DNA damage and repair

Definitions & Key takeaways

DNA damage is any abnormal change in the DNA sequence that may occur due to environmental factors, such as UV radiation or chemicals. The body's cells have mechanisms to repair this damage, which helps to ensure that damaged DNA doesn't accumulate and results in uncontrolled cell division and tumor formation. DNA repair mechanisms include mismatch repair, base excision repair, nucleotide excision repair, non-homologous end joining, and homologous recombination.

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Our DNA is like a library - found in the nucleus of our cells - with thousands of books. Some of these books - called genes - are extremely important, because they carry the recipes for every single protein found in the cell.
Now, on a molecular level, DNA is made up of two strands of nucleotides, so each gene is just a segment of this nucleotide sequence.
Nucleotides of DNA are made out of a sugar - deoxyribose, a phosphate, and one of the four nucleobases - adenine, cytosine, guanine, and thymine - or, A, C, G, T for short.
The nucleotides on one strand pair up using hydrogen bonds with nucleotides on the opposing strand, to create the double-stranded DNA: specifically, A bonds with T, and C bonds with G, so they’re called complementary bases.
Now, the goal of DNA is to store information and pass it onto their daughter cells, and to use this information to create proteins.
To do this, there are two critical processes - DNA replication and gene expression. DNA replication occurs during the cell cycle - more specifically, during the S phase of interphase.
So, the cell cycle is made up of interphase - when the cell prepares for division - and mitosis - or the actual splitting of the cell in two daughter cell.
Interphase has 3 subphases - G1, S and G2, and during the S subphase, the cell replicates its DNA, so that the two daughter cells get the exact same DNA during mitosis.
If we zoom onto the double- stranded DNA, we can see that during DNA replication, the two DNA strands are separated by an enzyme called DNA helicase.
Then another enzyme, DNA polymerase, uses each of the single strands as a template and adds complementary nucleotides to it.
Gene expression, on the other hand, is the process of decoding the information stored in the DNA in order for the cell to make proteins, and it includes transcription and translation.
Transcription is where RNA polymerase copies the nucleotide sequence of the gene and creates a messenger RNA molecule, or mRNA that has the same sequence, with one tweak: it has uracil nucleotides - or U - instead of thymine.
Now during translation, cell organelles called ribosomes “read” the mRNA molecule in 3 nucleotide “words”, called codons - with each 3 nucleotide sequence coding for an amino acid that will eventually become part of the protein.
So, for the cell to keep functioning, the DNA strands need to remain intact, or at least mostly intact, in order to pass on or express unaltered genetic information.
Unfortunately, the cell is exposed all the time to both endogenous, and exogenous or environmental factors that can damage the DNA.
Luckily, if DNA gets damaged, the cell can enter a special phase outside the cell cycle - the G0 phase - where DNA repair mechanism try to fix to the damage.
If the DNA damage starts to pile up - a cell will typically go down one of three paths. First, the cell might go into senescence - which is when the cell stops dividing.
Second, the cell might undergo apoptosis, which is programmed cell death. Third, the cell might begin to undergo uncontrolled cell division and develop into a tumor.
None of these paths are ideal, so it’s essential for cells to fix reversible DNA damage and prevent too many DNA mutations from occurring.
Broadly speaking, there might be single strand damage - or double strand damage, and the cell has mechanisms to address both situations.
Single strand damage can happen because of endogenous causes - like errors in DNA replication - or exogenous factors - like harmful chemical or physical agents.
Single strand damage is fixed by three repair mechanisms: mismatch repair, base excision repair, and nucleotide excision repair.
During replication, DNA polymerase can sometimes put in the wrong nucleotide - like pairing adenine up with a cytosine instead of a thymine.
This is called a mismatch, and it happens about 60,000 times per replication - so 1 out of 100,000 nucleotides. Now the first way to fix a mismatch is right after it happens - because DNA polymerase is quite a resourceful enzyme, and it can look over its shoulder to check for errors and see if it put the right nucleotide in.
Kinda like checking an essay for typos before sending it in. If DNA polymerase finds a mismatch, it goes back and acts as an exonuclease - meaning, it removes the wrong nucleotide from the newly synthesized DNA strand and replaces it with the correct nucleotide.
This is called proofreading - and while it still leaves some mismatches behind, it reduces the error rate to about 600 times per replication - so 1 out of 10 million nucleotides.
Next is mismatch repair - which relies on special proteins, called MSH proteins - and fixes the remaining errors after replication.
When MSH proteins see a mismatch in a newly synthesized strand, they recruit an enzyme - called endonuclease - that acts like a pair of scissors and severs the nucleotide bonds from the DNA strand.
Then another enzyme - called exonuclease - removes the damaged segment of DNA, leaving a gap in the daughter DNA strand.
Then, DNA polymerase can come in and fill this gap with new nucleotides - kinda like when you have to go back and rewrite a paragraph of your essay if your supervisor tells you there's an error in there.
And finally, once DNA polymerase is done matching correct nucleotides, another enzyme, DNA ligase, seals the bonds and the damage is successfully repaired.
But even mismatch repair leaves a tiny number of errors, which is six errors per cell division - so 1 out of 1 billion nucleotides.
Of course, nothing compared to the original 60,000. Now, base excision repair comes in when the cell's DNA suffers damage from exposure to harmful chemicals or physical factors.
For example, chemicals like nitrites and nitrosamines, which are found in cured or pickled foods, can cause deamination - or the removal of an amino group - from the nitrogen bases.
This changes the molecular structure of the nitrogen base. For instance, deamination transforms cytosine into uracil, guanine into xanthine, and adenine into hypoxanthine, and 5-Methylcytosine - which is a cytosine with a methyl group attached - into thymine.
So, nitrites can make quite a mess with DNA, a bit like a toddler grabbing your computer and changing letters in your document.
Base excision repair relies on enzymes called glycosylases that remove the damaged base and leave behind a tiny gap in the nucleotide, called an AP site - because it is apurinic or apyrimidinic.
In other words, all that’s left of the nucleotide is the sugar and the phosphate group, because it has no base there whatsoever.
Then, an enzyme called AP endonuclease severs the bonds between that phosphate and sugar and the other nucleotides. After that, an exonuclease removes them, leaving a gap in the damaged DNA strand.
Finally, DNA polymerase fills the gap with new nucleotides, and DNA ligase seals the bonds. Nucleotide excision repair, on the other hand, fixes DNA damage when it is caused by physical agents like harmful UV radiation.
UV radiation creates pyrimidine dimers - most often between two adjacent thymines on one of the DNA strands. These two thymines form bonds with each other, distorting the DNA strand in that region of the molecule.
So, nucleotide excision repair relies on endonucleases that make two incisions in the DNA strand - one on the 3’ side of the damage and one on the 5’ side - leaving a fragment of 12 to 24 nucleotides with free ends - so they’re exposed to exonucleases.
Exonucleases then remove these nucleotides. Finally, DNA polymerase inserts new nucleotides, and DNA ligase seals the bonds once again.
Lastly there’s ionizing radiation - like x rays and gamma rays - which can break the phosphate backbones of both DNA strands.
This causes double- strand breaks which are the most deleterious type of DNA damage. Generally, double- stranded DNA breaks aren’t clean, meaning that each end of the broken DNA has a single- stranded region that overhangs the rest of the double- stranded DNA.
It’s like when you break a pencil with your hands, the rest of the pencil remains intact, but the part where the break happened is jagged.
Now, to fix double- strand breaks, there’s two main repair mechanisms. The most common type is non-homologous end joining, where a protein complex called DNA protein kinase begins by binding to each end of the broken DNA.
Then it recruits another protein, called artemis - named after the Greek goddess! - to cut off the single- stranded ends.
It’s like using a tiny bit of sandpaper to smooth the broken ends of the pencil, so that the pencil can be more easily glued together again.
Finally, a ligase enzyme - which would be the glue - binds the two ends of DNA. Since artemis cuts off some nucleotides, non-homologous end joining is an error prone repair - that leads to a loss of genetic information.
The other repair mechanism called homologous recombination , which relies on homologous recombination. Our 46 chromosomes come in 23 pairs of two homologous chromosomes - which code for the same traits, and therefore have similar nucleotide sequences.
As a result, a double strand break on one of the chromosomes can be repaired by using the sister chromatid! First, a protein complex - called MRN - binds to each end of the broken DNA and recruits exonucleases that remove nucleotides from one strand of the DNA.
To make the process more clear, we can call the ends of the broken DNA “end 1” and “end 2”. So now, “end 1” is placed near a similar nucleotide sequence called homologous sequence - because it’s found in the same spot on the homologous sister chromatid.
“End 1” then pairs up with the complementary strand of the intact homologous DNA region, creating a loop in the homologous DNA.
Then, a DNA polymerase synthesizes nucleotides to extend “end 1”, until it reaches a sequence that is complementary to “end 2”.
Then, end 1 releases the homologous DNA and its last few nucleotides bind to the last nucleotides of end 2. Finally, DNA polymerase fills the gaps on both sides of the union, and DNA ligase seals the bond.
Since homologous recombination uses a sister chromatid as a template, this is a more reliable repair mechanism than non-homologous end joining, because there’s no loss of nucleotides.
Alright, as a quick recap… DNA repair mechanisms help prevent accumulation of DNA damage and avoid senescence, apoptosis, or uncontrolled cell division and tumor formation.
Single strand DNA breaks can be repaired using mismatch repair - which fixes nucleotide mismatches that DNA polymerase didn’t correct, base excision repair - which fixes deamination damage caused by chemicals, and nucleotide excision repair - which fixes pyrimidine dimers caused by UV radiation.
Double strand DNA breaks can be fixed by non-homologous end joining and homologous recombination.