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Gene Expression I: DNA Replication & Repair LE 5

DR. LOURDES L. BALCUEVA 03/22/2018

OUTLINE
Replication
I. Overview  DNA is duplicated to become the template to make DNA copies
A. Central Dogma B. Priming
 The genetic information (genes) in the DNA of chromosome
B. DNA Synthesis C. Elongation Process
can be transmitted by exact replication or it can be exchanged
C. Features of DNA IV. Termination by number of processes:
Replication A. Telomeres
 Recombination
II. Initiation B. Telomerase
 Crossing over: occurs in sex cells
A. Origin of Replication V. Replication Errors
A. Abnormalities and  Transposition: “jumping genes” important in evolution
B. Separation of Parental
Strands Repair  Conversion: important in DNA repair
C. Regulation of the B. Replication Repair
Initiation Process VI. Clinical Correlation
III. Elongation VII. Notes
A. Separated Parent VIII. Appendix
Strands

OBJECTIVES

 To discuss the general concept of replication

 Describe & give the functions of the enzymes involved in the
different steps of eukaryotic replication
 Initiation
 Elongation Figure 2. Mechanisms of DNA conversion
 Termination
 Explain the regulation of the initiation process
 Describe the elongation of the leading & the lagging strand
 Describe the process of termination
 Discuss DNA replication abnormalities & the different repair
mechanisms

I. OVERVIEW

A. CENTRAL DOGMA OF BIOLOGY

● The central dogma of molecular biology describes the flow of
genetic information within a biological system Figure 3. DNA transposition
● The central dogma of molecular biology describes the flow of Transcription
genetic information in cells from DNA to mRNA to protein. It
● DNA is used as a template to synthesize RNA
states that genes specify the sequence of mRNA molecules,
● Includes processing of non-coding and coding regions to introns
which in turn specify the sequence of proteins.
and exons, respectively
● Post-transcriptional modification of pre-mRNA to mRNA (exons)
by removing introns

Translation
● RNA provides the information to direct amino acid sequences
and synthesize proteins
● Translation is the process in which ribosomes in the cytoplasm
or ER synthesize proteins using mRNA template

Figure 1. The Central Dogma of Molecular Biology Figure 4. Interactions of RNAs during translation

Trans # 5 Group # 22: Cruz, J.N., Cruz, J.M., Cruz, K.J., Cruz, M.R. EDITOR: Sarah Kermina Chan 1 of 17
 Gene Expression I: DNA Replication and Repair
B. DNA SYNTHESIS Requirements for DNA Synthesis and Replication
● Requires enzymes to catalyze addition of mononucleotides to
DNA Molecule the growing chain
● Polynucleotides such as DNA and RNA are linear sequences of → DNA polymerases
nucleotides linked by 3’-to-5’-phosphodiester bonds between ▪ Add nucleotides to an existing primer that provides a 3’-
the sugars.The bases of the nucleotides can interact: OH group
→ Other bases: forming secondary structure of DNA (ladder ▪ These enzymes require both templates and primers and
appearance and helical conformation due to base stack) use 5’-dNTP precursors
→ With proteins that regulate activities in the molecule ▪ The “builder”
● The DNA substrate acted upon by the processes of replication, → Primase
recombination, and repair → making and breaking of ▪ Makes the initial primer made up of ribonucleotides
phosphodiester bonds; fundamental processes that are (RNA);
conserved in evolution ▪ “initializer”
● Double-stranded helix → Helicase
→ Two complementary, helical, polynucleotide chains coiled ▪ Unwinds the parental strands
around a common axis ▪ “Unzipping enzyme”
→ Major and minor grooves serve as attachment of regulatory → Ligases
proteins ▪ Puts DNA fragments together
● Antiparallel orientation of the strands ▪ Seals the gaps after addition of nucleotides after removal
→ One strand runs from 3’→5’ & the other from 5’→3’ of RNA primers
● Repeating sugar and phosphate molecules on the periphery ▪ “Gluer”
● Base-stacking interactions of purines and pyrimidines found at ● Requires precursors, particularly 5’-deoxynucleoside
the center of the molecule triphosphates (5’-dNTPs)
→ Base pairs
▪ A=T double H bonds
▪ G=C triple H bonds
→ Accurate pairing must exist between the bases otherwise
→ Mutations may occur (incorrect matching)

Figure 6. Mechanism of DNA Pol using dNTPs as substrate

Replication Fork and Bubble

● The separation of parental strands creates the replication fork
 Unwound DNA stabilized by proteins
 Site of DNA replication
● Replication can proceed in both directions
 Two forks created by the unwinding of DNA: Replication
Bubble: A bubble-like shape created by two replication
Figure 5. DNA molecule
forks that are close to each other and running in opposite
directions.
DNA Replication ● This requires a considerate input of energy
● The process by which DNA synthesis occurs ● Behind the fork, each newly synthesized strand of DNA base-
● DNA replication leads to the duplication of DNA, thus preserving pairs with its complementary parental template strand
genetic information that is carried as the sequence of bases ● Antiparallel strands at a replication fork present an immediate
● Occurs during the S phase of the cell cycle, which is followed problem for replication
by the G2 phase. The cell divides during the next phase (M), ● One daughter strand is oriented with the 3’-end toward the fork
and each daughter cell receives an exact copy of the DNA of and the other
the parent cell
● In all cells, replication can occur only from a single stranded
DNA (ssDNA) template. Therefore, mechanisms must exist to
target the site of initiation of replication to unwind the dsDNA
→ In eukaryotic cells, due to presence of nucleosome coiling of
DNA, it must be uncoiled from histones
● Requires a template to provide sequence information
→ Each of the two parental strands of DNA becomes the
template for synthesis of a new complementary strand
→ An A in one strand is always paired with a T in the other
strand
→ A C in one strand is always paired with a G on the other ●

Figure 7. Replication Fork

strand
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 Gene Expression I: DNA Replication and Repair
C. FEATURES OF DNA REPLICATION
Semiconservative
● Daughter chromosome = One parental DNA strand + One newly
synthesized complementary strand

Figure 8. Replication Fork vs Replication Bubble

Establishment of Replication Fork

● Requires various enzymes and proteins
→ Helicases
▪ bind to single-stranded DNA and ratchet along it in a fixed
direction with each step requiring hydrolysis of ATP → this
pushes apart the parental DNA to form a replication fork
▪ unwinds the parental strands
 Topoisomerases helps unwind the DNA
 by promoting negative supercoiling (or in some cases +
supercoiling)
 By cutting strands to prevent tension

Types of Topoisomerases Figure 10. Semiconservative Model of DNA Replication

 Topoisomerase I: acts by making a transient
single cut in the DNA bone, enabling the strands Other Models of DNA replication
to swivel around each other to remove the build-
up of twists
 Topoisomerase II (gyrase): acts by introducing
double stranded breaks enabling one strand of
DNA to pass thorugh another , removing knots
and entanglements formed by coiling

→ Single-stranded binding proteins

▪ Prevent reannealing of strands behind the helicase
▪ Keep the strands apart
▪ Reduce potential secondary structure and align the
template strands for rapid DNA synthesis

Bidirectional
● Synthesis begins at origin and occurs at two replication forks
moving away from the origin in opposite directions

Figure 9. Establishment of Replication Fork

Phases of DNA Replication

A. Initiation
1. Untwisting Parental Strands
2. Sliding Clamps
3. Priming
B. Elongation and Processivity
4. Strand Elongation
5. Primer Removal
6. Gap Filling
7. Ligation
C. Termination Figure 11. Bidirectional Synthesis of DNA

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 Gene Expression I: DNA Replication and Repair
Fixed Origin
● DNA replication is a very slow process
→ This is overcome by the presence of several origins and
replicons
● Eukaryotic DNA
→ Linear, bigger, and longer compared to that of the bacterial
DNA
→ Different fixed origins are needed where initial separation of
the strands occurs
→ Have multiple replication origins
● Replication begins at a specific site called the origin of
replication
● The origin of replication is fixed and directed by a particular
sequence of nucleotides
● Replication in prokaryotes begins at only 1 site (oriC) along the
single, circular chromosome
Simultaneous Strand Unwinding and Replication
● The two parental strands are unwound and replicated
simultaneously
→ Helicase separates parental DNA strands
→ DNA Polymerase adds complementary nucleotides to
daughter strands
Semi-discontinuous Elongation
● DNA polymerase can only synthesize in a 5 → ’3’ direction
● Since the strands in a double helix are antiparallel, one of the
new strands can be made continuously (leading strand), but the
other cannot (lagging strand)
→ Leading strand
▪ elongated continuously 5’-3’ direction toward the fork
→ Lagging strand
▪ elongated discontinuously 5’-3’ away from the fork but
are later joined together so that, overall, synthesis
proceeds toward the replication fork.
II. INITIATION
● DNA must be synchronized with cell division
→ Replication should occur only once per cell cycle
● In eukaryotes, replication starts in small portion termed as
replicons, which has its own origin.
→ Replicons
▪ Units of replication
● The origin is where the replication forks proceed outward in both
directions
● Replication stops when the replication forks meet
A. ORIGIN OF REPLICATION
Eukaryotes: Multiple points of origin
● In eukaryotic chromosomes, replication begins at multiple
points of origin (Figure 12). In comparison, replication in
prokaryotes begins at only one site along the circular
chromosome (Figure 14)
→ DNA of eukaryotes is linear, bigger, and longer compared to
bacterial DNA which is circular. This allows eukaryotic DNA
to have multiple points of origin.
● “Bubbles” appear at these points on the chromosomes (Figure
11 & 12)
→ Replication bubbles represent the two replication fork
proceeding in opposite directions
● A replication fork forms at the end of each bubble (Figure 11 &
12)
→ Since the bubble has two ends, each bubble will contain 2
replication forks, and DNA synthesis occurs at each of the
forks.
● The bubble will be enlarged until its end meets an end of
another bubble. The two bubbles will eventually merge and
replication is completed
● Since eukaryotic cells have multiple points of origin, replication
can occur at a faster rate such that duplication of large
chromosomes can occur within few hours.
● DNA synthesis occurs during the S phase of the cell cycle
resulting to rapid replication of entire chromosome
Figure 12. Replication of eukaryotic chromosome. A bubble forms in a point
of origin. The ends of the bubble contain replication forks where DNA
synthesis occur. Bubbles will grow larger until it fuses with one another,
thus completing replication. The red line represents the new DNA strand;
blue line represents the parental strands (Mark’s 4th edition)
● The best understood model for control of eukaryotic
replication is from yeast cell (Figure 6)
→ In yeast, the origins are termed as Autonomously
Replicating Sequences (ARS)
1. Replication is initiated by a multi-subunit protein called
the origin recognition complex (ORC), which binds to
the origin of replication.
2. The ORC marks the origin of replication and remains
bound to the origin throughout the cell cycle. It serves as
an attachment site for several proteins that help control
replication.
3. Next to bind to the ORC are highly unstable proteins
which include CDC6/18 and CDT1. These proteins
facilitate binding of a group of three additional proteins:
and MCM2, MCM3, and MCM5
a. CDCs are called replication activator protein
(RAP). In Devlin, they are called “matchmaker”
proteins.
 They allow the binding of MCMs to the ORC
 CDC stands for cell division cycle
b. MCMs are called the replication licensing factors
(RLF). MCM stands for mini chromosome
maintenance.
 They are termed as licensing factors because
replication cannot proceed until they are bound
 They have a weak helicase activity
 Includes MCM 2 to MCM7 (2020)
4. Binding of the RLFs will make the DNA competent for
replication.
a. After the combination of DNA, ORC, RAP, and
RLFs, the origin is now called pre-replication
complex (pre-RC) which is “licensed” to enter the S
phase of the cell cycle
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 Gene Expression I: DNA Replication and Repair
5. CDC7 kinase together with other cyclin-dependent
kinases trigger the initiation of DNA synthesis
a. When these cyclins combine with CDKs, they can
activate DNA replication and can also block
reassembly of a pre-RC after initiation
6. The activated MCM complex (pre-RC) then catalyzes
the initial separation of parental strands forming a
small replication bubble
c. Since the activated MCM complex has a helicase
activity, it then participates in unwinding the
replication fork and is thereby displaced from the
origin
7. After displacement, the origin forms a post initiation
complex and CDC6 is degrade. This step is important
in regulating the initiation process
a. Degradation of CDC6 prevents the reloading of the
origin with additional licensing factor
b. DNA must be synchronized with cell division
c. Replication should occur only once per cell cycle

Figure 14. Replication of a circular chromosome. Replication occurs

bidirectionally, beginning from a single point of origin (oriC). (Mark’s 4th
edition)

B. SEPARATING PARENTAL STRANDS

Separation
● DNA synthesis occurs at the replication fork, and the two
parental strands need to be separated to allow the fork to
progress and to serve as a template (Figure 8)
→ The separation of the parental strands creates the replication
fork
→ The separation requires considerable amounts of energy

Figure 13. Model for initiation of the DNA replication cycle in eukaryotes.
ORC is present at the replicators throughout the cell cycle. The pre-
replication complex (pre-RC) is assembled through sequential addition of
replication activator protein (RAP) and replication licensing factors (RLF).
Phosphorylation of the pre-RC triggers replication. After initiation, a post-RC
state is established and the RAP and RLFs are degraded (Biochemistry 8th
edition by Campbell & Farrell)
Prokaryotes: Single Point of Origin
● Replication in prokaryotes begins at only one point of
origin (oriC) along the circular chromosome
● DNA synthesis occurs at 2 replication forks at the point of origin.
The forks then move away from the origin in both directions
simultaneously (bidirectional) around the circle (Figure 7)
● oriC - point of origin
→ oriC has three 13-base repeats which are AT-rich, a feature
which aids strand separation (2020C)
→ AT base pairs have only two H-bonds, while GT base pairs
have three
Note: Doc. Balcueva mentioned to focus on eukaryotes
Figure 15. The two parental strand must be separated to allow the fork to
progress, act as a template, and synthesize new strands. (Mark’s 4th
edition)
● Cells use helicases, enzymes that separate the parental
strands at physiologic temperature
→ Helicase will bind to a single stranded DNA and capable of
separating annealed nucleic acid strands (Figure 9)
▪ This requires hydrolysis of ATP
▪ This pushes the parenteral DNA apart allowing the
formation of replication fork
Additional information:
→ Annealing means the complementary sequence of single
stranded DNA or RNA to pair by hydrogen bond to form a
double stranded polynucleotide.
→ DNA in its natural state, is annealed since it contains 2
strands paired by hydrogen bond.
● Single stranded DNA
→ In the absence of additional proteins, the separated parental
strands would quickly join with each other (reanneal),
forbidding the formation of replication fork

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 Gene Expression I: DNA Replication and Repair
→ To prevent reannealing, Single Strand DNA-Binding
proteins (SSBs) keeps the parental strands separated,
allowing formation of replication fork (Figure 16)
▪ SSB binds to the exposed single strands, helicases are
loaded onto the DNA, and the bubble is enlarged →
allows formation of replication fork
▪ It also reduces potential secondary structure that may
impede polymerization
▪ It also aligns the templates for rapid DNA synthesis
→ Single Strand DNA-Binding proteins (SSBs) keeps and
promotes separation in E. coli while Replication Protein A
(RPA) keeps and promotes separation in eukaryotes (2020)

Figure 18. Cell cycle regulation

Cell cycle (2020)

→ Life of a cell refers to the life of a cell from the time it is
produced by a dividing parent cell until its own division into
two daughter cells.
→ Consist of 4 distinct phases:
▪ G1 - growth and metabolism
▪ Synthesis (S) - DNA replication
▪ G2 - preparation for cell division
▪ Mitotic (M) - mitosis
→ G1, S, and G2 phase are known as interphase

Figure 16. Helicase separates the parental strands of DNA. SSBs prevents
reannealing and keeps the strands separated until they are copied. This
allows formation of replication fork → DNA synthesis

C. REGULATION OF THE INITIATION PROCESS

● In eukaryotes, the replication process is synchronous with the
cell division.
→ 1:1 ratio, replication occurs only once per cell cycle
● DNA replication regulation involves cyclins and CDKs
→ Cyclins are proteins that are produced in one part of a cell
cycle and degraded in another cell cycle
→ These cyclins are able to combine with CDKs forming Figure 19. Cell cycle (Khan academy)
Cyclin CDK complexes. The formed complexes then
activate DNA replication and also block reassembly of a pre- Table 1. Cyclins and cyclin-dependent kinases involved in cell cycle
RC after initiation. progression (2020)
→ The blocking of the reassembly of a pre-RC ensures that Cyclin Kinase Function
DNA replication only occur once for a single cell cycle. These D CDK4 and 6 Progression past restriction point at
happens by: boundary of G1 and S phase
▪ Cyclin-CDK complexes phosphorylate CDC6 → E, A CDK2 Initiation of DNA synthesis in early
inactivates CDC6 → blocks the reassembly of pre-RC → S phase
B CDK1 Transition from G2 to M phase
regulation of DNA replication initiation
▪ RAP and RLFs which are used in formation of pre-RC
complex are also degraded and a post-RC state is III. ELONGATION
established (Figure 13) A. SEPARATED PARENT STRANDS

● Each of the separated parental strands of DNA serves as a

template strand for the synthesis of a new complementary
strand
● Synthesis of both new strands of DNA occurs at the replication
fork that moves along the parental molecule
● Template strand: 3’ to 5’ direction
● New strand: 5’ to 3’ direction

Leading Strand
● Formed by continuous copying of parental strand that runs 3’
to 5’ toward the replication fork
● New strand of DNA that is synthesized continuously during
replication
Figure 17. Cyclin and CdK
● DNA polymerase ε continues addition of nucleotides

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 Gene Expression I: DNA Replication and Repair
Lagging Strand
● Formed by discontinuous copying of parental strand that runs
3’ to 5’ away from the replication fork
● New strand of DNA that is synthesized in short pieces during
replication and then joined later
● DNA polymerase δ completes the synthesis
→ Okazaki fragments
▪ Short fragments which are newly synthesized DNA
sequence that are formed on the lagging template
▪ RNA primers are removed by nucleases forming gaps.
These gaps are filled with appropriate base-paired
deoxynucleotide joined by DNA ligase
− Enzyme that catalyzes formation of phosphodiester
bonds between two polynucleotide chains

Figure 21. Sliding clamp

C. ELONGATION PROCESS
DNA Polymerase
● Synthesizes DNA in a 5’ to 3’ direction
● Requirements
→ Primer strand
→ Template strand
● Incoming deoxyribonucleotides are added to the hydroxyl group
(-OH) of RNA primers at the 3’ end of the growing DNA strands
● Needs a pre-existing 3’-OH on a sugar in order to attach a
nucleotide since it lacks the ability to initiate a new strand
● Deoxyribonucleoside triphosphates (dATP, dGTP, dCTP,
and dTTP) serve as substrates for the addition of nucleotides to
the growing chain

Figure 20. Mechanism of DNA synthesis at the replication fork

B. PRIMING

● DNA polymerases cannot initiate synthesis of new strands and

can only elongate pre-existing strand
● Therefore, it needs a primer
→ RNA primer
▪ Supplies free 3’-OH group to which the first
deoxyribonucleotide can covalently bond Figure 22. Action of DNA Polymerase
→ Primase ● Processivity
▪ RNA polymerase that synthesizes RNA primers in a 5’ to → Number of nucleotides incorporated before the enzyme
3’ direction dissociates from the template
▪ Provides a starting point for DNA polymerase to begin → The higher the processivity, the more efficient is the
synthesis of a new strand replication process
● Polymerase δ and ε require
→ sliding clamp Table 2. DNA Polymerases
▪ Proliferating cell nuclear antigen (PCNA) DNA Polymerase Function Processivity
▪ Tethers the enzyme to DNA while allowing it to move α initiate the synthesis of new Low
processively along the template strands together with primase
▪ Doughnut-shaped surrounding the DNA β DNA repair Low
→ sliding clamp loader γ replicates mitochondrial DNA High
▪ Replication factor C (RFC) δ elongation of lagging strand High
▪ Loads PCNA onto the DNA ε elongation of leading strand High

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 Gene Expression I: DNA Replication and Repair
Primer Removal
● Polymerase δ stops synthesizing one fragment when it reaches
the start of the previously synthesized Okazaki fragment
● The primer of the previously synthesized Okazaki fragment is
removed by flap endonuclease 1 (FEN1) and RNase H
→ Flap endonuclease 1 (FEN1)
▪ Hydrolyze the last ribonucleotide
→ RNase H
▪ Ribonuclease that specifically degrades RNA from RNA-
DNA hybrid
▪ 10 out of 11 ribonucleotides will be removed
● The gap left by the primer is filled by polymerase δ using
parental DNA strand as its template and the newly synthesized
Okazaki fragment as its primer
Figure 24. In a 3’ end template strand, replication of the lagging strand
Ligation requires short RNA primers to be added (green), and extended by DNA
● DNA ligase seals Okazaki fragments together polymerase. When the RNA primer is at the 5’ end of the lagging strand,
there is no nucleotide sequence to read in the next round of DNA
● 3’-hydroxyl group at the end of one fragment is ligated to the
replication. This result in a primer gap at the 5’ end of each strand
phosphate group at the 5’-end of the next fragment which (Biochemistry 8th edition by Campbell & Farrell)
forms a phosphodiester bond
Telomeres
● Located at the ends of eukaryotic chromosomes
● Series of repeated DNA sequences. 5’TTAGGG3’ in humans,
and is repeated more than 1000 times
● This repetitive DNA is noncoding and acts as a buffer against
degradation of DNA sequences at the ends, which would
occur with each replication as the RNA primers are
degraded and removed.
→ Loss of telomeres will not prevent the shortening of the linear
molecule during DNA replication → essential genes will be
lost → cell death.
● Some evidence shows a relationship between longevity and
telomere length. It is suggested that telomeres control how long
a cell can divide before dying

Figure 23. Action of DNA ligase

IV. TERMINATION

● Replication will end once a replication fork meets another

replication fork
● The ends of linear chromosome pose a unique problem during
DNA replication
● Recall that the two parental strands of DNA run at opposite
direction, at each end is a 3’ and 5’ DNA chain. 3’ end template
strand runs at 5’ to 3’ direction while 5’ end template strand runs
at 3’ to 5’ direction
● DNA polymerases can only elongate from a free 3’ hydroxyl
group and produce new strands in 5’ to 3’ direction
→ The 5’ end template strand (runs in 3’ to 5’ direction) is not a
problem because DNA polymerase copying it starts from its Figure 25. Telomeres
free 3’ hydroxyl end. This allows the continuous elongation of
the leading strand running in 5’ to 3’ direction, all the way to Telomerase
the end of the template. ● Even with long telomeres, cells will eventually die when their
→ It is the 3’ end template strand that poses a problem (Figure DNA gets shorter after each replication. The solution is
24) telomerase which provides the mechanism for synthesis of
▪ Remember that all DNA polymerases requires an RNA the telomeres
primer, and that the RNA primer would provide 3’ hydroxyl → Replaces lost telomere repeats
group. These 3’ hydroxyl group will allow the formation of → Adds new 6 deoxyribonucleotide repeats to the 3’ end of
DNA and replaced the RNA primer when they are telomere using its RNA portion as the template.
removed. ● In humans, the sequence of telomerase is 5’CCCUAA3’
▪ However, there is no sequence exist before the final RNA ● Telomerase is a special kind of reverse transcriptase enzyme
primer, thus it cannot be replaced by DNA resulting to a (Figure 16)
short gap and an incomplete lagging strand (Figure 24) → Binds to 5’ strand at the chromosome end and uses reverse
▪ Failure to complete the lagging strand in over many cycles transcriptase activity to synthesize DNA on the 3’ strand,
of replication will shorten the linear molecule until using its own RNA as the template.
essential genes were lost and the cell dies → This allows the template strand to be elongated → increase
▪ Therefore, it is essential to prevent continued loss of DNA the length of telomere
at the ends of chromosomes.

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 Gene Expression I: DNA Replication and Repair
Figure 26. Mechanism by which telomerase works. Telomerase acts both
as a template and a reverse transcriptase occurs using its own DNA to
produce RNA. This will allow elongation of telomere. (Biochemistry 8th
edition by Campbell & Farrell)
V. REPLICATION ABNORMALITIES AND REPAIR
A. REPLICATION ABNORMALITIES
Types of Replication Abnormalities
1. Single Base Alterations
a. Depurination
b. Deamination of Cytosine to Uracil
c. Deamination of Adenine to Hypoxanthine
d. Alkylation of Base
e. Insertion or Deletion of Nucleotide
f. Base-analog incorporation
2. Two Base Alterations
a. UV light-induced thymine-thymine (Pyrimidine dimer)
b. Bifunctional Alkylating Agent cross-linkage
3. Chain Breaks
a. Ionizing Radiation
b. Radioactive Disintegration of Backbone
c. Oxidative Free Radical Formation
4. Cross-linkage
a. Between bases in same or opposite strands
b. Between DNA and protein molecule (eg. Histones)
Mutations
→ Inheritable changes in the DNA sequence, result from
multiplication errors, from damage to the DNA, or from errors
introduced during repair of damage
▪ Mutation that are changes of a single base pair are called
point mutation
● Point Mutation can be categorized by
→ Nature of bases altered
▪ Transition
− purine/pyrimidine is substituted for another (ex. T for C
or C for T)
▪ Transversion
− purine is substituted for a pyrimidine or vice versa (ex.
A for C or C for A)
Figure 27. Point Mutation (from Introduction to Genetic Analysis)
→ Their effect on a coding sequence
▪ Missense mutation
− Point mutations when the change of a single base pair
causes the substitution of a different amino acid in the
resulting protein.
▪ Nonsense mutation
− Point mutation that results in a premature stop codon in
the transcribed mRNA.
− Has more severe effects because it can lead to
truncated (and generally unstable) polypeptides
▪ Silent or synonymous mutation
− Do not alter the amino acid encoded
− Includes changes in third nucleotide of the codon
− Some silent mutation will have serious consequence if
they alter the splicing pattern of the gene
Figure 28. Mutation effect to coding sequence (Devlin 6th edition)
● Frameshifts
→ Can disrupt the coding of a protein through insertions or
deletion of one or more base pairs (if the number of base
pairs is not a multiple of 3)
Figure 29. Frameshift mutation (from Introduction to Genetic Analysis)
Example: Cigarette smoking
Cigarettes contains a lot of poison in itself and what happens with
its smoke is that benzo pyrene is formed. There will be an
oxidation of benzo[a]pyrene and covalent binding to DNA.
Benzo[a]pyrene is not carcinogenic until it is oxidized within cells.
Then it can covalently bind to guanine residues in DNA,
interrupting hydrogen bonding in G-C base pairs and producing
distortions of the helix (Dra. Balcueva).
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 Gene Expression I: DNA Replication and Repair
B. REPLICATION REPAIR

● DNA replication is a highly accurate process, but mistakes can

occasionally occur as when a DNA polymerase inserts a wrong
base. Uncorrected mistakes may sometimes lead to serious
consequences, such as cancer.
● Repair mechanisms that remove potentially harmful alterations
to DNA and restore it to its original conformation

Important Terms
● AP endonuclease
→ Endonuclease that nicks DNA next to an AP site
● AP site
→ Site in DNA where a base is missing (AP site/apurinic
site/apyrimidinic site depending on the nature of the missing Figure 31. Exonucleases and Endonucleases
base)
● DNA glycosylase Table 3. Mechanism of DNA Repair
→ Enzyme that breaks the bond between a base and the Mechanism Problem Solution
deoxyribose of the DNA backbone Mismatch Repair Copying errors Methyl - directed
● DNA polymerase (single base or two- strand cutting,
→ Replaces and repairs the excised sequence; enzyme that to five- base exonuclease
forms DNA from deoxyribonucleotides on a DNA template unpaired loops digestion and
● DNA polymerase V replacement
→ Repair polymerase in bacteria that can replicate past Base Excision Spontaneous Base removal by N-
pyrimidine dimers and AP sites Repair chemical, radiation glycosylase, abasic
● DNA Ligase damage to a single sugar removal,
→ Joins the repaired DNA; enzyme that links separate stretches base replacement
of DNA
Nucleotide Spontaneous Removal of an
● Nick translation
Excision Repair chemical or approximately 30
→ A type of DNA repair that involves polymerase I using its 5’ to
radiation damage nucleotide oligomer
3’ exonuclease activity to remove primers or replace
to a DNA segment and replacement.
damaged nucleotides
Double-Strand Ionizing radiation, Synapsis,
● Origin of Replication
Break Repair chemotherapy, unwinding
→ The point at which the DNA double helix begins to unwind at
oxidative free alignment, ligation
the start of the replication
radicals
● Primer
→ A short stretch of RNA hydrogen-bonded to the template;
DNA to which the growing DNA strand is bonded at the start Excision Repair System
of the synthesis ● General mechanism that can very accurately repair many
● Primase different kinds of damage (Devlin)
→ Enzyme that makes a short section of RNA to act as a primer ● “Cut and patch” repair
for DNA synthesis ● Recognizes bulges in DNA double helix, removes damaged
● Proofreading strand and replace it
→ Process of removing incorrect nucleotides when DNA ● Defining characteristic is removal of damaged nucleotide(s),
replication is in progress leaving a gap in the DNA, followed by resynthesis using the
● Repair genetic information on the opposite strand, and then ligation to
→ Enzymatic removal of incorrect nucleotide from DNA and restore continuity of the DNA
their replacement by correct ones ● Repair most damaged due to UV radiation, such as thymine
● Replication Forks dimers and crosslinked products
→ Points at which DNA strands are formed ● Not as sensitive as mismatch system
● There are two major modes of excision repair
→ Base excision repair (BER)
→ Nucleotide excision repair (NER)

Table 4. Excision Repair

Basic Steps
1. Recognize the damage
2. Remove damage by excising part of one strand to leave a gap
3. Resynthesize to fill gap; uses genetic information from other
strand
4. Ligate to restore continuity of DNA back bone
Base Excision Repair Nucleotide Excision Repair
● Glycosylase removes base, ● Double excision removes
leaves back bone intact damage as part of an
● AP endonuclease cuts oligonucleotide ( 12-13 nt in
backbone, AP lyase E.coli, 27-29 nt in humans)
removes sugar ● DNA polymerase fills gap
Figure 30. Roles of Polymerases in Repair ● DNA polymerase fills gap ● DNA ligase seals nick
● DNA ligase seals nick
Note: Proofreading activity of 3’ exonucleolytic activity of α and ε

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 Gene Expression I: DNA Replication and Repair
B. Nucleotide-Excision Repair
Figure 32. DNA repair mechanisms
A. Base Excision Repair
● Bases that do not occur naturally are removed from DNA.
● Repair systems that recognize mutations in DNA that do not
cause distortion in the helix.
● Removes altered nucleotide from reactive chemicals in the diet
or produced by metabolism.
● Repairs many different types of damaged bases, including
methylated bases, deaminated bases, oxidized bases and
abasic (AP) site. (Devlin)
● Steps
→ DNA glycosylase recognizes alteration; enzyme cuts the N-
glycosyl bond between the sugar and base. (this does not
break sugar-phosphate backbone of the DNA; rather it
leaves an abasic deoxyribose in the backbone, an AP site
that must be removed)
→ Altered base is removed by cleavage of the glycosidic bond
between the base and the deoxyribose sugar of the DNA
backbone
→ AP endonuclease cleaves the DNA backbone ; cleaves the
phosphodiester bond at the 5’ side but leaves sugar attached
to the next nucleotide
→ AP lyase cuts 3’ to the PA site to remove the sugar. resulting
to nucleotide gap has a free 3’-hydroxyl. (Devlin)
▪ Polymerase β is the one that removes sugar phosphate
remnant that has been attached to the base (2020C & B)
→ A nucleotide complementary to the undamaged strand is
inserted. The gap is filled by DNA Polymerase (Devlin)
▪ Polymerase β is the one that fills the gap (2020B)
→ DNA ligase III seals the strand
Figure 33. Base excision (from Introduction to Genetic Analysis)
● Activated when DNA is damaged in a way that produces a
“bulky” lesion
● Occurs when DNA interacts with polycyclic aromatic
hydrocarbons (i.e. from smoking & chemotherapeutic drugs) or
after UV light-induced dimerization of adjacent pyrimidines
● acts on wide variety of damage, typically involving large adducts
or distortion of the double helical structure of DNA. (Devlin)
● Damage is removed by an enzyme complex that cuts several
nucleotide away on both sides of the damaged base(s), so the
damage is released as part of oligonucleotide (Devlin)
● 2 Pathways
→ Transcription-coupled pathway
▪ directs immediate repair response to the template strand
of transcribed regions, so that lesions there are repaired
faster than lesion elsewhere and transcription can
resume. (Devlin)
▪ Template strands of actively transcribed genes are
preferentially repaired
▪ Presence of lesion may be signaled by a stalled RNA
polymerase
▪ Ensures that genes of greatest importance to the cell
(actively transcribed genes) receive the highest priority on
the “repair list”
→ Global genomic pathway
▪ Slower, less efficient
▪ Corrects DNA strands in the remainder of the genome
● Steps
→ TFII-H (Transcription factor II H) - general transcription factor
involved in the initiation of transcription
→ TFII-H has two subunits (XPB and XPD) both are ATPases
with limited helicase activity, separating the strands of the
duplex in preparation for the removal of lesion
→ Pair of endonucleases cuts the damaged strand on both
sides of the lesion
→ Then the DNA segment between the incisions is released
▪ Note: The gap in the DNA is bound by RPA (Replication
Protein A) and is filled by pol δ (polymerase delta or
epsilon) stimulated by PCNA (proliferating cell nuclear
antigen) (Devlin)
→ DNA polymerase fills the gap from the excised segment
→ DNA ligase seals the strand (Devlin presumes that the DNA
ligase use is called LIG1)
▪ Note: Latter steps resemble completion of an Okazaki
fragment (Devlin)
Figure 34. Nucleotide excision (Biochemistry by Campbell)
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 Gene Expression I: DNA Replication and Repair
Double Strand Breakage Repair (DSB)
● DNA potential lethal events (Devlin)
● This can stimulate genetic recombination that can lead to
● Caused by ionizing radiation from x-rays, gamma rays, particles
● Also introduced during replication of damaged DNA
● 2 Repair pathways
Figure 34. Base excision vs Nucleotide Excision
Mismatch Repair
● Specialized form of nucleotide excision repair that removes
replication errors. (Devlin)
● Mismatches are not like DNA damage; There is no damaged or
modified base present, just the wrong one of the four bases.
(Devlin)
● Removal of mismatched bases incorporated by the DNA
polymerase that escaped the proofreading exonuclease
● Mismatched bases cause structural distortions of the double
helix
→ Recognized by a repair enzyme during the methylation of
DNA
● In newly replicated DNA, removing the newly synthesized base
would preserve the genetic information, whereas excising the
base on the parental strand would permanently alter the DNA,
producing mutation. (Devlin)
● From 2020C:
● Key Enzymes and Steps
→ The MutSHL mismatch repair system consists of three
genes/proteins
▪ MutS protein
− Recognizes the distortion, binds to it, then recruits the
next two proteins; creates a loop that contains the
mismatch (Devlin)
▪ MutH protein
− Finds the nearest GATC site and nicks the non-
methylated strand; nicks the unmethylated, newly
synthesized strand on either side of the mismatch
(Devlin)
▪ MutL protein
− Stabilizes the DNA-binding protein complex; binds and
coordinates the subsequent cleavage and excision.
(Devlin)
→ Helicase (UvrD) unwinds the strand from the site of the nick
through the site of the mismatch; and the nicked single
strand is degraded by either a 5’ or 3’ exonuclease or 3’ to 5’
exonuclease, depending on free end
→ The gap is then filled in by DNA polymerase III (Pol III),
adding nucleotides to the 3’ end of the nicked strand, which
hopefully inserts the correct bases
→ DNA ligase seals the strand
chromosomal translocation, and unrepaired (DSBs) lead to
broken chromosomes and cell death (Devlin)
released by radioactive atoms, certain chemicals, and free
radicals
→ When these forms of radiation collide with a fragile DNA
molecule, they often break both strands of the double helix
→ Non-homologous End Joining (NHEJ)
▪ Complex of proteins bind to the broken ends of the DNA
duplex and catalyzes series of reactions that rejoin the
broken strands
▪ Mechanism is error prone and mutagenic
▪ May join lengths of DNA that were never previously
attached → generates new combinations of genetic
material → chromosomal rearrangement
▪ Predominant pathway in mammalian cells
Figure 35. Non-homologous end joining (from 2020B)
▪ Steps
− Lesion is detected by Ku, a heterodimeric, ring-shaped
protein
− Two Ku proteins bind, one on either side of the broken
ends of DNA
− DNA-dependent protein kinase (DNA-PK), which is a
catalytic subunit of a DNA-dependent protein kinase is
recruited by the Ku complex, then activates the XRCC4
protein
− XRCC4 directs DNA ligase IV to repair/joins the end of
the broken DNA
→ Homologous Recombination (HR)
▪ More accurate pathway
− fewer errors in the base sequence of the repaired DNA
▪ Can only be employed during the cell cycle after DNA
replication (late S or G2 phase) because it requires a
homologous chromosome in the nucleus
▪ Any DNA that was lost is replaced using homologous DNA
as a template
− Note: Defects in both repair pathways have been
linked to increased cancer susceptibility
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 Gene Expression I: DNA Replication and Repair
VI. CLINICAL CORRELATION
A. CANCER AND CHEMOTHERAPY
● Tumor cells have higher proliferation and replication rates
and so many of the chemotherapy medications is directed at
inhibiting DNA replication.
● Errors in recombination can lead to gene duplications or
deletions, and chromosomal rearrangements and the frequency
of recombinational events is increased by damage do DNA due
to the disruptions of replication forks or breaks in the DNA.
→ Recombination can occur between related (not identical)
sequences within a chromosome or between chromosomes.
→ Recombination can also occur by non-homologous end
joining following a double-strand break in DNA
→ Some forms of cancer can be triggered by chromosomal
rearrangements that result in gene fusions that form
oncogenes
▪ Chronic myeloid leukemia and acute lymphocytic
leukemia
− translocations between chromosomes 9 and 33 that
create fusion proteins between genes BCR and ABL
▪ BCR-ABL fusion
− an active tyrosine kinase that functions as an oncogene
● Mismatch repair
→ Defects in mismatch repair cause hereditary nonpolyposis
colon cancer (HNPCC) and important in other forms of
cancer → defect in mismatch repair genes MLH1 and MLH2.
● Actively dividing cells are much more sensitive to these
drugs and this characteristics opens up a therapeutic window
→ Normal actively dividing cells (e.g. cells of the intestinal tract,
bone marrow, and hair follicles) can also be affected by
these drugs → explains the common side effects of
chemotherapeutic drugs (i.e. vomiting, hair loss, increased
susceptibility to infections)
● Mechanisms of interruption of DNA replication
→ Interference of the pools of deoxynucleotides needed for
DNA synthesis
▪ Hydroxyurea
− Inhibits ribonucleotide reductase needed to make
deoxyribonucleotides from ribonucleotides →
decreases concentrations of dNTPs; commonly used to
treat melanomas and myeloproliferative diseases
● Drugs that interrupt with DNA replication
→ Antimetabolites
▪ Interfere with the synthesis of precursors of 5’-NTPs
→ Methotrexate
▪ Inhibits dihydrofolate reductase that is needed to maintain
reduced tetrahydrofolate required for nucleotide synthesis
→ 5-Fluorouracil
▪ Inhibits thymidylate synthase; when metabolized into its
triphosphate form can be incorporated into DNA and lead
to strand breaks
→ Vinca Alkaloids
▪ Inhibit microtubule assembly and thereby interfere with
mitosis and chromosome segregation
→ Topoisomerase inhibitors
▪ Slow or stop replication by preventing the untwisting of
parental strands
→ Topoisomerase poisons
▪ Inhibit resealing of the phosphodiester bonds
● Other chemotherapeutic drugs
→ Alkylating agents
▪ Cyclophosphamide, busulfan, nitrosoureas, cisplatin
(treatment of metastatic testicular and ovarian cancers)
→ Purine analogs
▪ 6-Mercaptopurine
− Acute leukemias and immunosuppression after organ
transplantation; inhibits purine synthesis and causing
toxicity after incorporation into DNA.
B. HIV THERAPY
● A key step in the HIV life cycle is the synthesis of a DNA copy
of the viral RNA genome catalyzed by a reverse
transcriptase
● Reverse transcriptase: major target of chemotherapy because
it is NOT essential for normal cells
● Zidovudine
→ First drug approved for the treatment of HIV infection
→ A nucleoside analog with an azido group on the sugar
→ Can be phosphorylated into the triphosphate form which
competes with dTTP for incorporation of the reverse
transcript; terminates the growing chain of the transcript once
it is incorporated
● Didanosine and Zalcitabine (dideoxycytidine)
→ Also function as chain terminators after incorporation of their
phosphorylated derivatives by the HIV reverse transcriptase
● Reverse transcriptases do not carry out proofreading which
makes their error rate much higher than DNA polymerases
→ Complicates the treatment for AIDS
→ Population of viruses carried by any one patient can contain
many mutants and these mutations can continue to increase
over time
→ Combination therapy attempts to circumvent this problem
C. ANTIBIOTICS
● Antibiotics that target either subunit of DNA gyrase rapidly stop
E. coli DNA replication → preventing the reduction in linking
number of the parental strands prevents the strands from
untwisting
● Topoisomerase inhibitors
→ prevent catalytic activity and target ATPase subunits
encoded by gyrB
● Topoisomerase poisons
→ freeze the DNA-covalent links and are lethal converted into
double-strands break, as in replication
→ Nalidixic acid
▪ used to treat UTI; targets swivelase subunits encoded by
gyrA
→ Ciprofloxacin
▪ one of the most effective oral antibiotics in clinical use
today; for the treatment and prevention of anthrax and
many other bacterial infections
D. XERODERMA PIGMENTOSUM
● Caused by defective DNA repair
● Characterized by photosensitivity and high susceptibility to skin
cancers in sun-exposed areas of the body, and neurological
problems
● A rare autosomal recessive disease that can be caused by
defects in any of 8 different genes, reflecting the complexity of
DNA repair of pyrimidine dimers, the most common damage
introduced by exposure to UV light.
● Mutations in 7 of the XP genes lead to defects in the initial
incision step in nucleotide-excision repair of pyrimidine dimers:
explains the extreme photosensitivity
VII. NOTES from Doc Balcueva’s Reading (from
2019B & 2020C)
A. INITIATION OF REPLICATION IN EUKARYOTIC CELLS
● E. coli (prokaryote) DNA replication begins at one site along the
single, circular chromosome
● Higher organisms’ polymerases incorporate into DNA at much
slower rates. But, eukaryotic cells replicate their genome in
small portions called replicons
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 Gene Expression I: DNA Replication and Repair
→ Replicons → They cannot reassociate with an origin that has already
▪ Each has its own origin from which replication forks “fired”
proceed outward in both directions (There are about 10K-
100K different origins)
▪ 10-15% of replicons are actively engaged in replication
during S-phase of cell cycle
▪ Closely related replicons undergo replication
simultaneously
▪ Active replicons at a particular time during one round of
DNA synthesis tend to be active at a comparable time in
succeeding rounds
● The timing of replication is related to the activity of the gene in
the region and its state of compaction
→ Highly compacted, low activity (least acylated) = last regions
to be replicated
● Autonomous replicating sequences (ARS)
→ promote DNA replication in yeast
→ Have conserved sequence of 11 base pairs
→ Function
▪ specific binding site of origin recognition complex (ORC)
● Replication of amphibian chromosome begins with random
selection
● Replication of mammalian chromosome begins within defined Figure 36. Steps leading to replication of yeast replicon (from 2020B)
regions of DNA
● Shorter cell cycles utilize a greater number of sites as origin of C. EURAKYOTIC REPLICATION FORK
replication
● Initiation site of replication is thought to be governed by ● All replication systems require the following:
epigenetic factors: → Helicases
→ Position of nucleosome → Single-stranded DNA-binding proteins
→ Types of histone modification → Topoisomerase
→ State of DNA methylation → Primase
→ Degree of supercoiling → DNA polymerase
→ Level of transcription → Sliding clamp and clamp loader
→ DNA ligase
● Large T-antigen
B. RESTRICTING OF ONCE PER CELL CYCLE → Viral helicase
→ Added to mammalian replication proteins to study initiation of
● Each portion of genome is replicated only once during each cell eukaryotic replication
cycle → Encoded by SV 40
● Initiation of replication at particular a origin requires them to ● DNA of eukaryotic cells is synthesized in a semi-
undergo the following phases: discontinuous manner
→ Step 1 ● Okazaki fragments of lagging strands (150 nucleotides) are
▪ ORC smaller than in bacteria
− described as "molecular landing pad”, is bound to the ● In E coli, the leading and lagging strands are synthesized in a
origin of replication. (in yeast cells, ORC remains coordinate manner by a single replicative complex
associated to the origin throughout cell cycle) (replisome)
→ Step 2 ● DNA polymerases of eukaryotic cells
▪ Assembly of protein-DNA complex called prereplication → Require a primer, elongate DNA strands in 5’ – 3’ direction
complex (pre-RC) during/following mitosis by addition of nucleotides to a 3’ hydroxyl group
− This is “licensed” to initiate replication →α
− Formation is associated with 6 MCM proteins (Mcm2- ▪ Tightly associated with primase to initiate the synthesis of
Mcm7). Interaction of proteins hexameric ring-shaped Okazaki fragments
complex that possesses helicase activity − The complex binds to unwound DNA coated by RPA (a
− 2 complexes per origin travel in opposite directions single-stranded DNA-binding protein)
later (step 4) − Primase initiates the synthesis by assembly of short
− Note: formation of pre-RCs on a site only makes the RNA primer then is extended by addition of 20
site a POTENTIAL ORIGIN deoxyribonucleotides by polymerase α
→ Step 3 →β
▪ Activation of key protein kinases leads to the ▪ For DNA repair
phosphorylation of MCM complex and other proteins and →γ
to the initiation of replication ▪ Replicates mitochondrial DNA
− Cyclin-dependent kinase (Cdk) →δ
o activity remains high from S-phase through mitosis, ▪ Primary DNA-synthesizing enzyme during replication of
which suppresses formation of new pre-RCs lagging strand
− Consequence of CYCLINdk ▪ requires a sliding clamp
o origins can only be activated once per cycle →ε
→ Step 4 ▪ Primary DNA-synthesizing enzyme during replication of
▪ once replication is initiated at the beginning of S phase, leading strand
the MCM helicase moves with the replication fork ▪ Requires a sliding clamp
● After replication in mammalian cells, MCM proteins are ● Polymerases γ, δ and ε all possess 3’ → 5’ exonuclease; has
displaced from the DNA, but can still remain in the nucleus. proofreading activity

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 Gene Expression I: DNA Replication and Repair
● Sliding clamp (aka PCNA in eukaryotes)
→ Tethers the enzyme to DNA allowing it to move processively
along a template
→ Thought to play a major role in orchestrating events that
occur during DNA replication, repair, and recombination
→ Referred to as a “molecular tool belt” due to its ability to
bind a diverse array of proteins
● Clamp loader (aka RFC)
→ Loads PCNA onto DNA
● Once RNA-DNA primer is formed, polymerase α is replaced by
PCNA-polymerase δ complex completes the synthesis of
Okazaki fragments
● When the complex reaches the 5’ end of the previously made
Okazaki fragment, it continues along the lagging strand
template, displacing the primer
● The displaced primer is cut from the newly synthesized DNA
strand by an endonuclease (FEN-1) and the resulting nick is
sealed by DNA ligase
→ The enzymes are recruited via interactions with PCNA
D. REPLICATION AND NUCLEAR STRUCTURE
● Active replication forks are localized within 50-250 sites called
replication foci
● The clustering of replication forks may provide a mechanism for
coordinating the replication of adjacent replicons
E. CHROMATIN STRUCTURE AND REPLICATION
● Chromosomes consist of DNA tightly complexed to regular
arrays of histone proteins that are present in form of
nucleosomes
● Nucleosomes that form during replication are comprised of
roughly equivalent amounts of histone molecules inherited from
parental chromosomes and newly synthesized ones
● Core histone octamer of a nucleosome contains (H3H4)2
tetramer with a pair of H2A/H2B dimers
● Stepwise assembly of nucleosomes
→ Facilitated by accessory proteins (histone chaperones)
▪ accept new or parental histones then transfer them to
daughter strand
→ CAF-1 is the best studied and interacts with sliding clamp
PCNA
● Classic Model
→ (H3H4)2 tetramers present prior to replication remain intact
and are distributed randomly between the two daughter
duplexes → old and new (H3H4)2 tetramers are intermixed
on each daughter DNA molecule → H2A/H2B dimers of a
nucleosome separate and bind randomly to new and old
(H3H4)2 tetramers present
● Recent Model
● o (H3H4)2 tetramers from parental nucleosome split into two
H3/H4 dimers, each combine with newly synthesized H3/H4
dimer → “mixed” (H3H4)2 tetramer
● Epigenetic Information
→ Not encoded within a chromosome's DNA sequence
→ Encoded in the patters of:
▪ Methylated cytosine residues in a cell's DNA
− transmitted via DNA methyltransferase (DNMT1)
▪ Post-translational modifications of the core histone
associated with the DNA
− modifications present in old histones will guide the
modifications of new histones within the neighboring
nucleosomes on the same DNA strand
▪ It is important for these patterns to be transmitted from
parent chromatin to daughter chromatin (very little is
known about the mechanism of transmission)
REVIEW QUESTIONS
1. What is the process that compensates for the slow
process of DNA replication in eukaryotes?
a. repeated replication of one ARS
b. one replicating fork per strand
c. one MCM complex per strand
d. several replicons in a single molecule
2. Which of these genes are most likely to be replicated
first?
a. compacted regions into heterochromosomes.
b. related to the DNA sequence of nucleotides
c. replicons that are located close together
d. presence of highly acetylated histones
3. What do you call the 11 conserved sequence of base
pairs in DNA recognized and bound by the origin
recognition complex (ORC)?
a. ARS
b. ORF
c. RFC
d. RPA
4. What is the direction of the leading strand during
replication in eukaryotic DNA?
a. 5’PO4 -> 3’OH
b. 3’PO4 -> 5’OH
c. 3’OH-> 5’PO4
d. 3’PO4 -> 5’OH
5. During DNA replication, the incoming nucleotide
triphosphate binds with the nucleotide in the daughter
strand by:
a. Hydrogen bonding with the template bases
b. Phosphodiester bonding between the 3’OH of previous
nucleotide & 5’PO4 of incoming nucleotide triphosphate
(NTP)
c. Phosphodiester bond between the 5’PO4 of the previous
nucleotide and the 3’OH of the incoming nucleotide
6. Which enzyme is responsible for unwinding of DNA
strands forming the replicating fork in eukaryotes?
a. MCM2-MCM7 protein
b. PCNA (Proliferating Cell Nuclear Antigen)
c. Pol β
d. primase
7. What reaction occurs between the base of the template
strand and the base of the incoming nucleotide?
a. cleavage of pyrophosphate from the entering nucleotide
b. covalent bond between the 2 phosphate molecules
c. hydrogen bond between the bases
d. phosphodiester bond between the 3’OH & 5’PO4
8. What is the correct enzyme: protein function pair?
a. Pol α: synthesis of DNA Okazaki fragments
b. Pol β: synthesis of DNA in mitochondria
c. Pol €: synthesis of leading strand
d. Pol ¥: for repair of damaged DNA
9. Which is the correct protein: DNA replication process
pair?
a. RPA: sliding clamp for processive movement of enzyme
b. RFC: loads the PCNA in the DNA strand
c. ORC: prevents reannealing of ssDNA molecules
10. Aside from primase, what is the enzyme responsible for
the synthesis of the initial part of the lagging strand?
a. Pol ¥ c. Pol €
b. Po lα d. Pol β
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 Gene Expression I: DNA Replication and Repair
11. MCM becomes active to start the separation of the DNA 15. What will prevent a previously replicated origin from
strands after what process? being replicated again?
a. binding to Cdc6 and Cdt1 a. Activation of several protein kinases
b. binding with the ORC b. Absence of telomerase base pairs
c. loading of PCNA c. Cyclins form S phase throughout mitosis
d. stimulation by Cdk & DDK d. Presence of PCNA up to the end of replication

12. At the end of replication of both DNA strands, what Answers: d, d, a, c, b, a, c, c, b, b, d, a, d, d, c

enzyme will remove the RNA nucleotides?
a. FEN1 (Flap Endonuclease) REFERENCES
b. Polβ
c. RNA endonuclease 1. Textbook of Biochemistry by Devlin 6th edition
d. Telomerase 2. Biochemistry by Campbell
3. 2020ABC transes
13. What extends the nucleotides that overhang after the 4. Dr. Balcueva’s lecture
removal of the primer RNA at the end of DNA replication? 5. Mark’s Basic Medical Biochemistry 4th edition
a. Appropriate nucleotides are incorporated by Pol d/ε 6. Online Journals
b. Appropriate nucleotides are incorporated by Pol α 7. Biochemistry Pearson (Appling et al)
c. Overhang nucleotides remain unpaired 8. Albert’s Molecular biology
d. Telomerases pair with overhanging nucleotides 9. Wiley Cell Biology
10. Essentials of Genetics by Krug an dCummings
14. Mutation caused by cigarette smoking damages DNA by
what reaction?
a. Benzo-a-pyrene causes a single gene deletion
b. Nicotine causes deletion of a segment of DNA
c. Nicotine causes excision of a nucleotide base
d. Oxidized benzo-a-pyrene binds with guanine

APPENDIX

Figure 37. Proteins in Replication Fork. Note: DNA Polymerase ε is used in leading strand not δ

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 Gene Expression I: DNA Replication and Repair
Table 5. Summary of Proteins in DNA replication
Table 6. Prokaryotic vs Eukaryotic DNA polymerase
In prokaryotes In eukaryotes Function
DnaA ORC proteins Recognition of origin of
replication
Gyrase Topoisomerase Relieves positive
I/II supercoils ahead of
replication fork
DnaB MCM DNA helicase that
unwinds parental duplex
DnaC Cdc6, Cdt1 Loads helicase onto DNA
SSB RPA Maintains DNA in single-
stranded state
γ-complex RFC Subunits of DNA
polymerase holoenzyme
that load the clamp onto
the DNA
Pol III core Pol δ (lagging Primary replicating
strand)/ϵ enzymes; synthesize
(leading entire leading strand &
strand) Okazaki fragments; have
proofreading capability
β-clamp PCNA Ring-shaped subunit of
DNA polymerase
holoenzyme that clamps
replicating polymerase to
DNA; works with Pol
III/δ/ϵ
Primase Primase Synthesizes RNA primers
-------- Pol α Synthesizes short DNA
oligonucleotides as part
of RNA-DNA primer
DNA ligase DNA ligase Seals Okazaki fragments
into a continuous strand
Pol I FEN-1 Removes RNA primers Figure 38. Direction of Replication in Two Strands

Figure 39. Summary of Replication

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