Term
| What are the 3 basic features of DNA replication? |
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Definition
1. Semiconservative
2. Specific origins of replication
3. Bidirectional |
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Term
| Describe the discovery of the semiconservative process |
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Definition
Messelson and Stahl
-made different densities of DNA using N
Taylor, Woods, Hughes
-radioactive H |
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Term
Describe origins of replication
Prokaryotes |
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Definition
1. OriC site
a. 13bp sequence in 3 consecutive repeats
-AT rich and is the bubble beginning
b. 9bp sequence 4 times interspersed
-binding site for proteins
*single replicon site |
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Term
Describe origins of replication
Eukaryotes |
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Definition
1. Autonomously Replicating Sequences
-about 50 bp
-11bp AT rich core
*multiple sites of replication |
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Term
Describe origins of replication
DNA viruses (SV40) |
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Definition
-double stranded DNA virus
-core origin is 64 bp sequence
-27 bp central site
1. AT rich 2. Binding region 3. random
*perfect palindrome is binding site for proteins |
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Term
| Describe Bidirectional Replication |
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Definition
Schnoss and Inman used bacteriophage lambda
-denatured DNA at AT rich regions
-could then determine it replicated both ways |
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Term
Describe DNA Polymerase I
1. Other name
2. Requirements
3. Enzymatic activities |
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Definition
1. Kornberg's Enzyme
2. a. 5' triphosphates of each DNA nucleoside
b. Mg 2+ ions
c. DNA template strand
d. primer with free 3-OH'
3. 5-->3 polymerase
5-->3 exonuclease
3-->5 exonuclease |
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Term
Describe the following polymerases in E. coli
1. I
2. II
3. III
4. IV
5. V |
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Definition
1. DNA repair
2. DNA repair (lacks 5-->3 exonuclease)
3. complex; only 5-->3 on ssDNA, primary in E. coli
4. DNA repair
5. DNA repair |
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Term
Describe the subunits of DNA Poly III in E. coli
1. alpha
2. beta
3. epsilon
4. theta
5. t |
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Definition
*contains at least 20 proteins = holoenzyme
Alpha: polymerization
Beta: forms a clamp
Epsilon: proofreading
Theta: required for core unit to work
t: binds to another t and holds core together |
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Term
| Describe the players of replication on the lagging strand |
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Definition
5-->3 direction
-Okazaki fragments
-started by RNA primase (provide 3' OH end)
-linked by DNA ligase |
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Term
| Describe the steps of replication on the lagging strand |
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Definition
*takes place on unwound ssDNA
1. initiation by DNA primase
2. 5-->3 synthesis and detachment
3. DNA poly 3 synthesize 5-->3 from free 3' OH ends
4. Simultaneous removal of RNA by 5-->3 exonuclease and then synthesis of missing bases by DNA poly 1
5. Covalent closure by DNA ligase |
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Term
| Describe the unwinding DNA |
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Definition
-in DNA beta form
-must spin at 3000 rpm
1. unzipped by DNA helicase
-changes ATP to ADP
2. will go to hairpin structures without SSB
3. single strand binding proteins prevent it from coming back together and in extended form |
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Term
Describe the following:
1. Topoisomerase 1
2. Topoisomerase 2 |
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Definition
*topoisomerases catalyze breaks in the DNA to relieve supercoils
1. nicks one strand; no ATP
-provides axis of rotation so one strand can rotate independently
2. nicks both strands; uses ATP
ex. DNA gyrase |
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Term
| Describe the differences in Eukaryotic Replication |
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Definition
1. replication limited to S phase
2. multiple replicons per chromosome
3. two or more polymerases at each fork |
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Term
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Definition
complex at the replication fork on lagging strand
1. DNA helicase
2. DNAb and DNAc complex
3. Primase |
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Term
| Describe the two theories of nucleosome assembly during replication |
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Definition
1. H2a and H2b are removed
-only 6 proteins that hold loosely are left
-this allows the DNA to be held loosely enough to replicate
2. All proteins leave and then reattach |
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Term
| What are the three proteins that assist in disassembly and assembly of nucleosomes? |
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Definition
1. Nap-1
-transports histones to the site of synthesis from cytoplasm to nucleus
2. CAF-1
-carries histones to the chromosomal sites
3. PNCA
-histones bind to this
-clamp that tethers DNA poly delta to the template |
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Term
| Describe the main problem with telomere replication on the lagging strand |
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Definition
there is no 3' OH needed for polymerization after the RNA fragment is removed
*the strand will get shorter and shorter after each replication |
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Term
| Describe DNA replication at the telomere |
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Definition
1. telomerase binds to the 5' end of the lagging strand that is being replicated
2. extends the 3' end by carrying RNA bases and binding the complementary bases
-RNA templated DNA synthesis
3. translocates up the 3' strand and continues to extend
-telomerase breaks off
4. completion of DNA replication occurs
-RNA primer is laid down and poly 3 adds bases |
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