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Midterm 5
Lecture 34: DNA replication
Questions:
What does DNA replication involve?
Proteins that involve 7 enzymatic activities
What is polymerase I?
Primer removal and DNA repair, 3 to 5, and 5 to 3 exonucleases
What is polymerase III?
Replication, 3 to 5 exonucleases
What is exonuclease activity?
Proofreading
What happens 5’ to 3’?
Polymerase activity
What is important about polymerases?
Processive enzymes - make long stretches without dissociation
Possess high fidelity - one mistake every 10^9 and 10^10
What is required for replicating DNA?
Template strand - 3’ to 5’
Primer with free OH
dATP, dGTP, dCTP, dTTP, and Mg2+
What is formed when the free hydroxyl attacks the triphosphate nucleotide?
Phosphodiester bond
Midterm 5 1
, What does polymerase III consist of?
2 copies of each core enzyme(dimer), exonuclease, clamp, and other subunits
What does dNTP binding do?
Induces conformational change at active site to form a tight pocket that fits
certain bases
What is editing of nuceotides promoted by?
Instability of a mis matched nucleotide in the active site, causing migration of
base to exonuclease active site
What are helicases?
Uses the energy of ATP hydrolysis to separate strands of double helix to make
DNA available for DNA polymerase → separate DNA strands
What do helicases contain?
6 subunits that form a ring like structure around DNA
How does ATP hydrolysis by helicase relate to DNA?
Causes conformational change that pulls DNA through helicase center
What disease can occur with a defect in helicase?
Werner
What is topoisomerase?
Relieves the strain of the over winding, uses ATP hydrolysis to relax DNA
structures, I(single strand break) and II(double strand break)
What is DNA ligase?
Joins the free OH of one base with the phosphate of another base,
phosphodiester bond, seals nicks
What is the start of DNA replication?
Origin of replication
What is DNA A and DNA B?
A identifies origin, binds 4 sites in element, and oligomerizes, wrapping
origin DNA around itself
Midterm 5 2
, B unwinds DNA using energy of ATP, helicase
What prevents reannealing at the origin and what does it form?
Single strand binding(SSB) proteins; prepriming complex
What is primase?
Synthesizes RNA to establish primer to start replication
What is the replication fork?
Site of DNA synthesis, leading strand and lagging strand
What is Okazaki fragment?
Lagging stand is made up of these pieces to synthesize in 5’ to 3’
What removes the primer on the Okazaki fragments?
5’ to 3’ exonuclease activity from DNA polymerase I(replaces RNA with DNA),
DNA ligase fills in the gaps between the fragments
What allows coordination of leading and lagging strand?
Dimerization of polymerase
What are differences with E.coli and human replication?
E.coli - small genome, single circular chromosome, one ori, DNA poly I/III,
primase, and SSB
Human - large genome, 23 pairs of linear chromosome, lots of oris, DNA
poly alpha and epsilon, and RPA
Lecture 35: DNA Repair and Recombination
Questions:
What prevents DNA from being lost when primer gets removed on linear DNA?
Telomerase - adds hundreds of G rich repeats onto the end of chromosomes
What are telomeres?
Ends of chromosomes
What does telomerase do?
Midterm 5 3
Midterm 5
Lecture 34: DNA replication
Questions:
What does DNA replication involve?
Proteins that involve 7 enzymatic activities
What is polymerase I?
Primer removal and DNA repair, 3 to 5, and 5 to 3 exonucleases
What is polymerase III?
Replication, 3 to 5 exonucleases
What is exonuclease activity?
Proofreading
What happens 5’ to 3’?
Polymerase activity
What is important about polymerases?
Processive enzymes - make long stretches without dissociation
Possess high fidelity - one mistake every 10^9 and 10^10
What is required for replicating DNA?
Template strand - 3’ to 5’
Primer with free OH
dATP, dGTP, dCTP, dTTP, and Mg2+
What is formed when the free hydroxyl attacks the triphosphate nucleotide?
Phosphodiester bond
Midterm 5 1
, What does polymerase III consist of?
2 copies of each core enzyme(dimer), exonuclease, clamp, and other subunits
What does dNTP binding do?
Induces conformational change at active site to form a tight pocket that fits
certain bases
What is editing of nuceotides promoted by?
Instability of a mis matched nucleotide in the active site, causing migration of
base to exonuclease active site
What are helicases?
Uses the energy of ATP hydrolysis to separate strands of double helix to make
DNA available for DNA polymerase → separate DNA strands
What do helicases contain?
6 subunits that form a ring like structure around DNA
How does ATP hydrolysis by helicase relate to DNA?
Causes conformational change that pulls DNA through helicase center
What disease can occur with a defect in helicase?
Werner
What is topoisomerase?
Relieves the strain of the over winding, uses ATP hydrolysis to relax DNA
structures, I(single strand break) and II(double strand break)
What is DNA ligase?
Joins the free OH of one base with the phosphate of another base,
phosphodiester bond, seals nicks
What is the start of DNA replication?
Origin of replication
What is DNA A and DNA B?
A identifies origin, binds 4 sites in element, and oligomerizes, wrapping
origin DNA around itself
Midterm 5 2
, B unwinds DNA using energy of ATP, helicase
What prevents reannealing at the origin and what does it form?
Single strand binding(SSB) proteins; prepriming complex
What is primase?
Synthesizes RNA to establish primer to start replication
What is the replication fork?
Site of DNA synthesis, leading strand and lagging strand
What is Okazaki fragment?
Lagging stand is made up of these pieces to synthesize in 5’ to 3’
What removes the primer on the Okazaki fragments?
5’ to 3’ exonuclease activity from DNA polymerase I(replaces RNA with DNA),
DNA ligase fills in the gaps between the fragments
What allows coordination of leading and lagging strand?
Dimerization of polymerase
What are differences with E.coli and human replication?
E.coli - small genome, single circular chromosome, one ori, DNA poly I/III,
primase, and SSB
Human - large genome, 23 pairs of linear chromosome, lots of oris, DNA
poly alpha and epsilon, and RPA
Lecture 35: DNA Repair and Recombination
Questions:
What prevents DNA from being lost when primer gets removed on linear DNA?
Telomerase - adds hundreds of G rich repeats onto the end of chromosomes
What are telomeres?
Ends of chromosomes
What does telomerase do?
Midterm 5 3
