Lecture 7: Stress response Flashcards
What does cellular stress cause?
DNA damage and accumulation of unfolded proteins
What is a cellular response to DNA damage and accumulation of unfolded proteins?
DNA repair through e.g. SOS response and protein folding control through e.g. heat-shock response.
Why is repair of DNA necessary?
DNA damage can lead to mutations or cel death. A cell with damaged DNA can also transform into a cancerous cell (when mutations affect the cell-cycle or signal transduction routes).
Name three general DNA repair mechanisms.
- Editing of DNA during replication
- Removal of damaged DNA
- Join double-stranded breaks
How are mistakes prevented (or corrected) during DNA replication?
By proof-reading by DNA polymerase III
Normally, the 3’ prime end is extended by incorperation of a correct base. The sugar phosphate bond is then formed, the DNA is now stable. How does proof-reading by DNA polymerase III work when a wrong base is incorperated into the DNA?
When an incorrect base is incorperated at the 3’ prime end, it cannot base pair with the template strand. This causes a loose 3’ prime end and DNA replication stops because of this. Luckily DNA polymerase III can solve this problem, by breaking the OH-bonds between base pairs at the 3’ prime end. The loose 3’ prime end is now longer and more flexible, so that it can interact with the exonuclease site of DNA polymerase. Here, 4-5 nucleotides of the loose end are cut off. The 3’ prime end is then extended with new (and correct) nucleotides.
Why does DNA polymerase III stop when there’s a loose 3’ prime end?
Because unpaired 3’-OH end of the lagging strand blocks further elongation, so 3’-to-5’ exonuclease activity chews back the DNA untill it gets to a base paired 3’-OH end.
What base is typically wrongly base paired?
Cytosine, due to having only one benzene ring and thus being prone to chemical modifications (that occur naturally or due to damaging agents). But other nucleotides also are prone to changes.
Sometimes DNA polymerase III misses a wrongly incorporated base pair or mutations occur after DNA polymerase III has passed by. How does mismatch repair work?
Mismatch repair consists of the proteins MutS and MutL. MutS recognizes mismatches base pairs. MutS is in contact with MutL, where MutL will scan the rest of the DNA for a gap (where a nucleotide is missing). When MutL finds this gap, it will remove the entire strand from the mismatched base pair to the gap. Then the DNA is repaired by DNA synthesis.
In the case of heavy damage, extra DNA polymerases are used to restore DNA structure and/or replication. What is the downside of these DNA polymerases?
They are not as precise at proofreading the DNA as e.g. DNA polymerase III. They allow errors and with this introduce mutations in the DNA.
After DNA replication, it’s still possible that nucleotides change. What can cause these changes in nucleotides?
- Spontaneous (depurination or deamination)
- Mutagenic agents
- UV light
What’s is the difference between the purine nucleotides and the pyrimidine nucleotides?
Purine nucleotide bases are guanine and adenine and have a double ring structure. Pyrimidines are cytosine and thymine and have a single ring structure.
What is deamination and depurination?
Depurination is when purines lose their complete base (the double ringed structure), so the bare phosphate sugar is left over. Deamination means that the amine group in the base is lost, which changes the base (in this way cytosine can change into uracil).
1What will happen after DNA replication in double stranded DNA with a deaminated cytosine?
Where first cytosine can bind to guanine, deaminated cytosine will turn into uracil. Uracil cannot bind to guanine, so after DNA replication either uracil is changed back into cytosine or guanine is changed into adenine so that it can bind to uracil. So deamination will result in substitution.
What will happen after DNA replication in double stranded DNA with a depurinated nucleotide, like adenine?
Depurinated adenine means that there’s complete base loss. Where adenine was first able to bind with thymine, it can no longer base pair. DNA replication of this strand will result in deletion, where either both nucleotides (adenine and thymine) are deleted or adenine is deleted and replaced with a new adenine.
What types of nucleotide directed repair is there?
- Base excision
- Nucleotide Exchange (NER)
What is base excision repair?
DNA glycosylases are enzymes that scan the DNA for non-paired bases (due to deamination, alkylation, oxidation, methylation etc.). Here, they remove the deaminated/alkylated/etc. base. The non-paired base that is left over is recognized by AP endonuclease and removes the sugar phosphate of the removed base. This creates a single-nucleotide gap. DNA polymerase adds in a new nucleotide and DNA ligase seals the nick.
How is a mutated nucleotide recognized?
A mutated nucleotide is recognized by its flexibility, due to H-bonds that are changed/disrupted. Glycosylases monitor the ability to flip and flipping leads to excision.
What are UV-induced mutations?
UV can result in cross-linking of two nucleotids. Cross-linking occurs between pyrimidines (T-T/C-C/C-T/T-C).
What is nucleotide excision repair?
This type of repair occurs when pyrimidines are cross-linked by e.g. UV. These pyrimidines need to be removed as nucleotides. An excision nuclease makes cuts in the strand containing the cross-linked pyrimidines, DNA helicase then cuts out this strand. A new strand is then made by DNA polymerase and ligase.
What is nonhomologous end joining? Is there a downside to this mechanism?
Nucleotides around the double-stranded break are degraded until the gap matches on both strands. The end on both sides of the break are then joined together. The downside is that part of the DNA sequence is deleted in the process. This can introduce gene mutations that can result in tumor cell formation.
What is homologous recombination?
First, nucleotides around the double-stranded break are degraded until the gap matches on both strands. But now, the double stranded DNA with the gap is compared to the DNA sequence on the sister chromatid. This results in accurate repair using the information from the sister chromatid.
During DNA replication there’s a nick in the DNA. When the replication fork encounters this nick, the fork will break and a double stranded break is created with two doubled stranded DNAs. How is this DBS repaired (through homologous recombination)?
An exonuclease will degrade the 5’ end of the template strand where the DBS is located, which creates a 3’ prime overhang. RecA/Rad51 protein can bind to the 3’ prime overhang. These proteins can then find the homologous DNA strand (closeby due to replication fork) and can bind the two strands together. DNA synthesis can be proceeded by moving on the replication fork.
What kind of proteins are RecA/Rad51?
They are single stranded binding proteins (SSBs) and upon activation (ATP binding) can bind single stranded DNA. When this happens, strand invasion will occur. Here, a double stranded DNA (DNA duplex) is matched to the single stranded DNA through homology and so strands are exchanged. The RecA/Rad51 proteins are then hydrolysed, which creates a new DNA heteroduplex.
Summary of homologous recombination to repair a double-stranded break in pictures.
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What does it mean that protein folding occus co-translational?
mRNA is translated into proteins in ribosomes. As the polypeptide chain is growing during translation, the polypeptide chain folds itself in a certain way (like folding of N- and C-terminal domain). Folding of the protein is then completed after release from ribosomes.
How does Hsp60 function and correct misfolded proteins?
The Hsp60 form a hsp60-like protein complex. There’s a trap in this complex that contains a hydrophobic domain that attracts misfolded proteins. When a misfolded protein enters the ‘trap’ of the Hsp60-like protein complex, it uses ATP to attract GroES. GroES is a cap that seals of the trap so that the misfolded protein can only leave when it has folded correctly. Upon ATP hydrolysis GroES is released and with this also the now correctly folded protein.
How is the proteasome build up? Also explain how the proteasome recognizes misfolded proteins and how the proteasome functions.
The proteasome is composed of unfoldases at the top, a central cylinder with proteolytic activity and a cap at the bottom. It recognizes misfolded proteins by their polyubiquitine chain. The misfolded protein moves down the proteasome, first being unfolded by the unfoldases and consquently being degraded by the proteolytic centre.
How is ubiquitin tagged/attached to a misfolded protein?
An ubiquitin-activating enzyme (E1) binds and activates ubiquitin via hydrolysis of ATP. E1 bound to ubiquitin can then bind to the ubiquitin ligase complex (consisting of E2 and E3). E1 can leave the complex. And the ligase complex then scans for proteins that need to be degraded. The hydrophobic patch of these misfolded proteins are recognized, where it will then bind to the ubiquitin ligase. In this way the protein can be tagged with ubiquitin.
How is amyloid formed in prion disease?
Normally amyloid has a globular formation, but when denaturing and renaturing of this globular formation can result in a very rare conformational change (beta sheets). These beta sheets can form heterodimers with the globular form, which causes the globular form to change into the beta sheet as well. A homodimer is now formed and when this process happens multiple times, amyloid is formed.
