DNA Replication Flashcards
What were people uncertain genetic material was in the early 20th century?
Carbohydrates
protein
lipid
something else
Recognition that _ was genetic material was a key insight
DNA
What did DNAs structure immediately suggest?
DNA could serve as a blueprint and that blue print could be passed down through generations
Where are genes carried?
chromosomes
What are chromosomes comprised of?
DNA and proteins
Experiements suggested that the _ component of chromosomes is the genetic material
DNA
What was Fredrick Griffith’s experiement with?
Mice and streptococcus pneumoniae
What cells killed inoculated mice?
Smooth cells
S-strain
What cells did not kill inoculated mice
Rough cells
R-strain
How do you eliminate the virulence in the s-strain
with heat
What happens to a mouse with live R-strain cells and heat-killed S-strain? and what was recovered?
Mouse dies
Live S-strain was recovered
What does recovering live S-strains from dead mice tell us?
Cell debris from dead S-cells had converted R cells into S-cells
Avery-Mcarty-Mcleod
What material in the cell debris was responsible for the transformation of the S cell DNA into the live R cells?
How do we know?
DNA
The mouse did not live until the DNA debris was destroyed
What was the first evidence that DNA was genetic material?
(From Griffith, Avery-mccarty-mcleod experiment)
The mouse with live R-cells and dead S-cells did not live until the S-cell DNA debris was destroyed
What was the Hershey-Chase experiement?
Phages with radiolabeled protein and phages with radiolabeled DNA were used to infect hosts. The host cells were then processed to see if the radiolabel was within cells or outside of cells. Radioactive DNA was recovered in bacteria due to the radiolabeled DNA.
What was recovered in the Hershey-Chase experiment?
Bacteria with radioactive DNA
Structural features of DNA must allow for _ _
Faithful replication
Genetic material must have _ _
informational content
Genetic material _ be able to change on rare occaision
must
What 3 pieces of information about DNA
were crucial in determining its structure
The chemical composition of DNA
Chargaff’s rule
X-ray crystallography
What are the 3 components of DNA?
Tri-hosphate
a deoxyribose sugar
A nitrogenous base
What are the 4 nitrogenous bases of DNA
Purines: adenine and guanine
Pyrimidines: cytosine and thymine
Purine structure?
double ring
Pyrimidine structure?
single ring
What is chargaffs rule?
T=A, C=G but A+T does not necesarily = C+G
Who conducted x-ray crystallography of DNA
Rosalind Franklin and Maurice Wilkins
What did x-ray crystallography suggest?
DNA is long, skinny, helical and comprised of two similar parts
What happened in approx 1953?
James watson and francis crick inferred that DNA is a double helix
Watson and Crick
“_ must always pair with _ because…”
Purine must always pair with a pyrimidine because it is most consistent with xray data
What are DNA’s complementary base pairs?
A-T
C-G
What holds the strands of DNA together
Hydrogen bonding between the nucleotides
How many Hydrogen bonds does A-T have?
C-G?
AT-2
CG-3
Where do base pairs stack in DNA?
In the center of DNA as planar structures
Do major and minor grooves impact its function?
yes
Alternating minor and major
Meselson-Stahl experiment found that DNA replication is _
Semi-conservative
What does semi-conservative replication mean?
1 old strand and 1 new strand
When do you see the replication fork?
Second round of replication
Replicating DNA is mixed between _ and _ states
unwound and overwound states
_ _ removes twists and supercoils to make replication possible
DNA gyrase
_ unwinds double helix at the replication fork and SSB binds
Helicase
_ _ cuts DNA strands and rotates them to prevent supercoiling
DNA gyrase
What rejoins the strands
DNA gyrase
_ help relive tension in DNA
Topoisomerases
(gyrase)
What do topoisomerases do?
Break the phosphodiester bonds between nucleotides and then reseal these breaks
E.coli
Initiation of replication begins at the ___
Origin (oriC)
E.coli
DnaA binds to _ _ which are specific _ _ _
DnaA binds to DnaA boxes, which are
specific, short DNA sequences
E.coli
What is adjacent to DnaA boxes?
AT-rich sequences that melt easily which opens up the DNA
E.coli
What do additional DnaA molecules do once DNA is opened up a bit?
additional DnaA molcules bind to the DNA less specifically
E.coli
Opening up of the DNA leads to the recruitment and loading of _
Leads to the recruitment and loading of DnaB (helicase)
E.coli
Once bound _ (DnaB) further _ _ _
Once bound helicase (DnaB) further opens the DNA
E.coli
After helicase further opens up DNA this leads to the recruitment of the _
replisome
E.coli
What does the replisome do?
It allows for DNA polymerase to come in and synthesize a complementary DNA strand
E.coli
What is DNA polymerase?
The enzyme that synthesizes a complementary strand of DNA
E.coli
Who isolated DNA polymerase from E.coli and demonstrated its function in vitro
Arthur Kornberg
E.coli
How many DNA polymerases does E.coli have?
5
E.coli
DNA pol I catalyzes chain growth _ - _ end
5’ to 3’
E.coli
DNA pol I additional 3’ to 5’ exonuclease activity
Removes mismatched bases
E.coli
DNA pol I additional 5’ to 3’ exonuclease activity
Degrades single strands of DNA or RNA
E.coli
Which DNA pol does most of the work in replication in E.coli ?
DNA pol III
E.coli
What are the two types of strands at the replication fork?
Leading and Lagging
E.coli
Which is the leading strand?
Which is the lagging strand?
Leading strand (3’) is going into the fork
Lagging strand (5’) is going away from the fork
DNA synthesis is primed using _ copied from DNA generated by _
Using RNA copied from DNA generated by primase. This gives DNA pol III a place to start
Which DNA pol extends the with new DNA?
DNA pol III
What does DNA pol 1 do to the RNA at the 5’ end?
Remove the RNA and fills the gap
What connects the adjacent fragments after the RNA (primers) are removed?
DNA ligase
Normal base pairing results in…
faithful transmission of the DNA
Mistmatches between bases can occur, sometimes resulting in
mutations
Point mutations
What is a transition?
When one purine changes to the other
or when one pyrimiding changes to the other
Point mutations
What is a transversion?
When a pyrimidine transitions to a purine or vice versa
What activity enables error correction?
3’ to 5’ exonuclease activity
What facilitates DNA replication on both strands?
Replisome, accessory proteins and looping
Eukaryotes
How do eukaryotic origins differ from e. coli origins?
Eukaryotes have multiple replication origins. Different parts of the chromosome will be replicated simultaneously
Eukaryotes
How does replication extend from these origins?
Bidirectionally
Eukaryotes
Eventually contigious, fully replicated _ are produced
Chromatids
Eukaryotes
When does DNA replication occur in eukaryotes?
During S phase
Eukaryotes
Initiation of DNA replication at the eukaryotic involves _ of specific .
Such as _
Involves recruitment of specific proteins.
Such as Cdc6
Eukaryotes
What happens after the specific proteins are recruited?
Helicase will unwind the DNA, allowing DNA polymerase to bind
Eukaryotes
What melts the AT regions to help binding to chromosome?
Helicase + Cdt1
Eukaryotes
How do you fill the gap at the teleomere in linear chromosome?
Telomerase helps extend the telomeres, fixing the gap
(most of the gap)
Eukaryotes
What happens after the telomere is extended by telomerase?
Example in humans?
Proteins bind to the end to form a cap that stabilizes the structure at the end.
TRF 1 and TRF 2
