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DNA REPLICATION CELL BIOLOGY: Note #1. 1 of 6
# DNA REPLICATION
Cell Biology | DNA Replication Medical Editor : Aldrich Christiandy
OUTLINE
I) FUNDAMENTALS
II) PROCESS OF DNA REPLICATION
III) TELOMERASE
IV) CLINICAL SIGNIFICANCE
V) APPENDIX
VI) REVIEW QUESTIONS
VII) REFERENCES
I) FUNDAMENTALS (A) FUNCTION OF DNA REPLICATION
Figure 1. Cell cycle [Alberts et.al.,2014]
In order for cells to replicate (1 parent cell 2 daughter cells) you need to replicate the DNA within the parent cell
DNA replication occurs in the S-phase of the cell cycle
The purpose is to take 1 double stranded DNA molecule (dsDNA)2 double stranded DNA molecules (dsDNA)
Figure 2. Cell replication - DNA replication
This allows the DNA/ genetic material from the 1 parent cell to be passed on to the 2 daughter cells
(B) DNA REPLICATION IS SEMICONSERVATIVE
Figure 3. Semiconservative replication [Urry et.al.,2020]
Parental dsDNA contains two strands that are referred to as the parental strands
When dsDNA is replicated the DNA polymerases use the parental strands to build new identical daughter strands
With each parental strand in a dsDNA molecule there is a complementary daughter strand essentially mixing old (parental strand) with new (daughter strand)
(C) DNA REPLICATION OCCURS IN A
5 3 DIRECTION
Figure 4. DNA replication direction [Urry et.al., 2020]
DNA polymerase adds nucleotides in a particular
direction
It uses the 3 OH group of deoxyribose sugar on preceding nucleotide as its starting point
When it adds nucleotides, it does this by adding the 5 Phosphate end to the 3OH end forming a phosphodiester bond between them
Additional Information
Terminology [Rodwell et.al., 2018]
o Electro philic = electron -poor
o Nucleophilic = electron -rich
Figure 5. Nucleophilic attack [https://chem.libretexts.org/@go/page/106337]
The DNA replication direction is associated with nucleophilic attack / nucleophilic substitution (S N2)
o Nucleophilic substitution reaction happens when a
nucleophile attacks the central atom (electrophile )
Both bond -breaking and bond -forming occurs simultaneously [Tim, 2021]
Figure 6. Nucleophilic attack on DNA elongation [Nelson and Cox, 2017]
o The nucleophile is the 3 -OH group of the nucleotide at the 3 end of the growing strand [Nelson and Cox, 2017]
o Nucleophilic attack occurs at the phosphorus of the incoming deoxynucleoside 5 -triphosphate [Nelson and Cox, 2017]
Last edited: 9/25/2021 2 of 6 CELL BIOLOGY: Note #1. DNA REPLICATION
(D) DNA REPLICATION OCCURS BIDIRECTIONALLY
> Figure 7. Bidirectional replication
DNA polymerase uses the two parental strands as a template to synthesize daughter strands.
Two replication forks occur at the replication bubble and
DNA polymerase synthesizes new daughter strands on the leading and lagging strand again in a 53 direction
II) PROCESS OF DNA REPLICATION
DNA replication occurs in three phases:
o Initiation
o Elongation
o Termination
(A) INITIATION OF REPLICATION
> Figure 8. Initiation of replication [Alberts et.al.,2015]
(1) Origin of replication
This is where DNA replication begins
A pre-replication protein complex recognizes the origin of replication based on an A-T (adenine and thymine) rich area in DNA.
This area is preferred because there are only two hydrogen bonds between A-T as compared to three hydrogen bonds between G -C
This A -T area is similar to the promoter region (TATA box) during DNA transcription
(2) Replication bubble
The pre-replication protein complex binds to the origin of
replication (A-T rich area) and separates the two parental strands from one another
o Creating a replication bubble
o Take a look at Figure 15 for better visualization
(3) Maintaining replication bubble
> Figure 9. Single stranded binding proteins
After the replication bubble is formed the separated parental strands want to rebind one another very badly
o So, we prevent this via single stranded binding proteins (SSBP) which keep the strands separated
In addition, the separated template strands are highly susceptible to nuclease enzymes that want to break the
separated strands apart
o So , the SSBPs prevent nuclease enzymes from breaking down the separated parental strands
(4) Replication fork
This a Y -shaped region that is found on both ends of the replication bubble
An enzyme called helicase works in the replication fork
unwinding DNA in front of it to enable replication.
Helicase unwinds the dsDNA into separate parental strands that can be used as templates for DNA polymerase to make new DNA strands in the 53 sequence
The replication fork subsequently creates a leading and
lagging strand
The activity of helicase unwinding is highly ATP dependent (5) Regulation of supercoils
> Figure 10. DNA supercoils [Nelson and Cox, 2017]
As helicase continues to unwind the DNA at the replication forks it can create an overwinding (positive supercoils) of DNA ahead of the replication fork
o This can be problematic as helicase wouldn't be able to continue to unwind DNA if its not fixed
Enzymes called topoisomerases fix these supercoil issues
o Topoisomerase I
Uses a nuclease domain to cleave phosphodiester bonds in the DNA supercoils
this relieves the supercoiling ahead of replication fork
Uses a ligase domain to refuse the phosphodiester bonds after the supercoiling has been relieved
This enzyme can cleave phosphodiester bonds on one or both DNA strands and this process
does NOT require ATP
o Topoisomerase II (DNA gyrase) and IV
Uses a nuclease domain to cleave phosphodiester bonds in the DNA supercoils
this relieves the supercoiling ahead of replication fork
Uses a ligase domain to refuse the phosphodiester bonds after the supercoiling has been relieved
This enzyme can cleave phosphodiester bonds on both DNA strands and this process does require ATP
One other interesting thing this enzyme can do is insert negative supercoils into the DNA to relax the DNA supercoils and make it easier for helicase to unwind DNA DNA REPLICATION CELL BIOLOGY: Note #1. 3 of 6
(B) ELONGATION OF REPLICATION
Figure 11. Elongation of replication
(1) RNA primer synthesis
An enzyme called primase creates RNA primers on
the leading and lagging strand
RNA primers are a sequence of approximately 10 nucleotides that are complementary to the parental
strands that are formed in replication bubble
The function of these primers is to create a starting point for DNA polymerases
DNA polymerases need a 3OH end in order to
synthesize DNA in the 53 sequence
(2) DNA synthesis
DNA polymerases use the RNA primers on leading and lagging strands to create new daughter strands in the 53 sequence
DNA polymerases
o DNA polymerase I & III are in prokaryotes
Additional Information
List of prokaryotes and eukaryotes DNA Polymerase type s
o Prokaryotes [Fuchs et.al.,2013]
DNA polymerase I
DNA polymerase II
DNA polymerase III
DNA polymerase IV
DNA polymerase V
o Eukaryotes [Rodwell et.al., 2018], [Zheng et.al.,2020]
B F amily [Zheng et.al.,2020]
DNA polymerase
DNA polymerase
DNA polymerase
DNA polymerase
DNA polymerase
DNA polymerase III uses the 3 OH group of deoxyribose sugar on preceding nucleotide as its starting point
It adds nucleotides by adding the 5 Phosphate end to the
3OH end forming a phosphodiester bond between them
It does this one at a time constantly synthesizing in the 53 direction
Every time a nucleotide is added a pyrophosphate is released which is then broken down into two phosphate groups by pyrophosphatase
Figure 12. Leading strand, lagging strand, and Okazaki fragments [Nelson and Cox,2017]
Leading Strand
o DNA polymerase III synthesizes DNA in a continuous fashion on leading strand because it only needs one RNA primer to get going
Lagging Strand
o DNA polymerase III synthesizes DNA in a discontinuous fashion on lagging strand because
multiple RNA primers are being formed at the moving replication fork to get going
This discontinuous DNA replication on the lagging strand
creates fra gments of daughter DNA complementary to parental DNA strand these are called Okazaki fragments
(3) Proofreading
DNA polymerases I & III have a unique proofreading ability
DNA polymerases I & III can read the nucleotide they are going to add or just added to the daughter strand via a 35 proofreading domain
o If the nucleotide they added wasn't the correct complementary nucleotide they use their 53 exonuclease domain to remove the previously incorrectly added nucleotide
Then they can add on the correct complementary nucleotide using its 53 synthesizing domain.
(4) Removal of RNA primers
Now that the DNA polymerases have used the RNA primers to help synthesize DNA, they don't need these RNA primers anymore so we remove them
DNA polymerase I in prokaryotes can remove the RNA primers in a 53 fashion
(5) Filling the gaps due to removed RNA primers
After RNA primers are removed there are gaps between newly synthesized DNA on lagging strand
DNA polymerase I fills these gaps by inserting nucleotides in these gaps in a 53 fashion
Remember it uses its 35 proofreading domain to make sure nucleotide is correct and then uses its 53 domain to add nucleotides.
Then an enzyme called DNA ligase takes all the DNA fragments on the lagging strand and fuses them together creating a continuous daughter strand.
Ligase activity is ATP dependent 4 of 6 CELL BIOLOGY: Note #1. DNA REPLICATION
(C) TERMINATION OF TRANSCRIPTION (1) DNA polymerase reaches telomeres
> Figure 13. Termination of transcription
DNA polymerases continue to replicate the DNA and approach replication fork where they meet another DNA polymerase and they hop off the DNA at that point
Another time is when DNA polymerases continue to replicate the DNA and approach the end of the chromosomal DNA called telomeres
At the telomeres there is a particular nucleotide sequence on the 3 OH portion of telomeres
o Where the DNA polymerases are released from the DNA and their synthesis function has come to an end
o Meaning they don't replicate the 3OH region of telomere
That nucleotide sequence that causes the DNA polymerases to hop of DNA is TTAGGG
> Figure 14. Telomere length and cell division [Melk and Halloran, 2001]
There's significance to this step
o As DNA replication continues to occur over time those
telomeres will continue to shorten with each replication event
Because they aren't being replicated
That's why there is a maximum limit to the number of replication cycles DNA can go through because those telomeres will continue to shorten this is called the Hayflick Limit III) TELOMERE & TELOMERASE (A) STRUCTURE
> Figure 15. Telomere
Telomeres are a noncoding DNA fragment located at the
3 ends of chromosome
Telomeres contain a tandem repeat of nucleotides TTAGGG
(B) FUNCTION
Prevents gene loss during continuous DNA replication
o With each replication the lagging strand gets
shorter
o This is because DNA polymerases cannot synthesize the last part of the 5 end on lagging strand
o Because there is no 3OH group for them to build off
of this leads to a couple hundred nucleotides not being replicated shortening the telomeres
But thankfully the telomeres dont code for RNA
so you don't lose transcription function
(C) REGULATION
> Figure 16. Telomere replication
To avoid gene loss on 3 end of DNA strand, the telomeres use an enzyme called Telomerase
Telomerase is a Ribonucleoprotein which contains an RNA template of particular sequence of nucleotides
AAUCCC
It uses reverse transcription then to synthesize complementary DNA to that RNA template TTAGGG
This process helps to continue to elongate the telomeres so that as replication occurs it shortens the telomeres and not the genes on 3 end of DNA
This enzyme is highly expressed in cells that perform a
lot of cell replication stem cells IV) CLINICAL SIGNIFICANCE (A) HELICASE DEFECT
Bloom syndrome
o Deficiency in helicase enzyme BLM gene
mutation
o Clinical features
Short stature
Butterfly rash on nose and cheeks
Cafe au lait spots
Risk of leukemia
(B) DRUGS THAT MODULATE TOPOISOMERASE ENZYMES (1) In eukaryotic cells
Topoisomerase I is inhibited by irinotecan & topotecan
o These are chemotherapy drugs nuclease domain on these enzymes phosphodiester bond breakage DNA destruction in cancer cells
Topoisomerase I can be targeted by antibodies in
Scleroderma
o In scleroderma, the plasma cells make antibodies
(scl-70) against topoisomerase I
Topoisomerase II is inhibited by etoposide and
teniposide
o These are chemotherapy drugs nuclease domain
on these enzymes phosphodiester bond breakageDNA destruction in cancer cells DNA REPLICATION CELL BIOLOGY: Note #1. 5 of 6
(2) In prokaryotic cells
Topoisomerase II & IV is inhibited by fluoroquinolones
o These are antibiotics like ciprofloxacin nuclease domain on these enzymes phosphodiester bond breakage DNA destruction in bacterial cells
Table 1. Summary of topoisomerase enzyme modulation
Topoisomerase
Enzyme Location Can be Targeted by
I Eukaryotes
Irinotecan &
topotecan
Antibodies (in
scleroderma )
II Prokaryotes & Eukaryotes
Eukaryotes :Etoposide &
Teniposide
Prokaryotes :
Fluoroquinolones (e.g., ciprofloxacin)
IV Prokaryotes Fluoroquinolones (e.g., ciprofloxacin)
Irinotecan , topo tecan , and fluoroquinolones:
o Similarity:
Mechanism of action
Nuclease domain on these enzymes phosphodiester bond breakage DNA destruction in bacterial cells
o Difference:
Irinotecan , topo tecan are chemotherapy drugs
Fluoroquinolones are antibiotic s
(C) DRUGS THAT MODULATE DNA POLYMERASE III
Figure 17. Nucleoside analogues [Andreeva et.al.,2021]
Figure 18. HIV and Nucleoside reverse transcriptase inhibitors
Anti-Retroviral drugs like nucleoside reverse transcriptase inhibitors can be used in HIV
o This includes drugs like Didanosine , lamivudine ,
zidovudine , abacavir , etc.
These drugs act like nucleoside analogues
o DNA polymerase III can't tell the difference between them and real nucleo tides
o However, these drugs don't have a 3OH end so you
cannot add nucleotides to them this terminates DNA replication in cells infected with HIV
(D) TELOMERASE ACTIVITY IN CANCER
Figure 19. Telomerase activity in cancer
Cancer cells have one goal replicate uncontrollably
However, remember that as you shorten telomeres
replication stops
o Cancer cells find a way around that they increase telomerase activity
o This elongates telomeres increasing the number of replications cycles the cell can go through
V) APPENDIX
Figure 20. Origin of replication in prokaryotes and eukaryotes [Urry et.al.,2020] 6 of 6 CELL BIOLOGY: Note #1. DNA REPLICATION
VI) REVIEW QUESTIONS
When does the DNA replication occurs?
a. G 0 phase
b. G1 phase
c. S phase
d. M phase
What does the DNA polymerase use in order to add nucleotides in a particular direction?
a. 5Phosphate end of deoxyribose sugar on preceding nucleotide
b. 3 OH group of deoxyribose sugar on preceding nucleotide
c. Hydrogen bonds from the nitrogen bases
d. Hydrolysis in ATP that leads to release of pyrophosphates
How many replications forks are there inside 5 replication bubbles?
a. 2
b. 6c. 10
d. 14
What does the pre-replication protein complex recognize at the origin of replication?
a. G-C rich area
b. A-T rich area
c. A-A rich area
d. T-T rich -area
Which protein that prevents the strands from rebinding during replication?
a. SSBP
b. Helicase c. Topoisomerase
d. Ribozyme
Why topoisomerases can cleave phosphodiester bonds in the DNA supercoils?
a. It can melt the DNA strands
b. It has ability to alter the hydrogen bonds
c. It has phosphodiesterase inhibitor
d. It has nuclease domain
What enzyme that creates RNA primers on both leading and lagging strand?
a. Primase
b. Primary enzyme
c. DNA polymerase
d. Reverse transcriptase
Which DNA polymerase that synthesize DNA during elongation of replication?
a. DNA polymerase I
b. DNA polymerase III
c. DNA polymerase IV
d. DNA polymerase V
Which DNA polymerase that has unique proofreading ability?
a. DNA polymerase I & III
b. DNA polymerase IV & V
c. DNA polymerase I & II
d. DNA polymerase III & V
In which disease that topoisomerase can be targeted by antibodies?
a. Systemic lupus erythematosus
b. Xeroderma pigmentosum
c. Scleroderma
d. Atypical mole
CHECK YOUR ANSWERS VII) REFERENCES
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