Lec5-7 Flashcards
what was Arthur Kornberg’s goal
study DNA replication in vitro
describe Kornberg’s experiment steps
- develop an in vitro assay for DNA synthesis
- grind up e.coli cells to get cell extract containing all cellular proteins (in theory DNA pol.)
- add radioactive thymine (C14), all 4 dNTPs, and DNA
- incubate to allow DNA synthesis
- add perchloric acid to stop rxn and precipitate DNA, found some radioactive nt in the pellet therefore successful in vitro synthesized DNA
- add DNase enzyme to break up DNA and radioactive nt, precipitated again, and found DNA in supernatant, realized something in crude cell extract is synthesizing DNA - purify DNA synthesizing “activity” from crude protein fraction
- separate crude protein into fractions and test each one for DNA synthesizing ability
how did Kornberg discover DNA synthesis is 5’ to 3’
- he started with DNA, all 4 dNTPs and radioactive thymine again
- allowed the rxn to go in vitro without the radioactive nt then he added it briefly (pulse)
- then continued with the addition of perchloric acid and a pulse of 3’-5’ exonuclease
- discovered radioactivity is lost from DNA, becomes acid soluble
-added pulse of 5’-3’ exonuclease and discovered radioactivity is retained on DNA - therefore nts are added to 3’ end of DNA
how did Kornberg prove DNA involves the release of pyrophosphate from dNTPs
- he started with a template, primer, all 4 dNTPs, and radioactive carbon
- shot gamma rays at terminal phosphate group
- saw radioactive carbon goes into precipitate (incorporated in DNA) but radioactive phosphate goes in supernatant
- therefore phosphate gets removed
what would occur if DNA synthesis was random
radioactivity would be found in both pellet and supernatant for both exonuclease treatments
what direction is continuous DNA synthesis
5’-3’
what direction is discontinuous DNA synthesis
5’-3’
what experiment provided evidence for lagging strand synthesis
- a pulse of radioactive thymidine was used to label DNA in vivo with E.coli
- alkali denaturation to separate the strands
- sucrose gradient separated DNA according to size
- measured radioactivity in each fraction
- when pulsed shortly (5-10secs), almost all radioactivity was found at the top (small pieces of ssDNA) but when pulsed longer (60 sec), they found some radioactivity in bigger pieces as ligase eventually joined fragments together but they didn’t know this
how did they discover ligase joins fragments?
used temp. sensitive e.coli mutants for ligase gene and found e.coli raised in permissive temp, their ligase worked but when temp was raised (restrictive), ligase denatured and they found lots of radioactivity in small DNA pieces due to ligase not able to join them in longer pulse
describe lagging strand synthesis
- DNA primase synthesizes a short RNA primer on the lagging strand as it’s exposed
- DNA pol extends until it meets the older primer
- old RNA primer is replaced by DNA
- ligase joins the Okazaki fragments on the growing chain
how did Okazaki discover DNA synthesis on the lagging strand requires RNA
- added RNase to the rxn which degrades the primer so no synthesis of the lagging strand and only saw radioactivity in fractions corresponding to large DNA pieces
- added radioactive uridine, which is only incorporated in RNA, instead of H3-dT and saw radioactivity corresponding to small DNA pieces
DNA replication machinery
- Mcm helicase: opens up double helix at replication fork by denaturing DNA as it moves
- RPA: stabilizes ssDNA on the lagging strand to prevent ssDNA tendency to make hairpins by complementarity
- PCNA: encircles DNA to keep DNA pol from sliding off by forming a ring around DNA, ATP binding to clamp loader opens PCNA which binds to DNA duplex and ATP hydrolysis locks PCNA around DNA and releases the clamp loader
- DNA polymerase, binds to PCNA
DNA replication mechanism
- clamp loader binds to PCNA to open PCNA ring
- PCNA ring encircles DNA/RNA hybrid
- primase adds primers to ssDNA
- PCNA-clamp loader moves along DNA util it finds 3’ end
- PCNA closes around DNA
- PCNA binds to DNA pol
- DNA pol now stably associated with DNA
what are the 3 mechanisms that ensure DNA replication accuracy?
- bp precedes covalent attachment of nt
- 3’-5’ exonuclease activity, if the wrong nt is incorporated into the primer strand, it changes the configuration at the replication fork and DNA pol stalls, chews back the wrong nt on 3’OH of primer strand and resumes to add the correct nt
- mismatch repair, MSH2, and MLH1 scan newly synthesized DNA, detect mismatches, make nicks on the new DNA strand, the strand is removed by DNA pol I, the large chunk of ssDNA left is used for repairing and families with mutations in these genes have high cancer incidence
true or false: polymerase and exonuclease activity are found in different parts of DNA pol
true
what bacteriophage first identified exonuclease function
T7, mutations in this DNA pol produce a mutator phenotype
how many replication origins do proks and euks have
one, many
what phase does DNA replication take place in eukaryotes?
S phase
how is DNA synthesis on the lagging strand completed?
- sheltrin recognizes telomere repeat sequences at chromosome ends and binds to protect from repair machinery and attract telomerase
- telomerase extends 3’ end by using its own RNA as a template
- then DNA pol and primase can extend and complete replication on lagging strand
what is DNA replication-dependent on
DNA-dependent DNA polymerases
what are telomerase and RT from retroviruses dependent on
RNA-dependent DNA polymerases
describe RNA-dependent DNA polymerase
when a retrovirus infects a cell, it brings its RNA genome and RT that makes cDNA (RNA-DNA hybrid) copies of its RNA, then the dsDNA can be incorporated into the cell and replicated in its genome
give examples of an RNA-dependent RNA polymerase
SARS-CoV-2, Influenza
give an example of DNA-dependent RNA polymerase
transcription
how did they identify genes required for DNA repair
used a classical, forward genetics approach by experimenting w/ haploid yeast, subjected yeast cells to mutagen so there could be random mutations in every gene, then plated out single cells to grow colonies and took replica plates to grow in low-level of UV radiation so it wouldn’t kill the yeast cells as they have the repair machinery, used 1 control, then they looked for the colony that didn’t grow in low UV radiation and discovered Rad mutants which were also unable to survive other known DMA damaging agents at levels that the wild type cells could tolerate
what are the 2 types of DNA damage
- chemical alteration of a nt ex. deamination, depurination (change in DNA sequence at next S phase), pyrimidine dimers (failure of DNA + RNA pol to go past the altered nt)
- ss or ds break in DNA backbone, results in DNA + RNA pol unable to go past the break
what DNA damaging agent increases pyrimidine dimers
UV light
what DNA damaging agent increases cytosine deamination
nitrous acid
what DNA damaging agent increases ds breaks
x-rays
what is depurination
loss of a purine base (G,A), leads to deletion
what is deamination
loss of cytosine becomes uracil, changes DNA sequence
what are the error-free pathways?
- base excision repair for altered base
- nt excision repair for pyrimidine dimers
what is the error-prone pathway
translesion repair
how does BER work
for deaminated C:
1. double helix scanned to detect altered bases
2. glycosylase which is specific to each type of altered base removes it
3. AP endonuclease and phosphodiesterase break the phosphodiester bond to remove backbone
4. DNA pol adds new nt and ligase seals nick
for depurination:
- glycosylase not required, AP endonuclease and phosphodiesterase break the phosphodiester bond to remove backbone and DNA pol adds new nt and ligase seals nick
what is a pyrimidine dimer
2 pyrimidines (C-C or T-T) next to each other bp and DNA pol can’t go through them, creating a barrier for RNA synthesis but it doesn’t directly cause a mutation however if it’s not repaired by NER it causes xeroderma pigmentosa due to translesion repair pathway which is highly active, leading to high mutation rate and cancer
how does NER work
- XPC-Rad23 scans DNA to detect damage
- recruits XPG (excision nuclease) for excision of the damaged strand
- DNA helicase helps unwind DNA so ss with pyrimidine dimer comes off
- DNA pol adds new nts and ligase seals the nicks
how does translesion repair work
- DNA pol reaches the pyrimidine dimer
- replication stalls
- PCNA is modified to eject replicative DNA pol so translesion DNA polymerase can bind
- translesion DNA pol goes through damage but adds random nt by best guess
- cell is saved but introduces mutation
what is Cockayne syndrome
UV sensitivity syndrome but not as severe as xeroderma pigmentosa b/c the thymine dimers can still be repaired however it’s associated with growth defects, neurological disorders, and premature aging all due to high cell apoptosis which is also seen in XPG but not XPC
how does TCR work
- RNA pol II runs into pyrimidine dimer
- transcription stalled at DNA damage
- CSB identifies stalled RNA pol and binds
- CSB recruits XPG excision nuclease
- then repaired by DNA pol and ligase
why is coupling NER to transcription important
ensures the cell’s most important DNA is efficiently repaired and ensures transcription can proceed through damaged DNA
how are ds breaks repaired?
- NHEJ which is error-prone, results in a small deletion, ends are recognized by Ku heterodimers
- HR which uses the sister chromatid as a template for repair, Mre11 recruited to DNA ends, it recruits BRCA1 to digest 1 strand then Rad51 mediates exchange with sister chromatid then repair DNA pol uses the sister chromatid as a template, the invading strand is released and DNA pol continues to add nts on the damaged strand and ligase seals everything
how can we use Cas9/CRISPR for gene editing
Cas9 mediates a ds break in DNA which is repaired by NHEJ and produces a small deletion that was wanted
how is CRISPR used to introduce specific point mutations in a gene
Cas mediates a ds break then a plasmid sequence containing a gene sequence is altered to introduce a specific mutation in the DNA and is useful to test for putative domains and significance of specific a.a.
how is CRISPR used to introduce an epitope tag
a plasmid containing tag sequence plus 1kb of flanking sequence on either side is introduced to gRNA and Cas9 which mediates a ds break near one end of the coding sequence and then repaired by HR
