DNA Structure and Replication in Eukaryotes and Prokaryotes Flashcards
Define DNA replication.
This is the biological process of producing two identical replicas of DNa from one original DNA molecule.
What are the three major steps in DNA replication.
◾ Initiation
◾ Elongation
◾ Termination
DNA replication: Initiation
Can DNA replication initiate randomly at any point in DNA?
No
DNA replication: Initiation
Where does DNA replication begin?
DNA replication begins at a specific site termed as the origin of replication.
DNA replication: Initiation
What role does DnaA play in DNA replication?
It binds to the DNA molecule at the origin sites, flagging it for the docking of other proteins and enzymes essential for DNA replication.
DNA replication: Initiation
What enzyme is recruited to unwind the DNA double helix into single strands, and how does it function?
The enzyme helicase is recruited to unwind the DNA double helix into single strands. Helicases break the hydrogen bonds between base pairs in an energy-dependent manner (ATP hydrolysis is required).
DNA replication: Initiation
What is the replication fork, and what role do single-strand binding proteins (SSB) play in DNA replication?
The replication fork is the region of DNA where the hydrogen bonds are broken, and DNA is unwound. Single-strand binding proteins (SSB) bind to the unwound regions to prevent them from annealing, allowing the replication process to proceed in two opposite directions along the DNA molecule.
DNA replication: Initiation
What enzyme is responsible for the synthesis of a new, complementary strand of DNA, and what additional roles does it play?
DNA polymerases are responsible for the synthesis of a new, complementary strand of DNA using the existing strand as a template. In addition to replication, they also play an important role in DNA repair and recombination.
DNA replication: Initiation
Why can’t DNA polymerases start DNA synthesis independently, and what enzyme provides the necessary 3′ hydroxyl group?
DNA polymerases cannot start DNA synthesis independently because they require a 3′ hydroxyl group to start the addition of complementary nucleotides. This is provided by an enzyme called DNA primase, which synthesizes a short stretch of RNA (a primer) complementary to the existing DNA strands.
DNA replication: Initiation
What is a primer in DNA replication, and what role does it play?
A primer is a short segment comprising 9-12 nucleotides. It provides DNA polymerase the required platform to begin copying a DNA strand. Once the primers are formed on both strands, DNA polymerases extend these primers into new DNA strands.
DNA replication: Initiation
What causes supercoiling during DNA replication, and how is it resolved?
The unwinding of DNA during replication may cause supercoiling in the regions following the fork. These DNA supercoils are relaxed by a specialized enzyme called topoisomerase, which binds to the DNA stretch ahead of the replication fork.
DNA replication: Elongation
What role does DNA polymerase III play during the elongation phase of DNA replication?
DNA polymerase III starts synthesizing a complementary sequence in each of the separated strands, using the parental strands as templates for the newly synthesizing daughter strands.
DNA replication: Elongation
How does the directionality of DNA polymerization affect the replication process?
Elongation is unidirectional, meaning DNA is always polymerized in the 5′ to 3′ direction. This results in continuous replication on the template strand that runs 3′ to 5′, and discontinuous replication on the template strand that runs 5′ to 3′.
DNA replication: Elongation
What is the leading strand in DNA replication?
The leading strand is the DNA strand that is synthesized continuously in the 5′ to 3′ direction, using the template strand that runs in the 3′ to 5′ direction.
DNA replication: Elongation
How does DNA polymerase III contribute to the synthesis of the leading strand?
DNA polymerase III recognizes the 3′ OH end of the RNA primer and adds new complementary nucleotides, continuously generating the new leading strand as the replication fork progresses.
DNA replication: Elongation
How is the lagging strand synthesized during DNA replication?
How are RNA primers removed and replaced during DNA replication?
RNA primers are removed by the enzyme DNA polymerase I (DNA pol I) via its 5′ ⇒ 3′ exonuclease activity. DNA pol I then replaces the RNA primers with new deoxyribonucleotides using its 5′ ⇒ 3′ DNA polymerase activity, ensuring the newly synthesized DNA strands are continuous and free of RNA.
How does DNA ligase contribute to the completion of DNA replication?
After primer removal, the lagging strand still contains gaps or nicks between the adjacent Okazaki fragments. DNA ligase identifies and seals these nicks by creating a phosphodiester bond between the 5′ phosphate and 3′ hydroxyl groups of adjacent fragments, ensuring the newly synthesized DNA strand is continuous and complete.
State the function of DNA Polymerase I.
It is primarily involved in the removal of RNA primers and filling in the gaps with DNA. It has both 5’ to 3’ exonuclease activity and 5’ to 3’ polymerase activity. It also has 3’ to 5’ exonuclease activity for proofreading and error correction.
State the function of DNA Polymerase III.
It is the primary enzyme responsible for DNA synthesis during replication. It has high processivity and is capable of synthesizing long stretches of DNA. It processes 5’ to 3’ polymerase activity for adding nucleotides to the growing DNA strand and 3’ to 5’ exonuclease activity for proofreading and correcting errors during replication.
Briefly discuss termination of replication in eukaryotes.
🧬 Replication forks from adjacent replication origins meet and converge.
🧬 The replication machinery, including DNA polymerases and helicases is disassembled.
🧬 Topoisomerases resolve any intertwined DNA to ensure proper segregation of the replicated chromosomes.
🧬 Telomerase extends the telomeres to prevent loss of genetic material at chromosome ends.
Further notes:
Telomerase is an enzyme that adds repetitive nucleotide sequences to the ends of chromosomes, known as telomeres. Telomeres protect the ends of chromosomes. During DNA replication, the very ends of chromosomes cannot be fully copied, leading to gradual shortening with each cell division. Telomerase counteracts this shortening by adding telomeric repeats, ensuring that the genetic material is preserved and preventing the loss of important DNA sequences.
DNA replication in Prokaryotes
Where and how does DNA replication begin in Prokaryotes (E. coli)?
🧬 The OriC locus is the origin of replication in prokaryotes, specifically in E. coli.
🧬 It is a 245 base pair (bp) region.
🧬 It contains sequences that are the preffered binding site for the initiator protein DnaA.
🧬 Additionally, the OriC locus contains a tandem repeat of 13 bp sequences that are AT-rich (adenine-thymine rich). These AT-rich regions are highly conserved and are easier to unwind compared to GC-rich (guanine-cytosine rich) regions due to fewer hydrogen bonds between adenine and thymine. This unwinding is essential for the replication process to begin.
🧬 The binding of DnaA causes localized unwinding of the DNA, creating a single-stranded region.
Discuss the process of DNA replication in E. coli, from the binding of DnaA.
🧬 The binding of DnaA causes localized unwinding of the DNA, creating a single-stranded region in the following manner:
(1) DnaA oligomerizes and the DNA wraps around this DnaA complex.
(2) DnaB helicase is loaded around the DNA with the help of helicase loader DnaC.
(3) This results in the formation of complex, which makes the ssDNA accessible to other proteins.
(4) DnaB unwinds the DNA at the OriC forming a replication fork.
🧬 Topoisomerase prevents supercoiling of the DNA.
🧬 Following the formation of the replication fork, DnaG (primase) is then able to synthesize and attach the RNA primer.
🧬 DNA polymerase III assembles on the prepriming complex signaling initiation of replication.
🧬 Elongation and termination is similar to replication in Eukaryotes.
What are some mechanisms used in Prokaryotes to prevent reinitiation of replication within the same cell cycle?
🧬 Physical sequestration of the origin
🧬 Methylation of the origin of replication (delayed methylation)
🧬 Inhibition of DnaA binding (Prokaryotes only)
🧬 Represssion of DnaA transcription (Prokaryotes only)
Compare replication in Eukaryotes and Prokaryotes in terms of origin of replication.
Prokaryotes typically have a single origin of replication (OriC) on their circular DNA molecule, whereas Eukaryotes have multiple origins of replication on their linear chromosomes to ensure the entire genome is replicated efficiently.
The difference between the leading and lagging strands of DNA replication is that ________.
(a) synthesis of the leading strand is continuous but discontinuous on the lagging strand
(b) a single primer is used in the leading strand while many primers are used in the lagging strand
(c) DNA synthesis proceeds in the same direction as unwinding DNA while it is in the opposite direction of unwinding of DNA, for leading and lagging strands respectively.
(d) All of the above.
(e) None of the above.
(d) All of the above.
DNA gyrase is ________.
(a) absent in proliferating cells of eukaryotes
(b) used to unwind DNA helix
(c) used in the process of transcription
(d) a type II topoisomerase and potential target of anticancer drugs
(e) none of the above
(d) a type II topoisomerase and potential target of anticancer drugs
Unwinding of DNA double helix is catalysed by ________.
(a) DNA gyrase
(b) DNA helicase
(c) DNA polymerase
(d) DNA ligase
(e) ribonuclease H
(b) DNA helicase
A ________ fills in the gap using the undamaged complementary strand of DNA as a template.
(a) DNA ligase
(b) DNA repair endonucleases
(c) DNA polymerase
(d) gyrase enzyme
(e) helicase enzyme
(c) DNA polymerase
Which of the following enzymes repairs nicks in DNA by forming a phosphodiester?
(a) helicase
(b) DNA polymerase
(c) DNA ligase
(d) DNA gyrase
(e) topoisomerase
(c) DNA ligase
Type II topoisomerases are ________.
(a) confined to proliferating cells in eukaryotes and are targets of anticancer drugs
(b) used to unwind DNA helix
(c) used in the process of transcription
(d) a class that doesn’t include DNA gyrase
(e) none of the above
(a) confined to proliferating cells in eukaryotes and are targets of anticancer drugs
Which of the following statements is not true about DNA replication in prokaryotes?
(a) requires a primer
(b) produces a double-stranded DNA
(c) both DNA strands serve as a template
(d) utilizes DNA polymerase
(e) adenine base pairs with uracil
(e) adenine base pairs with uracil
