Download DNA Replication and DNA Repair Study Guide Focus on the

DNA Replication and DNA Repair Study Guide
Focus on the following.
1. Meselon and Stahl experiments showing semiconservatism.
a. N-15 labeled DNA is heavier than N-14 DNA.
b. N-15 bacteria placed in medium (which only contains N-14 DNA).
c. CsCl density gradient used to note difference between N-15 (lower in tube) and N14(higher in tube) DNA.
i. Transfer of N-15 DNA to N-14 media produces an intermediate layer of ½ N-14
and ½ N-15.
d. Therefore, each new DNA molecule consists of one old strand (N-14) and one new
strand (N-15).
i. Semiconservatism.
2. Origin and Direction of Replicatin (Replication Fork
a. Origin of Replication
i. Beginning point of replication
ii. Prokaryotes (bacteria)- 1 origin of replication
iii. Eukaryotes- 1 to 2000 origins of replication per chromosome
b. Direction- two forks proceed in opposite directions
c. Forks
i. Replication sites
ii. Proceed in one direction (one for each direction)
iii. Replication can only proceed in ϱ͛ƚŽϯ͛ĚŝƌĞĐƚŝŽŶ
3. Role of RNA primers
a. Short strand of RNA that is complementary to the template strand and contains a free
OH group for attachment
i. Can be 1 to 60 bases long.
b. Leading Strand- replication is continuous
i. DNA synthesized uninterrupted from 1 RNA primer
c. Lagging Strand- replication occurs from multiple primers
i. Synthesis is discontinuous
ii. Short strand eventually joined together
1. ƌƌĂŶŐĞĚϯ͛ƚŽϱ͛;ďƵƚƉƌŽĐĞĞĚƐϱ͛ƚŽϯ͛)
d. RNA Primase (RNA Polymerase in DNA)
i. Lays down RNA primer during DNA replication
ii. DNA polymerase needs free OH to start (which RNA primer has).
4. Okazaki Fragments. Describe Formation
a. Short sections of RNA primer and DNA on a lagging strand.
b. Discontinuous.
i. WƌŽĐĞĞĚƐŝŶϱ͛ƚŽϯ͛ĚŝƌĞĐƚŝŽŶ;ŽŶϯ͛ƚŽϱ͛ƐƚƌĂŶĚͿ
ii. Very fragmented
5. Role of helicases, topoisomerases,and single stranded binding proteins
a. Helicases
i. Unwind DNA
ii. Require ATP
1. Hydrolyzed in order to function
iii. Supercoiling-increased or decreased torsional strain put on molecule
1. Caused by the formation of multiple closed loops.
b. Topoisomerases
i. Relieve tension ahead of the replication fork.
1. Type I: DNA topisomerase
a. Nicks (breaks) one strand
b. Passes other strand through break
c. NO ATP!!!
2. TyperII: Topoisomerases
a. Enzyme-bridged break
b. Another duplex passes through
c. Takes out 2 supercoils at once
d. REQUIRES ATP!!!
3. Type II: Topoisomerase II
a. TARGET FOR MANY MEDICINES!!!!
c. Single Strand Binding Proteins (SSB)
i. Keep strands as single entities.
ii. Reusable!
iii. 1000x greater affinity for single strands
iv. Protects against nucleases.
6. Activity of DNA polymerase III
a. Read over how it was discovered.
b. Extends growing DNA
i. ^ƚŽƉƐĂƚϱ͛ŶƵĐůĞŽƚŝĚĞŽĨZEƉƌŝŵĞƌ
ii. CAN GO NO FURTHER!!!
1. Think of the nun ĨƌŽŵ͞dŚĞĂsŝŶĐŝŽĚĞ͊͟
iii. Once bound to template, it never dissociates.
iv. 2 at each replication fork (4 in the bubble)
7. Activities of DNA polymerase I, including proof reading and error repair
a. 3 Functions
i. džŽŶƵĐůĞĂƐĞϱ͛ƚŽϯ͛ĂĐƚŝǀŝƚLJ
1. Cuts out primer
ii. Fills in spot with dNTP that matches exposed template
iii. džŽŶƵĐůĞĂƐĞϯ͛ƚŽϱ͛ĂĐƚŝǀŝƚLJ
1. Proofreads
2. ůĞĂǀĞƐϯ͛ƚĞƌŵŝŶĂůĞŶĚƐ͘
iv. DNA Ligase (extra)
1. Seals in nicks between fragments
a. Uses DNA
b. Also links with phosphodiester bond
b. ATP necessary for replication and repair!!!!
8. Mechanism of Replisome
a. Helicase separates into leading and lagging strands
i. SSBS maintain stability of single strands
ii. Primase lays down RNA primers for DNA polymerase III
iii. DNA polymerase III lays down nucleotides for leading and lagging strand
iv. DNA polymerase I replaces RNA primers with DNA
v. DNA ligase links fragments in lagging strand
9. Eukaryotic DNA Polymerases
a. Alpha
i. Location-nucleus
ii. Function-synthesis and priming of lagging strand
1. RNA primers and DNA synthesis
b. Beta
i. Location-nucleus
ii. Function-DNA repair
c. Gamma
i. Location-Mitochondria
ii. Function-replicates mit. DNA
d. Delta
i. Location- nucleus
ii. Function-synthesis of leading strand (DNA synthesis)
10. Dissociation of histones from DNA and cooperative mechanism of nucleosome synthesis
a. Histones
i. At initiation sites
ii. Weakened by acetylation and phosphorylation
iii. Allows replication to begin
b. Nucleosome
i. Increased synthesis of histones required for new nucleosomes.
ii. Occurs with DNA replication
iii. Distributed only to 1 daughter strand
11. DNA damage: Thymine Dimers and Deamination
a. Physical Agentsi. High temperatures
ii. Radiation at different wavelengths
1. 240-300nm most effective
iii. X-rays
b. Chemical Agents
i. Methylating agents
ii. Nitrous acid
iii. Nitrosamines
iv. Acridine dyes, etc
c. Types of Damage
i. Thymine Dimer
1. Covalent linkage of two adjacent thymines in the same strand
2. H-bonding disrupted
3. Inhibits replication forks
ii. Deamination
1. Loss of amino group
2. Results in conversion
a. C to U
b. A to hypoxanthine
12. Nucleotide excision repair mechanism
a. Removal of the following
i. Bulky chemical modifications of DNA
ii. Pyrimidine (thymine) dimmers
b. Recognition of problem by protein complex
c. Endonuclease
i. EŝĐŬŝŶŐŽĨEĂƚϱ͛ĂŶĚϯ͛ĞŶĚƐ
d. DNA Polymerase epsilon
i. Fills in gap
e. DNA Ligase
i. Forms phosphodiester bonds.
f.
In summary
i. Endonuclease-cutting
ii. Polymerase-replacement of damaged DNA
iii. Ligase-seals
13. Base Excision Repair: Uracil DNA glycosylase ; SOS Repair
a. Uracil DNA glycosylase
i. Hydrolyzes the bond between uracil and deoxyribose
1. Results in the removal of uracil from DNA
2. Apyrimidine formed
ii. Nicked by endonuclease
iii. Repaired by DNA polymerase and ligase
b. SOS Repair
i. Not very well understood
ii. Found in eukaryotes
iii. Many enzymes are induced in response to high DNA damage
iv. SOS-SAVE OUR SUBUNITS!!!!!
14. Xeroderma pigmentosum and other repair defects, lethality of DNA damage
a. Xeroderma pigementosum
i. Increased sensitivity to light
1. More prone to skin cancer
2. Due to defects in repair of UV problems
ii. Autosomal recessive inheritance
1. Mutation on 1 gene
iii. Defect in excision repair system
1. XP variants
a. Symptoms but normal excision repair system
b. Polymerase problem
b. Other Repair Defects-See chart in notes
c. Lethality of DNA Damage
i. D37 (only 37 % survive)
1. Damaging agent that kills 63% of cells in culture
2. Average Damage per cell
a. Ionizing Radiation- Single strand breaks-1000 per cell
b. Ionizing Radiation- Double strand breaks- 40 per cell
c. Ultraviolet Radiation- Thymine dimmers-400,000 per cell
REMEMBER, THE FORCE WILL BE WITH YOU. ALWAYS!