Lec19 Flashcards
(37 cards)
Meselson and Stahl
tested the three hypothesizes for DNA replication by:
- Grew E. Coli in 15N media for many generations then they sampled the DNA
- Then transfer these cells to 14N Media
- Let the sample replicate for 1 generation then sample the DNA
- Let the sample replicate another generation then sample the DNA
- Find let the sample replicate a 3rd time and sample the DNA
DNA samples were analyzed by density gradient centrifugation (determined ratio between N14 and N15)
CONCLUDED DNA is SEMICONSERVATIVE
Taylor, Woods, and Hughes
Different way to show that DNA was semiconservative
-used Autoradiography to examine chromosomes during (metaphase of mitosis) of replication
-3H Labeled Thymine only present for first cell division then pattern of radioactivity observed as cells continue to divide.
After the first cell division both chromatids show 3H, while after the second division only 1 chromatid contained the 3H labeling. WHICH SUPPORTED SEMICONSERVATIVE
Theta replication
common in bacteria and other circular DNA molecules
Rolling circle replication
with conjugation in the F factor and some viruses (ex. lambda)
Linear Eukaryotic Replication
for eukaryotic chromosomes, which have multiple origins of replication along their length (eukaryotic replicons are 20,000-300,000 bp long)
Where does all replication start at?
in the replication origin
When does replication stop (proceeds to?)
until the entire REPLICON is replicated
Replicon
unit that is replicated together
What does Theta replication result in?
replication begins and then goes bidirectionally which results in 2 circular double stranded DNA molecules.
BIDIRECTIONAL REPLICATION
Characteristics of rolling circle replication?
replication fork can continue around many times
-producing many strands that can then be used as templates such as double stranded DNA molecule
Characteristics of linear eukaryotic replication?
- Multiple origins
- bidirectionally from each origin
- rate of 500-5000 nts/min
- Replicons typically 20,000 to 300,000 base pairs long
- unique procedure for replicating ends involving telomerase
Telomerase
?
DNA polymerase I in E. Coli
- discovered first
- requires template DNA, 4 deoxyribonucleoside, MG++ (used as cofactor)
- adds nucleotides 5’ to 3’ about 650 nts/min (thought to be slow)
- also has exonuclease function
DNA polymerase II in E. Coli
- Adds 5’ to 3’
- Fills in gaps less than 100 nts (repairs DNA and is slower than polymerase I)
DNA polymerase III in E. Coli
-Adds nucleotides 65,000 nt/min
-adds only in 5’ to 3’ direction
FASTEST
What does a phosphodiester bond formation reaction require?
nucleoside triphosphates, not just nucleotides
What catalyzes a phosphodiester bond formation reaction
catalyzed by the enzyme DNA polymerase
Leading Strand
replication starts at the bottom and movies 3’-5’ of the template strand (continuous replication)
lagging strand
replication starts at the top of the fork moving 3’-5’ of the template strand (discontinous replication)
-contains okazaki fragments
Initiation: Replication process in E. Coli
-initiator protein (DnaA) binds to origon of replication causing local unwinding and a short stretch of single-stranded DNA
Unwinding: replication process of E. Coli
- Helicase attaches to lagging template at replication fork and moves 5’-3’ breaking H-bonds as the replication fork moves along the DNA
- SSB binds to single stranded DNA stabilize it and prevent hairpin formation
- Gyrase rellieves supercoiling ahead of replication fork
Topoisomerases
allows supercoiling to occur
-Gyrase is a type of topoisomerase
Elongation Process
- DNA polymerase cannot start from scratch: They must have a 3’ OH that they can add nucleotides to.
- Primers are made by Primase, an RNA polymerase: ie. the primer is RNA
- DNA pol III adds nts primer
- The segment of RNA is eventually removed and replaced with DNA
Primosome
helicase and primase??
