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What is the primary function of DNA polymerases during DNA replication?
Which mechanism allows for the replacement of one DNA polymerase with another during DNA replication?
Which process is primarily involved in correcting errors that occur during DNA replication?
What is the role of telomeres in linear chromosome replication?
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What is the main challenge encountered during linear chromosome replication?
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Which statement about DNA repair mechanisms is correct?
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Why is it important for telomeres to maintain their structure during DNA replication?
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What generally occurs when a DNA polymerase encounters a damaged nucleotide during replication?
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What is the primary function of DNA polymerase ε?
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What occurs during the polymerase switch mechanism?
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Which type of polymerases are involved in DNA repair mechanisms?
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What process does flap endonuclease perform in DNA replication?
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What characteristic do telomeres possess?
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Which statement is true regarding the replication problem at the ends of linear chromosomes?
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What is a feature of telomeric DNA sequences?
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What happens if a flap is too long during DNA replication?
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Which DNA polymerases are primarily responsible for normal replication?
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What is the function of the β subunit in DNA polymerase III?
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What is the primary role of DNA pol I during DNA replication?
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Why is the proofreading function of DNA polymerase important?
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How are Okazaki fragments synthesized on the lagging strand?
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What is the role of DNA ligase in DNA replication?
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What mechanism do eukaryotes use to facilitate rapid DNA replication on their long chromosomes?
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What happens when the tus protein binds to the ter sequences?
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What feature of DNA pol III allows it to synthesize long stretches of DNA efficiently?
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In what direction do DNA polymerases synthesize new DNA strands?
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What is a key difference between leading and lagging strand synthesis?
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Why is catenation a challenge during DNA replication?
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During DNA replication, what is the primary function of the primase enzyme?
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What is the primary mechanism by which new DNA strands are synthesized during DNA replication?
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Which base pairing rule is crucial for the accuracy of DNA replication?
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During DNA replication, what are the newly synthesized strands referred to as?
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What is the role of the original DNA strands during the replication process?
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What is a key characteristic of DNA replication in terms of its efficiency?
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What is the role of telomerase in linear chromosomes?
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Which statement correctly describes the process of telomere lengthening by telomerase?
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How do telomeres affect cell division in elderly individuals?
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What is a common characteristic of cancer cells regarding telomerase activity?
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What happens to telomeres during each round of DNA replication if not stabilized by telomerase?
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What happens to the parental strands in the semiconservative model of DNA replication?
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Which model of DNA replication was experimentally validated by Meselson and Stahl?
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In the context of DNA replication models, what characterizes the conservative model?
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What experimental method did Meselson and Stahl use to differentiate between parental and daughter DNA strands?
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Which of the following accurately describes the dispersive model of DNA replication?
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What was the primary hypothesis regarding DNA replication based on Watson's and Crick's ideas?
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During the first round of replication in the conservative model, what occurs?
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What characteristic is specific to the dispersive model compared to the other models of DNA replication?
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What is an ARS element in simple eukaryotes?
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What characteristic is common to origins of replication in more complex eukaryotes?
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What specific role does the B1 and B2 sequence play in ARS elements?
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Where are origins of replication typically found in relation to nucleosome structures in eukaryotic cells?
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What is a unique feature of G-quadraplexes found in the replication origins of complex eukaryotes?
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What do the flanking histone modifications around eukaryotic origins favor?
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What does the separation of DNA occur within during the process initiated by ARS elements?
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How do origins of replication in simple eukaryotes compare to those in bacteria?
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What role does DNA pol ε play in DNA replication?
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Which of the following accurately describes a function of translesion-replicating polymerases?
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When Polymerase δ encounters a primer of an adjacent Okazaki fragment, what process occurs?
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What feature characterizes telomeric sequences in linear eukaryotic chromosomes?
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What challenge arises at the 3' ends of linear chromosomes during DNA replication?
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What occurs if a flap during DNA replication is too long?
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Which statement is true about the function of flap endonuclease in DNA replication?
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What is a unique characteristic of DNA polymerases regarding their synthesis direction?
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What is the primary function of the MCM helicase in the prereplication complex?
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Which type of origin of replication is most commonly found in complex eukaryotes?
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Which DNA polymerase is exclusively associated with primase to initiate DNA synthesis?
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What occurs during the exchange of DNA polymerase α for either polymerase ε or δ?
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How many nucleotides comprise the RNA-DNA hybrid primer synthesized by the DNA pol α/primase complex?
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What occurs to DNA after one generation in the semi-conservative replication model?
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What is indicated by the presence of equal amounts of light and half-heavy DNA after two generations?
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What structural feature characterizes the origin of replication in bacterial chromosomes?
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What is the process used to observe DNA within a CsCl gradient?
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What happens to DNA replication when the two replication forks meet on a bacterial chromosome?
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What method involves lysing cells to extract DNA for density gradient centrifugation?
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Which model of DNA replication was experimentally validated by the Meselson and Stahl experiment?
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What is the density of DNA typically around, which helps isolate it from other cellular molecules?
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Which of the following correctly describes the semiconservative model of DNA replication?
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What was the primary conclusion of the experiments conducted by Meselson and Stahl?
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What does the conservative model of DNA replication imply about parental strands after replication?
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In the dispersive model of DNA replication, what is true about the DNA strands after replication?
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What experimental method was used by Meselson and Stahl to distinguish between daughter and parental DNA strands?
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What is a key differentiating feature of the first round of replication in the conservative model?
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How did the results of the Meselson and Stahl experiment support the hypothesis on the nature of DNA replication?
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What is the main reason the semiconservative model is considered the correct mechanism of DNA replication?
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What is the main function of DNA polymerase δ during DNA replication?
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What occurs when DNA polymerase encounters a long flap during replication?
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Which of the following describes telomeric DNA sequences?
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What unique feature is associated with DNA polymerases that complicates linear chromosome replication?
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What is the primary role of translesion-replicating polymerases?
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What happens at the 3’ ends of linear chromosomes during DNA replication?
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Which of the following correctly describes the action of flap endonuclease?
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What distinguishes telomeres in linear eukaryotic chromosomes?
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What role do DnaA boxes play in the initiation of DNA replication?
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What happens to the daughter strand immediately after DNA replication regarding methylation?
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What is the primary function of DNA gyrase during DNA replication?
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In which direction does DnaB helicase travel along the DNA during replication?
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Why are multiple RNA primers needed on the lagging strand during replication?
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What is the primary role of single-strand binding proteins in DNA replication?
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What initiates DNA replication at the oriC site?
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What is a consequence of not having fully methylated DNA prior to the initiation of replication?
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Study Notes
DNA Polymerases
- Five main DNA polymerases with polymerase activity: DNA pol I, II, III, IV, and V
- DNA pol I and III are responsible for normal replication.
- DNA pol II, IV, and V are involved in DNA repair and replication of damaged DNA.
DNA Pol III
- Primarily responsible for DNA replication in E. coli.
- Composed of 10 subunits, each with a specific function.
- The alpha subunit catalyzes the formation of bonds between adjacent nucleotides during DNA synthesis
- The complex of all 10 subunits is called the DNA pol III holoenzyme.
DNA Pol I
- Composed of a single polypeptide.
- Removes RNA primers and replaces them with DNA.
DNA Pol III Subunit Composition
- Alpha (α) subunit - Synthesizes DNA.
- Epsilon (ε) subunit - Contains a 3’ to 5’ exonuclease site that removes mismatched nucleotides (proofreading).
- Theta (θ) subunit - Acts as an accessory protein, stimulating the proofreading function.
- Beta (β) subunit - Functions as a clamp protein, allowing DNA polymerase to slide along DNA without detaching.
- Tau (τ), Gamma (γ), Delta (δ), Delta prime (δ′), Psi (ψ), Chi (χ) subunits - Together they form the clamp loader complex, aiding the clamp protein in binding to the DNA.
Structure of DNA Polymerase
- Resembles a human right hand.
- Template DNA passes through the "palm" of the hand.
- "Thumb" and "fingers" wrap around the DNA.
- Variation in subunit composition across bacterial DNA polymerases, but all share a similar catalytic subunit.
Features of DNA Polymerase
- Inability to initiate DNA synthesis by linking two individual nucleotides on a bare template strand. This problem is solved by RNA primers, synthesized by primase.
- Ability to attach nucleotides only in the 5' to 3' direction. This is overcome by synthesizing new strands both towards and away from the replication fork, accommodating the anti-parallel nature of DNA strands.
Leading and Lagging Strand Synthesis
- Leading strand - Synthesized continuously towards the replication fork. Only requires one RNA primer at the origin of replication.
- Lagging strand - Synthesized in the 5' to 3' direction, but away from the replication fork. Requires multiple RNA primers.
- Short DNA fragments (Okazaki fragments) are synthesized on the lagging strand.
DNA Synthesis at the Replication Fork
- Removal of RNA primers by DNA pol I, followed by filling the gaps with DNA.
- DNA pol I utilizes 5' to 3' exonuclease activity to digest RNA and 5' to 3' polymerase activity to replace it with DNA.
- DNA ligase catalyzes the formation of a covalent bond to connect the DNA backbones, joining the Okazaki fragments.
DNA Replication Complexes
- Primosome - A complex formed by the physical association of DNA helicase and primase.
- Replisome - A complex formed by the primosome and two DNA polymerase holoenzymes.
Termination Sequences
- ter sequences - Located on the opposite side of the chromosome from oriC. Two termination sequences, T1 and T2, stop the counterclockwise and clockwise forks, respectively.
- Tus - Termination utilization substance binds to the ter sequences, arresting the movement of replication forks, ultimately halting DNA replication.
Termination of Replication
- Replication ends when opposing replication forks meet, typically at T1 or T2.
- DNA ligase covalently links the two daughter strands.
- Catenanes (intertwined circular molecules) are separated by DNA gyrase.
Isolation of Mutants
- Mutants are essential for understanding DNA replication.
- Temperature-sensitive (ts) mutants are crucial for studying essential genes. They can survive at a permissive temperature but cease to grow at a non-permissive temperature.
- Mutants that block DNA synthesis are lethal.
Bacterial DNA Replication Chemistry and Accuracy
- DNA polymerase forms a covalent (ester) bond between the inner phosphate group of the incoming deoxyribonucleoside triphosphate and the 3'-OH of the sugar of the previous deoxynucleotide.
- Pyrophosphate (PPi) is released as a byproduct.
Processive Nature of DNA Pol III
- DNA pol III remains attached to the template during daughter strand synthesis.
- The beta (β) subunit forms a ring around the template DNA, acting as a clamp protein.
- This allows the holoenzyme to stay attached to the DNA and increases the rate of DNA synthesis.
Fidelity Mechanisms of DNA Replication
- High fidelity is ensured by various mechanisms, including:
- Base pair stability
- Active site structure of DNA polymerase
- Proofreading function of DNA polymerase
Fidelity Mechanisms: Stability of Base Pairs
- Watson-Crick base pairs are more stable than mismatched pairs.
- However, base pair stability alone isn't sufficient, as error rates for mismatched base pairs are still relatively high.
Fidelity Mechanisms: DNA Polymerase Active Site
- The DNA polymerase active site is designed to only accept correctly paired nucleotides.
- Mispairing causes helix distortion, preventing the incorrect nucleotide from fitting properly in the active site. This induced-fit mechanism further reduces errors.
Fidelity Mechanisms: Proofreading Function of DNA Polymerase
- DNA polymerase possesses 3' to 5' exonuclease activity.
- Allows the enzyme to remove mismatched nucleotides from the newly synthesized strand.
- After removal, DNA synthesis resumes in the 5' to 3' direction.
Eukaryotic DNA Replication
- More complex than bacterial DNA replication.
- Includes features such as:
- Large linear chromosomes
- Chromatin organization within nucleosomes
- Complex cell cycle regulation
Multiple Origins of Replication in Eukaryotes
- Multiple origins of replication are necessary to replicate long linear eukaryotic chromosomes in a timely manner.
- Replication proceeds bidirectionally from these origins.
DNA Replication
- DNA replication is the process by which genetic material is copied.
- The original DNA strands serve as templates for the synthesis of new strands with identical sequences.
- It occurs quickly, accurately, and at the appropriate time in a cell's life cycle.
DNA Replication Mechanism
- DNA replication relies on the complementarity of DNA strands (A pairs with T and G pairs with C).
- The two complementary DNA strands separate.
- Each strand acts as a template for the synthesis of a new complementary strand.
- The newly synthesized strands are called daughter strands.
- The original strands are called parental strands.
Models of DNA Replication
- There were three proposed mechanisms for DNA replication in the late 1950s:
- Conservative Model: Both parental strands remain together after replication.
- Semiconservative Model: The resulting double-stranded DNA contains one parental and one daughter strand after replication (this is the correct model).
- Dispersive Model: Parental and daughter DNA segments are interspersed in both strands after replication.
- The Semiconservative Model was proven experimentally by Meselson and Stahl in 1958.
- They used isotopes of nitrogen (14N and 15N) to distinguish between parental and daughter strands.
Replication in Prokaryotes
- Replication begins at a specific origin of replication (oriC) on the circular bacterial chromosome.
- oriC's contain an AT-rich region, making it easier to separate the DNA strands.
- Replication proceeds bidirectionally from the origin.
- Two replication forks move in opposite directions.
- The process creates a "theta structure" as the DNA replicates.
Replication in Eukaryotes
- Replication occurs at multiple origins of replication on linear eukaryotic chromosomes.
- These origins are called ARS elements in simple eukaryotes like Saccharomyces cerevisiae.
- ARS elements are approximately 50 bp long, rich in A and T, and contain specific consensus sequences (ATTTAT(A or G)TTTA) and B1/B2 sequences.
- In complex eukaryotes, origins of replication are more dynamic and lack a consensus sequence.
- They often contain G-rich sequences (G4 motifs) that form G-quadraplex structures.
- G4 motifs are found in nucleosome-free regions and are often associated with promoters and CpG islands.
DNA Polymerases and their Functions
- DNA polymerases are enzymes responsible for synthesizing new DNA strands.
- They add nucleotides to the 3' end of a pre-existing strand.
- There are several types of DNA polymerases in both prokaryotes and eukaryotes, each with specific functions:
- DNA polymerase III (in prokaryotes): Main enzyme for replication, responsible for synthesizing new DNA strands.
- DNA polymerase I (in prokaryotes): Removes RNA primers and fills in gaps.
- DNA polymerase α (in eukaryotes): Initiates DNA synthesis and produces a short RNA-DNA hybrid.
- DNA polymerase δ (in eukaryotes): Responsible for elongating the lagging strand.
- DNA polymerase ε (in eukaryotes): Used for the processive elongation of the leading strand.
- Certain DNA polymerases can repair damaged DNA and perform translesion synthesis.
Okazaki Fragments and Lagging Strand Synthesis
- DNA replication is semi-discontinuous, meaning that one strand (leading strand) is synthesized continuously, while the other (lagging strand) is synthesized in short fragments called Okazaki fragments.
- Okazaki fragments are synthesized in the 5' to 3' direction, but the lagging strand is synthesized in the opposite direction of the replication fork movement.
- RNA primers are needed to initiate DNA synthesis on the lagging strand.
- DNA polymerase I removes these primers and fills in the gaps, and DNA ligase joins the fragments.
Telomeres and Telomerase
- Telomeres are specialized structures at the ends of linear eukaryotic chromosomes.
- They consist of repetitive DNA sequences and associated proteins.
- They protect the chromosome ends from degradation, fusion, and loss of genetic information.
- Telomeres shorten with each round of replication because DNA polymerase cannot synthesize DNA at the very end of a linear strand.
- Telomerase is an enzyme that adds DNA sequences to the ends of telomeres, preventing shortening.
- Telomerase contains both protein and RNA components.
- Its RNA component is complementary to the telomeric DNA sequence, allowing it to bind and lengthen the telomere.
Telomere Length and its Implications
- Telomere length is associated with aging, cell senescence, and cancer development.
- As cells age, telomeres tend to shorten, eventually leading to cell senescence (the loss of the ability to divide).
- Cancer cells often have mutations that increase telomerase activity, preventing telomere shortening and allowing for uncontrolled cell division.
- Telomerase activity is currently considered a target for potential anti-cancer drugs.
DNA Replication Models
- There are three models of DNA replication: Conservative, Semiconservative, and Dispersive.
- Conservative model: both parental strands remain together after replication, forming a separate new DNA molecule.
- Semiconservative model: each daughter DNA molecule contains one parental strand and one daughter strand, this is the correct model.
- Dispersive model: parental and daughter DNA fragments are interspersed in both strands, forming two hybrid DNA molecules.
Meselson and Stahl Experiment
- The Meselson and Stahl experiment used heavy nitrogen (15N) and normal nitrogen (14N) to label DNA strands and track their replication.
- They determined that DNA replication is semiconservative based on the results of the experiment.
Bacterial DNA Replication
- Bacterial DNA replication proceeds bidirectionally starting at a single origin of replication (oriC).
- The replication forks meet on the opposite side of the bacterial chromosome, ending replication.
- Three types of DNA sequences are important to oriC: DnaA boxes, AT-rich regions, and GATC methylation sites.
- DnaA proteins bind to DnaA boxes and cause the DNA to bend at AT-rich regions, initiating replication.
- DNA helicase breaks the hydrogen bonds in the DNA double helix to separate the DNA strands.
- DNA gyrase alleviates positive supercoiling ahead of the replication fork during unwinding.
- Single-strand binding proteins keep the separated DNA strands apart.
- Primase synthesizes short RNA primers, which are used to initiate DNA synthesis.
- DNA polymerases, specifically DNA polymerase III, are responsible for synthesizing DNA.
Eukaryotic DNA Replication
- Eukaryotic DNA replication involves multiple origins of replication.
- There are three main classes of origins: constitutive, flexible, and dormant.
- The pre-replication complex (preRC) assembles at origins, including the origin recognition complex (ORC) and MCM helicase.
- MCM helicase binding completes DNA replication licensing and allows origins to initiate replication.
- Eukaryotes have different DNA polymerases, with α, δ, and ε primarily responsible for nuclear DNA replication, and γ for mitochondrial DNA replication.
- DNA polymerase α, associated with primase, is involved in primer synthesis.
- Polymerase switch involves the exchange of DNA polymerase α for ε or δ for elongation of the leading and lagging strands.
- DNA polymerase ε is used for leading strand elongation, while DNA polymerase δ is used for lagging strand elongation.
- DNA polymerases also play a role in DNA repair, particularly translesion-replicating polymerases for damaged DNA.
Removal of RNA Primers in Eukaryotes
- The lagging strand in eukaryotic DNA replication has multiple RNA primers.
- Flap endonuclease removes the RNA primers by pushing a portion of the primer into a flap.
- Long flaps are cleaved by Dna2 nuclease/helicase.
Telomeres
- Telomeres are specialized structures at the ends of linear eukaryotic chromosomes.
- They consist of repetitive DNA sequences and associated proteins.
- Telomeric sequences are typically rich in guanine and thymine nucleotides.
- The 3' end of telomeres has a long overhang of 12 to 16 nucleotides.
- DNA polymerases cannot fully replicate the 3' ends of linear chromosomes, posing a replication problem.
End-Replication Problem
- DNA polymerase can only synthesize DNA in the 5' to 3' direction, and cannot initiate synthesis on a bare DNA strand.
- This leads to a shortening of the lagging strand in each round of replication, potentially losing genetic information.
- This can be addressed by telomerase, an enzyme that maintains telomere length by extending the 3' overhang.
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Description
Explore the five main DNA polymerases and their roles in DNA replication and repair. Understand the specific functions of DNA Pol I and III, including the unique subunit compositions of DNA Pol III involved in synthesizing and proofreading DNA. Test your knowledge on the mechanisms and importance of these essential enzymes in E. coli.