13.1 DNA Replication and Repair

Question 1

Watson and Crick Proposal

Answer 1

During rep, the \ unwinds and each of the parental strands serves as templates for synthesis of a new complementary stand

Question 2

3 Alternative Replication Proposals

Answer 2

Semiconservative Replication
Conservative Replication
Dispersive Replication

Question 3

Semiconservative Replication

Answer 3

• Watson and Crick Method
• each daughter duplex has one strand from parent structure
• by 2nd generation, only 2 of the 4 stands have 1 strand from the parental DNA

Question 4

Conservative Replication

Answer 4

• two original strand remain togehter after being a template
• 2 new stands stay together
• 1 daughter duplex has new strands one has parental
• 1 of 4 stands contain 2 strands from original parent

Question 5

Dispersive Replication

Answer 5

• parental strand broken into fragments
• new stand synthesized in short segments
• old fragments and new segments join together to form a complete strand
• 4 of 4 stands have 2 strands with parts of the original parent

Question 6

Meselson-Stahl Experiment

Answer 6

Meselson and Stahl found that DNA employs semi-conservative replication. They put the bacteria in an environment with a Nitrogen isotope. They first used N14. The bacteria then integrated this isotope into their DNA. Later they used an environment that contained N15. They then looked at which of the isotopes the bacterial DNA contained. They found that it contained both isotopes of nitrogen, which implies that conservative replication is not the correct conclusion. By having a closer look they could then also rule out disperse replication. Some helix strands were all N14. Other helix strands from the time in the N15 environment had N15.

Question 7

Semiconservative Replication in Eukaryotes

Answer 7

• experiment where cells transferred from thymidine medium to BRdU and completed 2 rounds of replication.
• resulted in one chromatid of each chromosome contains thymidine

Question 8

Bacterial Chromosome Replication: How and the unwinding problem

Answer 8

• 2 replication forks move in opposite directions from single origin. When replication forks meet at the opposite point on the circle, replication is ruminated and the 2 replicated duplexes detach from one another.
• circle becomes supercoiled easily
• uses DNA gyrase a TopoII removes positive supercoils ahead of DNA polymerase

Question 9

Templates and Nontemplates for Polymerase Activity

Answer 9

In order to be okay to be a template, there must be a template strand to copy, and a primer strand with a 3’ OH on which to add nucleotides. Therefore just a s.s template will not work.

Question 10

Incorporation of Nucleotides

Answer 10

•Enzyme selects nucleotides based on their ability to pair with nucleotide in template strand
•Mg+ ion draws H from OH group
•Creates nucleophilic O atom that attacks α-phosphate of incoming dNTP
•5’ to 3’, aniparellel
lagging and leading strand
leading = continuous
lagging needs to wait for replication fork to open up (Okasaki fragments)
DNA rep needs an RNA primer

Question 11

Evidence of Okazaki Fragments

Answer 11

Okazaki performed a DNA labeling experiment. Bacteria were incubated in 3H-thymidine for a brief period of time and immediately killed. The DNA in the bacteria was mostly small DNA fragments of 1000 to 2000 nucleotides. If bacteria were labeled for a longer period of time the shorter fragments would be transformed into longer fragments. Okazaki reasoned that the shorter fragments were being ligated together.

Question 12

Lagging strand synthesis

Answer 12

1. primer synthesis by primase (contrsuucts a short primer composed of RNA)
2. elongation by DNA polymerase III
3. primer removal and gap filling by DNA polymerase I
4. strand sealed by DNA ligase

likelihood of mistakes is greater for initiation than during elongation

Question 13

Replication Fork

Answer 13

• know how to label the picture
• helices breaks hydrogen bonds and helps unwind at DNA folk and helps unwind to avid supercoiling
• SSB maintains DNA in the s.s. form (keeps DNA from hydrogen bonding back together)
• SSB = single standee binding protein
• primase synthesizes the RNA primers that begin each okazaki fragments

Question 14

Leading and Lagging Strands in E. coli

Answer 14

• DNA polymerases on leading and lagging strands travel together
• Lagging strand template forms loop
• Area that replicated is actually flipped 180º . Picks up a loop, replicates it, lets it go, continues.
• Polymerase releases lagging strand when Okazaki fragment encountered
• DNA polymerase rebinds lagging strand template farther along

Question 15

Beta Clamp

Answer 15

• B clamp, clamps onto the DNA template and helps Pol locate itself onto the DNA strand, serves as a docking protein, also has a clamp loader that has a clamp ready to load onto the DNA. coordinated onto one large complex.
• Polymerase held to DNA by clamp as it moves along the template
• Enzyme disengages from β clamp cycles to a recently assembled clamp waiting at upstream region
• 2 protein subunit clamp structure

Question 16

DNA poly has 3 diffrent activités

Answer 16

• 5’ to 3’ Polymerase (responsible for synthesizing DNA)
• 5’ to 3’ Exonuclease (degrades RNA primers, when has to fill in the GAPs needs to get rid of the primer so chews through the primer and replaces with DNA)
• 3’ to 5’ Exonuclease (proofreading ability of DNA)

Question 17

How proofreading of DNA works

Answer 17

• as it replicated DNA, it can monitor for the correct geometry of the base pairs
• the width is about 11.1-10.8 armstrong that’s matched.
• That’s how the enzyme recognizes the incorrect base pairs. The mismatch base can’t fit in the active site properly
• incorrect nucleotide will fray and fall down into the site and cleave out.
• Incorrect nucleotides incorporated once for every 10^5 to 10^6 times
• Actual mutation rate is one in every 10^9 nucleotides
• Polymerase stalls when incorrect nucleotide incorporated- fraying of end

Question 18

Yeast Replicon

Answer 18

1) ARS autonomous replicating requence. ARS binds ORC (origin replication complex). ORC bound to origin
2)Liscening factors MCM bind to origin during/after mitosis . Makes Prereplication complex. Loading of MCM needs Cdc6 and Cdt1
3) DNA replication active from specific kinase, ex CDK
4) Replication happens in both directions. MCM form helicase that unwinds DNA. Mcm2-Mcm7 replicative helicase
5)CM proteins displaced from DNA.
Yeast: MCM proteins exported from nucleus

Question 19

Eukaryotic Replication Fork comparison to PROkaryotes

Answer 19

PCNA = sliding clamp
RFC = clamp loader (like gamma clamp loader in E.Coli)
RPA = SSB proteins

Question 20

Nuclear Matrix DNA replication

Answer 20

In this model, its not the replication machinery that moves along stationary DNA, but the DNA that is spooled through the replication apparatus which is formula attached to the nuclear matrix.