ATP cleavage to AMP + PP.

QUESTIONS:
 Mention each enzymes in each stages
o Initiation
 DnaA protein: binds to the nucleotides at the origin of replication and results with
strand separation with the formation of localized regions of ssDNA
 DNA helicase: separates the parental DNA strands by unwinding the double helix
 SSBP: prevents single strands of DNA from reassociating
 Topoisomerases: relieve torsional strain caused by unwinding
 Primase: synthesizes RNA primers
 RNA primer: has 3’-OH on the RNA strand and serves as the 1st acceptor of a
deoxynucleotide via DNA polymerase
o Elongation
 DNA polymerase: catalyzes the chemical reaction of DNA polymerization and
synthesizes DNA from 5’-3’ direction
o Termination
 5’-3’ exonucleases: DNA polymerase I has its 5’-3’ exonuclease activity which
hydrolytically remove RNA primers
 3’-5’ exonucleases: removes the error that the DNA polymerase III has detected due
to its own proofreading ability
 What happens to the lagging strand? The RNA primers are removed by nucleases (e.g., RNase H);
then the resulting gaps are filled with the appropriate deoxyribonucleotides by another DNA
polymerase.

4. Discuss (do not tabulate) the similarities and differences between prokaryotic and eukaryotic DNA
replication

Similarities between Prokaryotic and Eukaryotic DNA Replication:

1. Bidirectional Replication → Both prokaryotic and eukaryotic DNA replication initiate
bidirectionally from specific origins of replication.
2. Semiconservative Replication → Both types of replication follow a semiconservative mechanism,
where each daughter DNA molecule contains one parental strand and one newly synthesized
strand.
3. Unwinding of Parental Strands → Helicases unwind the parental DNA duplex ahead of the
replication fork in both prokaryotic and eukaryotic replication.
4. Action of DNA Polymerase → DNA polymerases catalyze the synthesis of new DNA strands in
both prokaryotes and eukaryotes. The synthesis occurs in a 5’-to-3’ direction, and
deoxyribonucleoside triphosphates serve as substrates.
 5. Function of RNA Primers → Both prokaryotic and eukaryotic DNA polymerases require RNA
primers synthesized by primase to initiate DNA synthesis.
6. Proofreading Mechanism → Both prokaryotic and eukaryotic DNA polymerases have
proofreading mechanisms to correct base-pairing errors during replication.
7. DNA Synthesis at the Replication Fork → Both prokaryotic and eukaryotic replication involve
synthesis of the leading and lagging strands, with the lagging strand synthesized discontinuously
in short fragments.
8. Function of DNA LigasE → DNA ligase seals the nicks between Okazaki fragments in both
prokaryotic and eukaryotic replication.

Differences between Prokaryotic and Eukaryotic DNA Replication:

1. Point of Origin of Replication → Prokaryotic chromosomes typically have a single origin of
replication, while eukaryotic chromosomes have multiple origins of replication.
2. Size and Complexity → Eukaryotic DNA replication is more complex and involves more proteins
due to the larger amount of DNA and its association with histones in nucleosomes.
3. DNA Polymerases → Prokaryotes have a few DNA polymerases, whereas eukaryotes have
numerous polymerases with specialized functions.
4. Replication Complex → The replication complex in eukaryotes is more elaborate and involves
additional proteins compared to prokaryotes.
5. Okazaki Fragment Size → Okazaki fragments are smaller in eukaryotes compared to prokaryotes.
6. Telomere Replication → Eukaryotes have specialized mechanisms, such as telomerase, for
replicating the ends of linear chromosomes, which is not present in prokaryotes.

→ These differences reflect the adaptations necessary to accommodate the different sizes and
structures of prokaryotic and eukaryotic genomes.

QUESTIONS:
 What phase of cell cycle for replication of eukaryotes and prokaryotes? In eukaryotes, S phase or
synthesis phase.
 Difference on how fast replication of eukaryote and prokaryote? Since prokaryotic cells typically
have only a single, circular chromosome, they can replicate faster than eukaryotic cells. In fact, a
prokaryotic cell can undergo two rounds of DNA replication before the cell, itself, has divided. This
means that DNA replication can occur during cell division in prokaryotes.

5. What is the role of telomeres in the aging process? Discuss the enzyme telomerase
 Telomeres, which naturally shorten with age, play a crucial role in cellular aging. The rate of
telomere shortening, approximately 24.8–27.7 base pairs per year in humans, is associated with
aging and age-related diseases. Various factors, including genetics, environment, lifestyle choices
(smoking, obesity, lack of exercise, unhealthy diet), and socio-economic status, influence telomere
length. Shorter telomeres, especially below a critical limit, lead to cellular senescence or apoptosis.
Accelerated telomere shortening is linked to health issues like coronary heart disease, diabetes,
cancer risk, and osteoporosis. Lifestyle factors can intensify telomere shortening, impacting health
and lifespan. Notably, cancer cells exhibit elevated telomerase activity despite shorter telomeres.
Understanding and managing telomere length is crucial for assessing aging-related risks and
potential interventions.
 Telomerase is an enzyme consisting of proteins and RNA, functioning as an RNA-dependent DNA
polymerase. It elongates the 3'-end of DNA strands by using its RNA as a template, complementary
to the repeating sequence in telomeres. The existing 3'-overhang serves as a primer for telomerase,
allowing synthesis of new DNA. This process repeats, and when the overhang reaches a sufficient
length, primase binds to complete the complementary strand. Despite lengthening, a 3'-overhang
remains, forming a protective structure with telomere-binding proteins, safeguarding chromosome
ends from damage and nuclease attacks.

QUESTIONS:
 If there are no telomeres, what will happen? Without telomeres, the ends of chromosomes would
look like broken DNA, and the cell would try to fix something that wasn't broken. That also would
make them stop dividing and eventually die.
 If it shorten, what can be affected? Coding sequence
 If maikli na si telomere, it is a sign of? Aging or old cells
 If inflamed na si telomere, will it shorten? Telomere shortening is a natural part of the aging process,
and inflammation can exacerbate telomere dysfunction by increasing the rate of telomere attrition,
leading to telomere dysfunction-mediated cellular senescence, and accelerating the aging process.
 Telomerase, where is it active? present in germ line, stem cells, most cancer cells and absent from
most somatic cells
6. Briefly describe the following repair mechanisms:
 Mismatch repair
o Involves the removal of the portion of the newly synthesized strand of recently replicated
DNA that contains a pair of mismatched bases. Bacteria recognize the newly synthesized
strand because, in contrast to the parental strand, it has not yet been methylated.
o Mismatch repair (MMR) is a crucial mechanism correcting errors in DNA synthesis,
particularly mismatches. In E. coli, Mut proteins facilitate this process, while humans have
homologous proteins. In prokaryotes, discrimination is based on methylation, with the
parental strand being methylated, and the repair targeting the unmethylated daughter
strand. In eukaryotes, recognition involves nicks in the newly synthesized strand. The repair
includes endonuclease nicking, exonuclease removing mismatched nucleotides, and DNA pol
III using the sister strand as a template to fill the gap. DNA ligase joins the newly synthesized
DNA to the original strand. Mutations in MMR proteins in humans are linked to hereditary
nonpolyposis colorectal cancer (HNPCC or Lynch syndrome), elevating the risk of colon and
other cancers.
 Nucleotide excision repair
o Involves the removal of a group of nucleotides (including the damaged nucleotide) from a
DNA strand
o Nucleotide excision repair (NER) is a DNA repair mechanism addressing distortions in the
DNA helix. Repair endonucleases cleave the abnormal chain, and a DNA polymerase fills the
gap using the complementary strand as a template. DNA ligase then connects the newly
synthesized segment, maintaining genomic integrity and stability.
 Base excision repair
o Involved a specific glycosylase that removes a damaged base by hydrolyzing an N-glycosidic
bond, producing an apurinic or apyrimidinic site, which is cleaved and subsequently repaired
o Base excision repair is a mechanism used to detect and remove certain types of damaged
bases. A group of enzymes called glycosylases play a key role in base excision repair. Each
glycosylase detects and removes a specific kind of damaged base.
o For example, a chemical reaction called deamination can convert a cytosine base into uracil,
a base typically found only in RNA. During DNA replication, uracil will pair with adenine
rather than guanine (as it would if the base was still cytosine), so an uncorrected cytosine-
to-uracil change can lead to a mutation
o To prevent such mutations, a glycosylase from the base excision repair pathway detects and
removes deaminated cytosines. Once the base has been removed, the "empty" piece of
DNA backbone is also removed, and the gap is filled and sealed by other enzymes

What diseases might ensue with defects in theses repair mechanisms?

 Mismatch repair
o Hereditary nonpolyposis colorectal cancer (HNPCC) or Lynch syndrome
 Autosomal dominant genetic disease resulting from mutations in one of four DNA
mismatch repair genes. This condition significantly elevates the risk of various cancers,
particularly in the colon and endometrium. Lynch syndrome, found in about 1 in 500
 individuals in the general population, contributes to 2% to 3% of all colorectal cancers.
Diagnosis involves analyzing tumor tissue for evidence of deficient mismatch repair,
including microsatellite instability and loss of mismatch repair protein expression.
 Nucleotide excision repair
o Xeroderma pigmentosum
 Xeroderma pigmentosum (XP) is a rare autosomal recessive genodermatosis resulting
from mutations in the nucleotide excision repair system. Individuals with XP exhibit
severe photosensitivity, skin pigmentary changes, heightened susceptibility to
malignant tumors, and occasional neurological degeneration. The condition arises
due to compromised nucleotide excision repair, leading to the accumulation of
unrepaired DNA damage, particularly from ultraviolet-induced lesions. XP manifests
in multiple subtypes (XP A-G) and variants, each linked to specific mutations in the
nucleotide excision repair system, contributing to diverse clinical presentations.
 Base excision repair
o MUTYH-associated polyposis
 MUTYH-associated polyposis is a rare genetic condition caused by specific changes in
the MUTYH gene, responsible for fixing errors in DNA caused by oxidative damage.
When this repair process fails, it can lead to specific changes in genes like APC and
KRAS, causing multiple colorectal adenomas. The severity and features of the
condition can vary among individuals due to factors like genetics, environment, and
epigenetics. It is an autosomal recessive syndrome, meaning individuals need two
abnormal copies of the MUTYH gene for the condition to manifest. Apart from
colorectal issues, MUTYH-associated polyposis may also affect other parts of the
body.
QUESTIONS:
 What discriminates the mismatch? MUE proteins
 How does it discriminate? ewan q