Lec19 Flashcards
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??
What is a nick?
nick is missing sugar phosphate bond
Gap
missing a nucleotide
purpose of DNA poly I
removes primer and fills in gap
purpose of DNA ligase
seals nick
ANALYZE CHART FOR REPLICATION FORK
!!!
Many differences of Eukaryotic replication from prokaryotic replication.
- many origins of replication exist
- origin must be licensed for replication to occur
- linear chromosomes instead of 1 circular chromosome
- Lots of different DNA polymerases molecules with various roles
- Replication of telomeres to make sure the ends are properly replicated
- Nucleosome assembly immediately follows replication
What is the objective of Licensing of DNA replication
to make sure each piece of each chromosome is replicated once and only once per cell division in eukaryotes
What doe the replication licensing factor do?
attaches to each origin of replication early in cell cycle
Where will replication only start during licensing of DNA replication
will only START AT LICENSED ORIGINS
-as replication proceeds from origin, licensing factor is removed
When is the licensing factor activated?
only active just after mitosis and before replication starts in cell cycle, so it will no be put back on an origin until early in the next cell cycle
What happens at the end of a chromosome when the primer pops off?
there is a gap left that a telomerase fills.
Telomerase
(a ribonucleoprotein) contains RNA which it uses to make several repeats of DNA to fill in the gap. WHICH means that the RNA is used as a template for DNA synthesis: REVERSE TRANSCRIPTION
Telomere length and Aging/Imortality?
studies show that the somatic cells of individuals with progeria have shorter telomeres and do not divide as well in culture, but this is not proof that the disease is due to a decrease in a telomere length.
