Genome Maintanance Flashcards
(29 cards)
- Describe the major forms of DNA damage and give a possible cause for the damage
Major source of DNA damage:
1. Metabolism within the cell
Types of damage to a single nucleotide or base in DNA:
1. Spontaneous oxidative damage on base or sugar
2. Hydrolytic attack
- Chemical reaction involving water - always occurs in cells
- If we have a hydrolytic attack on the bond between base and sugar, then base is no longer linked to the DNA backbone —> DEPURINATION (spontaneous loss of a purine base from DNA) —> base must be replaced
- more common in purines than pyrimidines
3. Uncontrolled methylation on the base
Size of arrow indicated frequency of such damage
EXAMPLE DAMAGE - Thymine dimers (large disruptive lesion)
4. UV light
- UV light interacts with bonds in DNA
- UV light induces bond formation between neighbouring nucleotides (pyrimidines in this case)
- We have two thymines next to each other with very strong bonds - no longer able to bind to opposite strand
- hugely distorted with thymidine dimers not base pairing with opposite strand adenines - complex lesion that must be repaired
- Gamma radiation (ionising radiation)
- exposed to this by the sun and naturally occurring isotopes
- causes breaks in the DNA backbone (single-stranded breaks or double-stranded breaks)
Note - chromosomes are very tightly packed in nucleus even when they’re being used - histones and proteins still protect them - they dont float around freely
- So if double-stranded breaks occur, the two seperate broken parts of DNA will not float away from each other but must be repaired if the DNA is going to be used
- Define DNA damage, and define mutation
- DNA damage
- DNA can’t be used properly (e.g. due to missing base, thymine dimer, DNA breaks) - DNA mutation
- A heritable difference in DNA compared to the wild type (normal version that exists in the population)
- Mutations provide the variation that allows natural selection to occur – some of the mutations that have occurred have given one particular part of the population a selective advantage, allowing evolution
- However, human genetic diseases are also caused by mutations
- The mutations have to occur in the gametes for it to be passed on
- Explain the implications for a cell of having unrepaired DNA damage
DNA polymerases are extremely accurate due to 2 mechanisms:
1. Binding of the nucleotides
2. Proofreading activity of DNA pol
- And if these fail, DNA repair may be able to fix it later
- When a wrong base is added it will lead to poor base pairing and if not fixed, each strand is used for replication and the two cells would have different DNA
> can become permanent mutation in one of the two cells
IMPLICATIONS OF UNREPAIRED DNA DAMAGE:
- DNA replication will fail to occur due to unpaired lesion and this will end up as single-stranded DNA and be degraded
- With an unrepaired break, when the replication fork gets to that it cant go further so it becomes a double-stranded break and causes loss of that chromosome
- Hence DNA lesions must be repaired before DNA replication occurs
- Explain how mismatch repair identifies the incorrect base after DNA replication
> Mismatch repair is specific to E.coli - humans dont have it
- E.coli DNA is methylated on the adenines of GATC sequences (different to methylation on CPG islands in eukaryotes)
- You can see methylated adenines on GATC palindromes
- As DNA is replicated, the new strand doesn’t have methylation on new GATC strands
- A is methylated on old strand but not on new strand - HEMI-METHYLATED DNA (marks which strand is old or new)
- Soon, the new strand is methylated via “Dam Methylase” and two strands can’t be distinguished anymore
> If there is a mismatch in DNA replication that DNA pol hasn’t repaired then E.coli can still identify new and old strands for a very short time
1. Mismatch gets recognised by several Mut proteins, in an expensive process (-ATP) they will scan along DNA until they hit a region that is hemi-methylated
2. Mut proteins make a cut on unmethylated side of DNA (new strand)
3. Helicases and exonucleases will then remove the section of new strand from single nick all they way to point of mismatch
4. DNA pol III then completes removed section and DNA ligase will ligate the nicks
Repairs single mismatches ONLY immediately after DNA replication
- Define depurination and deamination.
Two similar repair systems:
> If there is a damage to a single nucleotide
- Two similar repair systems for damage to nucleotides:
1. Base Excision Repair: for small damage
2. Nucleotide Excision Repair: for larger damage
hydrolytic attack targeting bond between base and sugar = DEPURINATION
Hydrolytic attack that removes whole base or removes amine group off the base = DEAMINATION
- Explain what happens when a cytosine or 5-Methylcytosine are deaminated
- Describe the roles of DNA glycosylates, endonuclease, DNA polymerase I and DNA ligase in base excision repair
BASE EXCISION REPAIR - REPAIRS DAMAGED/MISSING SINGLE BASES
- Deamination is a common damage to a single base
- E.g. Hydrolytic attacks removes amine group from base and disconnects base from backbone
- UNUSUAL RESULT:
- If cytosine is deaminated it turns into uracil —> only found in RNA
- if 5-methylcytosine is deaminated, it turns into thymine
Deamination changes the nucleotide into completely incorrect for that position
EXAMPLE - Uracil instead of cytosine present due to deamination of cytosine
1. DNA glycosylase (Uracil glycolysase here) recognises wrong base and removes it –> DNA backbone is unaffected
2. AP endonuclease cuts the DNA backbone at the gap and removes some of the nucleotides that was next to the uracil
3. DNA pol I replaces removed section of DNA with correct nucleotides
4. DNA ligase then ligates small nick in DNA backbone
- This is an error free pathway*
- Define thymine dimer and what causes them
- Describe the roles of nucleases, DNA helicase, DNA polymerase I and DNA ligase in nucleotide excision repair
NUCLEOTIDE EXCISION REPAIR - Thymine dimers
- Exinuclease enzyme makes a cut at either side of DNA lesion
- Then DNA helicase unwinds 2 strands in that section away from each other and because the section was cut from the backbone, it will go and get recycled
- This leaves a gap and so DNA pol I fills the gap
- DNA ligase then ligates remaining nick
- Contrast the mechanism of base excision repair and nucleotide excision repair
Yes - diagram
- Compare when base excision repair is needed and when nucleotide excision repair is needed
Base excision repair —> single nucleotide/base damage or missing base
Nucleotide Excision Repair —> thymine dimers due to UV light (pyrimidine)
- Explain the importance of repairing a double stranded DNA break
Double-stranded break repair:
- Double-stranded breaks are potentially dangerous
- Ionising radiation, errors of DNA replication, oxidising agents, and other metabolites can cause breaks across both strands of the DNA
- DS breaks are the most biologically significant lesion by ionising radiation
- Most DS breaks are repaired within 24 hours, but 25% of the repairs contain errors
- Explain why non-homologous end joining is considered error-prone, and why it is still a worthwhile repair mechanism
NON-HOMOLOGOUS END JOINING:
1. Small loss of nucleotides occurs because these ends are prone to degradation by any nucleases that come into contact with them
2. Repair pathway will remove some nucleotides to process the ends ready to join them together
- Explain how the following mechanisms together regulate the activity of Cyclin Dependent Kinases: binding of cyclin, phosphorylation of Threonine 160, phosphorylation of Tyrosine 15, degradation of cyclin, and binding of protein inhibitors
> When cells grow and divide, they go thru an ordered series of events = cell cycle
1. S phase = DNA synthesis
2. G2 phase = RNA + protein synthesis & growth
3. M phase = chromosomes and cells can be divided
4. G1phase = RNA + protein synthesis & growth
5. G0 phase = cell terminally differentiates and withdraws from cell cycle either temporarily or forever
What controls this orderly process?
1. Protein phosphorylation
- There are activating phosphorylation events and inactivating events
- Protein degradation
- Abundance of protein falls very quickly as its degraded - Protein synthesis
- Opposite of degradation action - Inhibitors
- Bind to one part of a protein to inhibit its activity
- Binding is reversible
> All eukaryotes use same system to control cell cycle:
- Kinases (CDKs) control cell cycle and proteins (CYCLINS) abundance fluctuate with cell cycle
- Explain the central role of Cyclin Dependent Kinases and Cyclins during cell cycle progression
CDKS:
- Family of protein kinases
- The kinase activity is cyclical
- Regulates the proteins that carry out cyclical cellular functions
- Are heavily regulated
- Require binding to a cyclin for activity
- Stable protein levels across cell cycle
- Animals have 8 CDKs
CYCLINS:
- Undergo a cycle of protein synthesis and degradation – protein levels are cylical
- Essential regulators of CDK activity
- Are also regulated
- Animals have 10 cyclins
- Can be divided into G1/S cyclins, S-cyclins and G2/M cyclins
> Mechanism 1 for regulating the cell cycle:
- phosphorylation of CDKs – activating and inhibiting
- Phosphorylation of Thr160 in the T-loop activates the CDK by allowing target binding
- Phosphorylating Tyr15, near the amino terminus, inactivates CDK2 by blocking the ATP binding site with its negatively charged
- Predict the activity level of a CDK based on its binding to cyclin, binding to inhibitors, phosphorylation of Threonine 160 and phosphorylation of Tyrosine 15
- CDK by itself is inactive, when bound by a cyclin protein, it becomes partly active
Not fully active because of the T loop blocking the active site of the CDK - Phosphorylation of Thr160 in the T-loop by CDK-activating Kinase (CAK) activates the CDK by allowing target binding
- importantly there is another phosphorylation event that can happen at Tyr15 near the amino terminus and this activatesd CDK
- CDK is fully active as long as Tyr15 is not phosphorylated
- Explain how regulation of transcription and regulation of ubiquitination controls the abundance of a cyclin during the cell cycle
> Abundance of cyclins is very important for the regulation of activity of CDKs
Mechanism 2 for regulating cell cycle - controlled degradation of cyclins:
- Initially CDKs are present but there is no cyclin –> CDK inactive
- When appropriate cyclin becomes synthesised it forms partially active or potentially active CDK-cyclin complex
- However, this has that inhibitory phosphorylation at Tyr15 –> still inactive, then phosphorylation of Thy160 occurs in T loop
> Why would you have both activating and inactivating phosphorylation at the same time?
- It makes sense if you think of it as a car; You stopped your car at a red light but the engine is still running (Activating phosphorylation). However you have your foot on the breaks (Inactivating phosphorylation) and this allows you to take your foot off the breaks quickly and put on the accelerator and you will be able to go very quickly rather than turning the car off at each stop light.
- So when inactivating phosphorylation is removed, the complex can become active very quickly
- A phosphatase will take off the inhibiting phosphorylation and some CDKs will become active
- The CDK will target many different targets but we will focus on two: allowing 2 feedback loops (positive & negative:
- Describe the positive feedback loop that causes a rapid activation of CDK/cyclin at a cell cycle transition point
- Positive feedback loop: CDK phosphorylates the phosphatase to activate it –> even more inhibiting phosphorylation is removed –> more CDK becomes active –> more CDK will phosphorylate more phosphatase –> repe
- Describe the negative feedback loop that causes a rapid drop of activity of a CDK/cyclin just after a cell cycle transition point
- Negative feedback loop: Simultaneously with the positive feedback loop, some active CDK will phosphorylate and activate another protein called DBRP
- DBRP attaches Ubiquitin proteins to the cyclin –> targets cyclin for degradation
- It is negative feedback loop because the more activities there is at CDK the more activity of DBRP, the more ubiquitin is added to the cyclins –> sends cyclins for degradation –> stops activity of CDK
- Inactive CDK is now ready for the next phase of the cell cycle to come around so cell cycle can happen again
- Hence, the phosphorylation that is activating the phosphatase and therefore activating the kinase allows for a big spike of CDK
- Define the role of Destruction Box Recognizing Protein
DBRP = Destruction Box Recognising Protein = targets cyclin for degradation
- There’s an amino acid sequence on cyclin called destruction box = 9 amino acid sequence near the amino terminus
- It attaches small proteins called Ubiquitin to the sequence - covalently attached proteins as a marker of degradation (or can be signalling something else)
- proteins tagged with ubiquitin is recognised by proteasomes = large protein complex that degrades proteins back to amino acids for recycling
REMAINING TWO MECHANISMS FOR REGULATING CELL CYCLE IS:
> MECHANISM 3:
- regulated synthesis of CDKs and Cyclins
> MECHANISM 4:
- Protein inhibitors of CDK activity
- E.g. p21 or p27 binds to CDK-Cyclin complex and inhibits their activity while bound to it
- Give two examples of CDK targets and the phase of the cell cycle at which they act
- There are 100s of proteins phosphorylated by CDKs
> EXAMPLE 1 - Target in Mitosis - Nuclear Lamins
- If we have a look at the inside of a nucleus we have a nuclear envelope within it there are proteins called nuclear lamins that make up a mesh called nuclear lamina
- when a cell transitions into M phase the nucleus is going to break down and the chromosomes are going to condense
- breakdown on envelope is partly assisted by the phosphorylation of lamins by the CDK, controlling entry into mitosis
- when phosphorylated nuclear lamins all fall apart instead of forming a mesh
- then the chromosome is able to be seperated by the spindle –> two identical copies of chromosome decondenses (early telophase) and nuclear envelope along with nuclear lamina forms around the chromatids
> EXAMPLE 2 - Target in Mitosis - Condensins
- One of the other things that has to happen for mitosis is the chromosomes will go to their most condensed state –> This is controlled by a large set of proteins called condensins
- If you mix condensins with DNA, then they will naturally condense that DNA in the test tube
- If there are mutations in the condensins, then the cell is no longer able to condense its DNA for mitosis
- There’s a theory that they bind to the chromosomes, loop out structures of already condensed DNA.
and allow those chromosomes to condense even more.,
- Explain the importance of checkpoints in maintaining genome integrity
It’s very important that any DNA damage in a cell is repaired before anything else happens to the DNA—especially before DNA replication (which happens in the S phase).
To make sure of this, the cell has checkpoints.
If DNA damage is detected, the cell halts entry into or progression through the S phase so that repairs can be made.
If the replication fork passes through damaged DNA before it’s fixed, that small damage could turn into a serious problem.
These checkpoints give the cell time to repair DNA before moving into the next phase of the cycle.
If DNA damage happens later in the cell cycle, such as before mitosis, the cell can also pause mitosis to allow for repair.
There are other problems that checkpoints can catch too:
If DNA hasn’t been fully replicated, the cell is stopped from going into mitosis because separating incomplete chromosomes would be harmful.
If a chromosome detaches from the spindle during mitosis, mitosis pauses until the chromosome is reattached correctly.
There’s also a checkpoint that checks the cell’s environment—to make sure it’s a good, supportive environment for the cell to begin dividing in the first place.
- Define the roles of the following proteins in the G1/S transition: E2F, pRB, p21, p53.
> How does S phase begin?
- there is a transcription factor E2F that can initiate transcription for enzymes used in DNA synthesis
- E2F is kept inactive by binding of pRB protein (retinoblastoma protein)
*pRB is an inhibitor of E2F transcription
- When the cell is ready to enter S phase, the CDK 2-cyclin E complex will activate to phosphorylate pRB
- pRB falls off and E2F is active - hurray :)
- E2F can now begin transcription of enzymes for DNA replication (G1–>S phase)
What if there is DNA damage?
- There are some enzymes that detect presence of DNA damage and these will send signal to p53 - the checkpoint controller
- p53 then causes an inhibiotor p21 to be transcribed
- p21 binds to CDK2-Cyclin E complex and inhibits its activity
- phosphorylation of pRB can longer occur so pRB stays bound to E2F and cell cycle cant go to S phase until DNA damage isnt repaired
- Describe the role of p21 in mediating the G1/S checkpoint response to DNA damage.
p21 binds to CDK2-Cyclin E complex so it cannot phosphorylate pRB and pRB stays intact to E2F so enzymes for DNA synthesis can’t be transcribed
