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33 Cards in this Set

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DNA Replication is conservative or semiconservative? What's this mean?
Semiconservative: means one of chains in "new DNA" helix was the original template
What is the basic enzyme involved in DNA replication? What are 3 of its important characteristics?
DNA Polymerase.
1. unable to unwind helix
2. unable to initiate transcription on new strand; it can only add nucleotides to preexisting chain
3. Directs chain growth only in 5-3 direction
What are the 3 basic "stages" of Replication
1. Initiation
2. Elongation
3.Polymerase Editing
In E.coli, how many origins are there? What are/is their name(s)?
1 origin; a segment of DNA that is 245 nucleotides long and called Ori-C (from Origin E. Coli)
Within the Ori-C sequence, there are two important sequences. What are they?
1. 9-mers: nine base pair sequence tt is repeated 4 times
2. 13-mers: three repeats of 13 base pair sequence. Ts AT rich region is easy to unzip
What are the 5 main genes and protein involved in initiation phase of transcription?
1. Dna A (protein)
2. Dna B (protein)
3. Dna C (protein)
4. Ssb (protein)
5. Dna G (Gene)
What is the function of Dna A protein?
It forms a multimeric complex tt binds to the 9-mers. Ts complex uses ATP to distort the DNA chain, melting the helix primarily at the weak 13-mer region
What is the function of Dna B and Dna C?
Dna C is a carrier protein tt delivers Dna B to the origin region. Dna B is a helicase. It moves along the helix using ATP to separate the two strands
What is the function of Dna G?
It produces Primase enzymes that synthesizes RNA primer. In E.coli, the helicase and primase (Dna B & G) bind tgtr forming the "Primosome". It's used in both initiation and elongation.
What is the function of Ssb?
Multiple copies of a "single strand binding protein (Ssb) keep the unwound DNA in an unpaired configuration to be read and dupilicated.
Describe the transcription of the lagging strand
-As the helicase moves forward it exposes new regions of DNA.
- The primase tn lays down a new primer
-DNA polymerase tn elongates the primer 5'-3' until it reaches the previous primer.
-Polymerase I replaces the RNA primer and DNA Ligase bonds the segments tgtr.
What are the 3 DNA Polymerases in E. coli?
1. Pol I
2. Pol II
3. Pol III
What does Pol I do?
Two functions;
a. 5'-3' exonuclease
b. 5'-3' polymerase
This allows it to remove and replace the RNA primer nucleotides on the lagging strand
What does Pol II do?
It's not used in replication. It is used in repair of damaged DNA.
What is the primary replication enzyme (specifically)?
Pol III; functioning on both leading and lagging strand.
*If mutated, cell will die.
What is the primary function of Pol III?
Synthesizes new DNA complementary to template strands while simu7ltaneously displacing the bound Ssb proteins.
What is the "Core Polymerase" made up of?
The α (polymerase proper),
ε (3'-5' exonuclease [editor]), and θ subunits constitute the "core polymerase"
WHat dimerizes the core polymerase?
τ subunit
What does the ß subunit do?
provides a clamp mechanism tt holds the core polymerase on the DNA
What is the "γ complex" and what does it do?
It is γσσ'χψ subunits function together to form the "γ complex". It loads the ß clamp and trfor the core polymerase onto the DNA
What does the core polymerase do?
It compares a free nucleotide to the next requied base and forms the phosphodiester bond wn the right base pair is recognized.
What does the ß clamp do?
Holds the core polymerase onto the DNA strand because the core tends to fall off after 10-15 linkages hv been made
What happens when the core polymerase stalls?
The ß clamp releases the core fm the DNA because the core came upon the previous primer. This allows the core to recycle to a new primer
What is the basic error rate for the synthetic rxn? How can this be reduced and wt's the reduced #?
1 base for every 10^3-10^4 polymerized. This is a high rate but Pol III has both proofreading and exonuclease activity and can "backup" over improper bases. This makes the error only 1 in 10^6.
In eukaryotes, what is the equivilant to each of the following: Dna A, Dna B, Ssb, Pol III, Combo of pimase, Pol III and Pol I?
-Dna A = ORC (origin recognition complex)
-Dna B = MCM
-Ssb = RPA (replication protein A)
-Pol III ~ Pol σ: it however works almost exclusively on leading strand
-Combo of pimase, Pol III and Pol I= Pol α combines w/eukaryotic primase to work on lagging strand
What is the solution in eukaryotes to the problem of shortening DNA strands after replication?
Telomeres: an enzyme called Telomerase acts as a reverse transcriptase, elongating the 3' end of the template DNA in the 5'-3' direction, directing short segments of RNA to be added to the end. The telomere segment can act as a template to replicate the final segment of the lagging strand and then fold over to protect DNA fm degredation damage
Eukaryotic chromosomal DNA has multiple origins of replication. What is the name for each segment of DNA replicated fm an individual origin?
Replicon
What is a Replication Unit?
Those replicons w/sufficiently similar regulatory sequences tt ty r replicated together
What is the pattern to the sequence of activation of replication units?
1. Replication units containing "housekeeping" genes are replicated early in S-Phase
2. Active euchromatin regions are replicated next
3. Inactive euchromatin is tn replicated
4. Heterochromatin regions of DNA are replicated
*This increases # of most important genes early in cell cycle
During DNA repair, how can the mismatch proofreading system distiguish between the old and new DNA strand?
By the degree of methylation: newly synthesized DNA are free of methyl groups
What follows behind the replication fork to insure proper base pair matching? What degree of fidelity does it have?
The Mismatch Proofreading system. The removal and replacement of misadded bases can raise the overall fidelity of the replication/proofreading process to only 1 in 10^9-10^10 bases.
What are the 3 steps involved in the repair mechanism of damaged DNA?
1. Recognition and removal of damaged nucleotides by a specific "DNA Repair Nuclease" leaving a gap in the effected strand of the DNA helix
2. A DNA repair Polymerase binds the free 3OH and fills the gap
3. DNA Ligase closes the remaining "nick"
What are the main DNA repair nucleases?
1. AP nucleases for depurination damage
2. DNA glycolases for base excision repair
3. A combo of 3' and 5' nucleases and helicases for nucleotide excision repair