Biochem Lecture 7

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Questions and Answers

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?

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?

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?

Slides backward into the exonuclease site for correction Signup and view all the answers

What inhibits the translocation of DNA polymerase I during proofreading?

Mismatched base pair Signup and view all the answers

Why is the proofreading activity of Pol I essential in DNA replication?

To improve the accuracy of polymerization Signup and view all the answers

How does the circular clamp formed by the two β subunits of E.coli polymerase III increase processivity?

By preventing dissociation from DNA Signup and view all the answers

What is the role of DNA ligases in the replication process?

Seal nicks in the DNA Signup and view all the answers

Which protein in E.coli replisome is responsible for primer removal?

DNA Polymerase I or III Signup and view all the answers

What do DNA Gyrases (DNA Topoisomerases II) do in DNA replication?

Relieve stress caused by unwinding DNA Signup and view all the answers

Which enzyme is responsible for making RNA primers in E.coli replication?

Primases Signup and view all the answers

Which protein in E.coli is responsible for recognizing the oriC sequence and opening the duplex at specific sites in the origin?

DnaA protein Signup and view all the answers

What is the function of the DnaB protein in E.coli?

Unwinds DNA Signup and view all the answers

Which protein in E.coli is required for the binding of DnaB at the origin?

DnaC protein Signup and view all the answers

What is the role of the FIS protein in E.coli?

Stimulates initiation Signup and view all the answers

Which protein in E.coli binds to single-stranded DNA?

Single-stranded DNA-binding protein (SSB) Signup and view all the answers

What is the role of the Mg2+ ion in facilitating DNA polymerase catalyzed reactions?

Stabilizing the structure of the pentacovalent transition state Signup and view all the answers

What is an important feature of a primer used in DNA polymerase reactions?

Contains a free 3’-OH group to initiate the reaction Signup and view all the answers

What is the function of the insertion site in DNA polymerase?

Binding of the incoming nucleotide Signup and view all the answers

Which part of a DNA polymerase resembles a human hand that wraps around the active site?

Core region Signup and view all the answers

What is the primary function of the post-insertion site in DNA polymerase?

Hold the newly made base pair before migration Signup and view all the answers

Why is processivity an important feature of DNA polymerase?

To add multiple dNTPs before dissociating from the template Signup and view all the answers

What leads to denaturation of nearby DUE sites during initiation of replication in E.coli?

Binding of DnaA proteins at R and I sites Signup and view all the answers

What facilitates the formation of the helical DnaA complex during replication initiation in E.coli?

HU, IHF, and FIS proteins Signup and view all the answers

What is the consequence of DnaA proteins binding at R and I sites during replication initiation in E.coli?

Denaturation of nearby DUE sites Signup and view all the answers

What is the role of DNA polymerase I in the replication process?

Removing DNA primers and replacing them with DNA Signup and view all the answers

Which protein serves as a trap for the replication fork during termination of replication in E. coli?

Tus proteins Signup and view all the answers

What is the function of Primase in DNA replication?

Adding primers periodically Signup and view all the answers

How do the Ter sequences contribute to the termination of DNA replication in E. coli?

Function as a trap that replication forks cannot leave Signup and view all the answers

What is the significance of the 20-bp Ter sequences in E. coli replication termination?

Cause replication forks to stop at specific regions Signup and view all the answers

What is the significance of DNA Polymerase I having a low processivity?

Its primary function is in clean-uo Signup and view all the answers

What is the primary function of DNA Polymerase III in E. coli replication?

Principal replication polymerase Signup and view all the answers

In E. coli, which DNA polymerase is involved in DNA repair processes along with DNA Polymerases II and IV?

DNA Polymerase V Signup and view all the answers

What is a distinct domain that comprises the 5' to 3' polymerase activity and the 3' to 5' proofreading exonuclease activity in E. coli?

Klenow fragment Signup and view all the answers

What activity does the 5' to 3’ exonuclease domain perform in E. coli DNA polymerase I?

Nick translation activity Signup and view all the answers

Which enzyme is responsible for sealing the nicks that remain after DNA polymerase I dissociates?

DNA ligase Signup and view all the answers

How does DNA polymerase III increase processivity during DNA replication in E. coli?

By forming a sliding clamp to prevent dissociation Signup and view all the answers

Which subunits make up the core domains of DNA polymerase III in E. coli?

Alpha, epsilon, theta Signup and view all the answers

What is the function of DNA Gyrases (DNA Topoisomerases II) in DNA replication in E.coli?

Relieve the stress caused by unwinding Signup and view all the answers

Which protein in the E. coli replisome is involved in stabilizing separated DNA strands?

DNA-binding proteins Signup and view all the answers

What is the primary function of the clamp-loader complex in DNA replication in E. coli?

Increase processivity of DNA polymerase III Signup and view all the answers

During initiation of replication in E. coli, what is the role of DNA ligases?

Seal nicks in the DNA backbone Signup and view all the answers

What is the key step in the initiation of replication in eukaryotes?

Loading of the MCM helicase complex onto the DNA Signup and view all the answers

What is the primary role of DNA topoisomerase IV in E.coli replication?

Breaking both DNA strands of one chromosome to allow another chromosome to pass through Signup and view all the answers

Which DNA polymerase in eukaryotes is used for DNA repair and has mitochondrial DNA replication activity?

DNA Pol gamma Signup and view all the answers

What is the function of the Autonomously Replicating Sequences (ARS) in yeast replication?

Serving as origins for DNA replication Signup and view all the answers

What is the approximate synthesis rate of nucleotides per second during eukaryotic replication compared to E. coli?

~50 nucleotides/sec (1/20th the rate seen in E. coli) Signup and view all the answers

What is the role of CDC6 and CDT1 proteins in eukaryotic replication initiation?

Promoting loading of the replicative helicase MCM2-7 Signup and view all the answers

Which protein in eukaryotes functions as a Single-Strand Binding Protein similar to E. coli SSB?

RPA Signup and view all the answers

What facilitates the synthesis of telomeres found at the end of linear chromosomes in eukaryotes?

Telomerase Signup and view all the answers

What facilitates the assembly of a pre-replicative complex at a eukaryotic origin?

Binding of AAA+ ATPases like ORC, CDC6, and CDT1 to the origin Signup and view all the answers

What is the role of DNA polymerase in DNA replication?

Synthesizing a new DNA strand using a template strand Signup and view all the answers

Which ions are involved in making the 3’-OH group a more powerful nucleophile during DNA replication?

Mg2+ ions Signup and view all the answers

What happens during the elongation of a DNA chain in DNA replication?

The DNA chain is lengthened using dNTPs Signup and view all the answers

Which enzymes in E. coli have 3' to 5' exonuclease activity?

All of the above Signup and view all the answers

Only DNA Pol I has 5' to 3' exonuclease activity

True Signup and view all the answers

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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