Session 7 -DNA repair

How our DNA repairs itself?

• DNA damage is detected

• Remove/ correct the damaged DNA

• Restore the DNA sequence accurately

This occurs in G2 checkpoint: has replication been completed properly?

1. The DNA repair mechanisms fix, cell continues normally

2. Cell-cycle arrest, too much persistent damage, cell remains alive, but no longer divides

3. Too much DNA damage, Apoptosis

4. Damage escapes repair, mutation passes through can lead to cancer

• As mutations occur spontaneously, DNA endogenous repairs these mutations, so DNA replicates normally.

• Where the ability to repair mutations needs to balance against rate of mutations.

  • If DNA repair machinary is too efficienct then evolution cannot occur

  • If reduction in DNA repair, leads to excess DNA damage, gives rise to cancer or death

What are some sources of DNA damages?

Exogenous factors: From the outside environment => free radicals that can react with DNA to cause oxidative damage, where free radicals are generated by UV light, inflammation, changes in metabolism, air pollution, ionising radiation and carbingogenic chemicals (radon gas - colourless)

How can mistakes happen during DNA replication?

Process of going wrong: Intrinsic errors, since problem arises from processes inside the cell.

DNA helicase → Primase → DNA polymerase → Occasionally something goes wrong → DNA sequence may change → Mutation

• Stalled fork replication: DNA replication stops unexpectedly, stalled fork is not stabilised and repaired, it can collapse, causing DNA to break or lead to errors in DNA replication

• Repetitive DNA: leads to fork slippages, the replication machinery can lose track of which repeat it is copying.

  • This can lead to more repeats than origional DNA (slipped-strand structure/ loop) => repeat expansion mutations

What is the replication fork and what reasons for it to stall?

The replication fork forms when DNA double helix opens for replication, forming a Y-shape

• Not enough nucleotides: DNA polymerase struggles to continue, so replication fork stalls

• Transcription machinery: DNA replication and transcription can interfere with each other, for example if RNA polymerase and associated structures are trying to copy DNA

• DNA-protein complexes: Proteins attach to the DNA which can sometimes create an obstacle

• DNA lesions: Area of DNA is damaged, so DNA polymerase encounters damaged DNA, DNA polymerase is unable to copy the region normally

• Heterochromatin: tightly packed, condensed chromatin, so replication machinery has a difficult time in accessing and copying some regions

Information of Huntington disease

• Within the HTT gene, there is a repeated base sequence of CAG, so if there is a slip up during fork replication, CAG = glutamine, leads to abnormal amounts of glutamine

• This can lead to protein misfolding accumulating in neurons, neuronal dysfunction and degeneration

• Reduced penetrance: Sometimes if individual has the genotype (HD), but does not show phenotypes (in HD it is 36-39 CAG repeats)

Everything about how different types of DNA defects require different repair mechanisms:

When one strand is effected, and the other DNA strand is intact, provides information to restore the damaged strand. [single strand damage is easier to repair than double strand]

When both strands are broken: If not repaired correctly, leads to mutations, deletions, chromosome rearrangements, translocation.

What are DNA adducts?

When a chemical group becomes covalently attached to DNA, leading to altered/ bulky DNA structure. Interferes with replication and transcription. Which are repaired by NER (nucelotide excision repair)

What happens when DNA polymerase adds incorrect base pair?

1. stops

2. Moves backwards

3. Removes incorrect nucleotide

4. Adds correct nucleotide

5. continues DNA replication

What is exonuclease activity?

DNA polymerase can remove the incorrect nucleotide, but so can exonuclease, while proofreading, it detects and exonuclease removes it. So, DNA polymerase can go back to add in correct nucleotide

Advantage: If the DNA polymerase misses to recognise the error, exonuclease can remove error.

What is deamination? How do DNA repair mechanisms correct this mistake?

Deamination: removal of amino group

• Cytosine removes amino group, becomes uracil

DNA glycosylase recognises the abnormal uracil, and removes the Uracil, but leaves the sugar and phosphate. The enzymes cut the damaged DNA site, and there is a small gap in DNA strand. DNA polymerase uses the complementary strand and adds in correct base. DNA ligase then forms sugar-phosphate backbone

What is Base excision repair compared to nucleotide excision repair?

BER: Repairs small, individual damaged bases, usually starting by removing one damaged base.

NER: removes a short stretch of several nucleotides surrounding a bulky/ distorting lesion.

What does nucleotide excision repair do? (NER)

Recognise damaged region, cut around the damaged DNA, DNA polymerase uses the complementary DNA strand to bring complementary base pairs, DNA ligase seals the sugar phosphate backbone.

How a double-strand DNA break is repaired, particularyly by non-homologous end joining (NHEJ)?

Step 1: Proteins recognise the broken DNA ends and hold them closer to one another.

Step 2: The two opposite strands, might not be evenly cut, so the proteins modifies/ trims the ends to make them suitable for joining

Step 3: DNA ligase joins the DNA backbone, however key thing is that some DNA might have been lost in the process.

Just another key point/ information:

• If one chromosome has lost some genetic information, this means the other homologous DNA can be used as a template, so can restore DNA more accurately

Why accumulating mutations can eventually contribute to cancer?

Mutagenesis: Formation of mutations

Cancer results in multiple mutations over time, rather than arising from one genetic change.

An example: Lynch syndrome, hereditary non-polyposis colorectal cancer, where inherited pathogenic variants affect DNA mismatch repair genes. Which leads to increased risk of getting cancer (careful individual not born with cancer)

Why can cancer re-occur once being treated with chemotherapy?

First, not all cancer cells or tumours have exactly the same mutations.

Heterogeneity: Variation or differences

Intertumour heterogeneity: Genetic differences between different tumours

Intratumour heterogenetiy: Genetic differences between cancer cells within same tumour

• This occurs, when tumours continue to divide (DNA replication), there is opportunity for additional mutations

So, if there is intratumour heterogeneity, then the drug given to kill tumour A, will be resistant to tumour B in the same tumour. So, after treatment tumour B survives and continues to grow.

Key note: Chemotherapy can create selective pressures, where changing environment leads to tumour cells trying to survive => leading mutations withing the tumour, can lead to resistance

What is genetic redundancy?

The cell contains two genes, performing the same function. So if gene A is mutated the function can still be carried out by gene B. However if both are lost can lead to cell death.

How can doctors use genetic redundancy to exploit cancer cells?

• If cancer cells do not have a function gene B

• Give a drug that inhibits gene A, leading to cell death

Genetic redundancy in BRCA and PARP:

BRACA 1 and BRACA2 are proteins important for accurate repair of double-stranded DNA breaks, by homologous recombination.

PARP: proteins involved in repairing single-stranded DNA strands

So, if PARP is inhibited, then BRACA1 and BRACA2 can be used instead.

If both PARP and BRACA are inhibited, then normal cells have lost both ways of dealing with damaged DNA, leading to accumulation of damaged DNA, increasing risk of cancer cell developing, and too much DNA damage leading to cancer death.

Normal cell:

DNA gets damaged all the time, so there are multiple DNA repair systems: PARP and BRACA1 and BRACA 2

PARP: proteins that deal with single strand DNA damage

BRACA1 and BRACA2: important for accurate repair of double-stranded breaks, particularly by homologous recombination.

We have multiple DNA-repair systems, so there is cell backup capacity. If there is loss of one repair mechanism, cell can deal using the other. For example, if BRCA lost function, PARP repair mechanisms are still working.

Synthetic lethality:

If a normal cell has BRCA defect, but PARP working normally, but turns into cancer cell due to multiple mutations. By giving drug that inhibits the PARP can lead to severe DNA damage, that is not repairs - leads to cancer death.