Podcast
Questions and Answers
What is the purpose of the 3' to 5'-exonuclease activity in DNA polymerases?
What is the purpose of the 3' to 5'-exonuclease activity in DNA polymerases?
- Proofreading synthesis for mismatched base pairs (correct)
- Facilitating DNA replication initiation
- Enhancing DNA polymerase binding to the template
- Assisting in DNA unwinding
What happens when there is a mismatched base pair during DNA synthesis?
What happens when there is a mismatched base pair during DNA synthesis?
- The enzyme skips the mismatch and continues synthesis
- The DNA polymerase detaches from the template
- Enzyme translocation to the next site is inhibited (correct)
- The DNA undergoes unwinding
Where is the exonuclease activity located in comparison to the polymerase activity in DNA polymerase I?
Where is the exonuclease activity located in comparison to the polymerase activity in DNA polymerase I?
- Separate from the polymerase activity
- Behind the polymerase activity (correct)
- Ahead of the polymerase activity
- Adjacent to the polymerase activity
How does DNA polymerase I correct a mismatched base pair?
How does DNA polymerase I correct a mismatched base pair?
What inhibits the translocation of DNA polymerase I during proofreading?
What inhibits the translocation of DNA polymerase I during proofreading?
Why is the proofreading activity of Pol I essential in DNA replication?
Why is the proofreading activity of Pol I essential in DNA replication?
How does the circular clamp formed by the two β subunits of E.coli polymerase III increase processivity?
How does the circular clamp formed by the two β subunits of E.coli polymerase III increase processivity?
What is the role of DNA ligases in the replication process?
What is the role of DNA ligases in the replication process?
Which protein in E.coli replisome is responsible for primer removal?
Which protein in E.coli replisome is responsible for primer removal?
What do DNA Gyrases (DNA Topoisomerases II) do in DNA replication?
What do DNA Gyrases (DNA Topoisomerases II) do in DNA replication?
Which enzyme is responsible for making RNA primers in E.coli replication?
Which enzyme is responsible for making RNA primers in E.coli replication?
Which protein in E.coli is responsible for recognizing the oriC sequence and opening the duplex at specific sites in the origin?
Which protein in E.coli is responsible for recognizing the oriC sequence and opening the duplex at specific sites in the origin?
What is the function of the DnaB protein in E.coli?
What is the function of the DnaB protein in E.coli?
Which protein in E.coli is required for the binding of DnaB at the origin?
Which protein in E.coli is required for the binding of DnaB at the origin?
What is the role of the FIS protein in E.coli?
What is the role of the FIS protein in E.coli?
Which protein in E.coli binds to single-stranded DNA?
Which protein in E.coli binds to single-stranded DNA?
What is the role of the Mg2+ ion in facilitating DNA polymerase catalyzed reactions?
What is the role of the Mg2+ ion in facilitating DNA polymerase catalyzed reactions?
What is an important feature of a primer used in DNA polymerase reactions?
What is an important feature of a primer used in DNA polymerase reactions?
What is the function of the insertion site in DNA polymerase?
What is the function of the insertion site in DNA polymerase?
Which part of a DNA polymerase resembles a human hand that wraps around the active site?
Which part of a DNA polymerase resembles a human hand that wraps around the active site?
What is the primary function of the post-insertion site in DNA polymerase?
What is the primary function of the post-insertion site in DNA polymerase?
Why is processivity an important feature of DNA polymerase?
Why is processivity an important feature of DNA polymerase?
What leads to denaturation of nearby DUE sites during initiation of replication in E.coli?
What leads to denaturation of nearby DUE sites during initiation of replication in E.coli?
What facilitates the formation of the helical DnaA complex during replication initiation in E.coli?
What facilitates the formation of the helical DnaA complex during replication initiation in E.coli?
What is the consequence of DnaA proteins binding at R and I sites during replication initiation in E.coli?
What is the consequence of DnaA proteins binding at R and I sites during replication initiation in E.coli?
What is the role of DNA polymerase I in the replication process?
What is the role of DNA polymerase I in the replication process?
Which protein serves as a trap for the replication fork during termination of replication in E. coli?
Which protein serves as a trap for the replication fork during termination of replication in E. coli?
What is the function of Primase in DNA replication?
What is the function of Primase in DNA replication?
How do the Ter sequences contribute to the termination of DNA replication in E. coli?
How do the Ter sequences contribute to the termination of DNA replication in E. coli?
What is the significance of the 20-bp Ter sequences in E. coli replication termination?
What is the significance of the 20-bp Ter sequences in E. coli replication termination?
What is the significance of DNA Polymerase I having a low processivity?
What is the significance of DNA Polymerase I having a low processivity?
What is the primary function of DNA Polymerase III in E. coli replication?
What is the primary function of DNA Polymerase III in E. coli replication?
In E. coli, which DNA polymerase is involved in DNA repair processes along with DNA Polymerases II and IV?
In E. coli, which DNA polymerase is involved in DNA repair processes along with DNA Polymerases II and IV?
What is a distinct domain that comprises the 5' to 3' polymerase activity and the 3' to 5' proofreading exonuclease activity in E. coli?
What is a distinct domain that comprises the 5' to 3' polymerase activity and the 3' to 5' proofreading exonuclease activity in E. coli?
What activity does the 5' to 3’ exonuclease domain perform in E. coli DNA polymerase I?
What activity does the 5' to 3’ exonuclease domain perform in E. coli DNA polymerase I?
Which enzyme is responsible for sealing the nicks that remain after DNA polymerase I dissociates?
Which enzyme is responsible for sealing the nicks that remain after DNA polymerase I dissociates?
How does DNA polymerase III increase processivity during DNA replication in E. coli?
How does DNA polymerase III increase processivity during DNA replication in E. coli?
Which subunits make up the core domains of DNA polymerase III in E. coli?
Which subunits make up the core domains of DNA polymerase III in E. coli?
What is the function of DNA Gyrases (DNA Topoisomerases II) in DNA replication in E.coli?
What is the function of DNA Gyrases (DNA Topoisomerases II) in DNA replication in E.coli?
Which protein in the E. coli replisome is involved in stabilizing separated DNA strands?
Which protein in the E. coli replisome is involved in stabilizing separated DNA strands?
What is the primary function of the clamp-loader complex in DNA replication in E. coli?
What is the primary function of the clamp-loader complex in DNA replication in E. coli?
During initiation of replication in E. coli, what is the role of DNA ligases?
During initiation of replication in E. coli, what is the role of DNA ligases?
What is the key step in the initiation of replication in eukaryotes?
What is the key step in the initiation of replication in eukaryotes?
What is the primary role of DNA topoisomerase IV in E.coli replication?
What is the primary role of DNA topoisomerase IV in E.coli replication?
Which DNA polymerase in eukaryotes is used for DNA repair and has mitochondrial DNA replication activity?
Which DNA polymerase in eukaryotes is used for DNA repair and has mitochondrial DNA replication activity?
What is the function of the Autonomously Replicating Sequences (ARS) in yeast replication?
What is the function of the Autonomously Replicating Sequences (ARS) in yeast replication?
What is the approximate synthesis rate of nucleotides per second during eukaryotic replication compared to E. coli?
What is the approximate synthesis rate of nucleotides per second during eukaryotic replication compared to E. coli?
What is the role of CDC6 and CDT1 proteins in eukaryotic replication initiation?
What is the role of CDC6 and CDT1 proteins in eukaryotic replication initiation?
Which protein in eukaryotes functions as a Single-Strand Binding Protein similar to E. coli SSB?
Which protein in eukaryotes functions as a Single-Strand Binding Protein similar to E. coli SSB?
What facilitates the synthesis of telomeres found at the end of linear chromosomes in eukaryotes?
What facilitates the synthesis of telomeres found at the end of linear chromosomes in eukaryotes?
What facilitates the assembly of a pre-replicative complex at a eukaryotic origin?
What facilitates the assembly of a pre-replicative complex at a eukaryotic origin?
What is the role of DNA polymerase in DNA replication?
What is the role of DNA polymerase in DNA replication?
Which ions are involved in making the 3’-OH group a more powerful nucleophile during DNA replication?
Which ions are involved in making the 3’-OH group a more powerful nucleophile during DNA replication?
What happens during the elongation of a DNA chain in DNA replication?
What happens during the elongation of a DNA chain in DNA replication?
Which enzymes in E. coli have 3' to 5' exonuclease activity?
Which enzymes in E. coli have 3' to 5' exonuclease activity?
Only DNA Pol I has 5' to 3' exonuclease activity
Only DNA Pol I has 5' to 3' exonuclease activity
Study Notes
DNA Replication
Initiation of Replication in E. coli
- Initiation occurs at a specific region called oriC
- Eight DnaA proteins bind to R and I sites in the origin
- DNA wraps around the DnaA complex, forming a right-handed helical structure
- The A=T-rich DUE region is denatured due to strain imparted by adjacent DnaA binding
DnaB Helicase
- DnaB Helicase is a hexamer ring structure that unwinds DNA strands
- DnaC protein helps in the binding of DnaB Helicase to the origin
- DnaB Helicase migrates along ssDNA, separating the strands in preparation for DNA synthesis
Elongation
- DNA Polymerase III is the primary enzyme for DNA synthesis
- It has a processivity of over 500,000 nucleotides
- The enzyme forms a circular clamp around the DNA, preventing its dissociation
Primer Synthesis
- A primer is a short RNA or DNA strand complementary to the template
- It contains a free 3'-OH group to begin DNA synthesis
- RNA primers are synthesized by the primase enzyme
DNA Polymerase I
- Has a 5' to 3' exonuclease activity for nick translation
- Removes RNA primers and replaces them with DNA
- Has a slow rate of synthesis (10-20 nucleotides/sec) and low processivity (3-200 nucleotides)
Proofreading and Error Correction
- Almost all DNA polymerases have a 3' to 5' exonuclease activity for proofreading
- Errors during synthesis are corrected by the 3' to 5' exonuclease activity
- This improves the accuracy of polymerization by 10^2 to 10^3 fold
Proteins Involved in Replication
- Over 20 enzymes and proteins are involved in DNA replication
- These include DNA-binding proteins, helicases, primases, DNA polymerases, and DNA ligases
Replisome
- A set of proteins involved in replication, including DNA-binding proteins, helicases, primases, DNA polymerases, and DNA ligases
- The replisome includes DNA polymerase III, which is the primary enzyme for DNA synthesis
Replication in Eukaryotes
- More complex than in bacteria
- Yeast has ~400 well-defined origins (ARS or replicators)
- Regulation is due to cyclin proteins and cyclin-dependent kinases (CDKs)
Initiation of Replication in Eukaryotes
- The Origin Recognition Complex (ORC) protein binds to the replication origin
- The helicase is a hexamer of Mini-Chromosome Maintenance proteins (MCM2-7)
- Loading of the MCM helicase complex onto the DNA forms the pre-replicative complex### Nick Translation and DNA Repair
- Nick translation is a process in which a break or nick in the DNA is effectively moved along with the enzyme DNA polymerase I.
- This process has a role in DNA repair and in the removal of RNA primers during replication.
DNA Polymerase III
- DNA Polymerase III is a complex structure with 10 types of subunits.
- It has three core domains made of a, e, and q subunits.
- The core domains are linked by the clamp-loader complex τ3γδδ’.
- Each core domain interacts with a dimer of b subunits that increases the processivity of the complex.
- The complex forms a sliding clamp that prevents dissociation.
- The processivity of DNA Pol III is >500,000 bp.
Architecture of DNA Polymerase III
- The circular clamp (2 subunits) surrounds the DNA.
- The enzyme has three core domains composed of subunits α, ε, and θ.
- These core domains are linked by a five-subunit clamp-loading complex (γ complex) with the composition τ3γδδ’.
DNA Metabolism
- DNA metabolism consists of a set of tightly regulated processes that achieve DNA replication, repair, and recombination.
- These processes are necessary for the stable storage of genetic information.
DNA Replication in Bacteria
- DNA replication in bacteria involves DNA elongation chemistry.
- Elongation of a DNA chain involves the reaction: (dNMP)n + dNTP → (dNMP)n+1 + PPi.
- The parental DNA strand serves as a template, and nucleoside triphosphates serve as substrates in strand synthesis.
- The nucleophilic OH group at the 3’ end of the growing chain attacks the α-phosphate of the incoming trinucleotide.
- Two Mg2+ ions are required for the reaction.
- Pyrophosphate is a good leaving group.
Mechanism of Action of DNA Polymerases
- DNA polymerase requires a template and a primer (and dNTPs) to function.
- The catalytic mechanism involves two Mg2+ ions, coordinated to the phosphate groups of the incoming nucleotide triphosphate, the 3’-hydroxyl group that will act as a nucleophile, and three Asp residues.
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