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Peer Essay #6
It is commonplace for a medical student to associate homologous recombination with genetic diversity and inheritance. However, an additional role that this mechanism plays in the cell is to repair DNA, specifically when both strands of DNA have been damaged. These particularly severe breaks are caused by exogenous, environmental factors, such as exposure to chemical agents, radiation, or ultraviolet light, or endogenous processes such as the accumulation of reactive oxygen species as by-products of cell metabolism, or the replication of DNA across a nick. These kinds of breaks occur most often when the cell is in the S phase of the Cell Cycle, when the cell is duplicating its DNA. This is because DNA replication happens very quickly and is therefore prone to making errors (Negritto, M.C.).
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Two separate mechanisms exist for repairing double stranded DNA breaks: homologous recombination (HR) and nonhomologous DNA end joining (NHEJ). Whether the cell chooses one method or the other depends on the proximity of a homologous piece of DNA. The cell is most likely to use HR once the it has undergone the S phase of the cell cycle. A cell in the G1 phase, which has not yet replicated the DNA, will most likely use NHEJ to repair double stranded breaks, while cells in the S or G2 phases can easily access the recently produced sister chromatid and will use HR as the primary pathway. NHEJ is preferred when double stranded breaks occur without the convenience of a nearby chromatid (Lieber).
In the NHEJ mechanism, the protein Ku binds to the DNA ends and serves as a dock for other repair proteins including a nuclease known as Artemis, ligase, and polymerase. These can be recruited in any order, attributing to the mechanism’s flexibility and ability to repair many different DNA breaks. As a general reminder, nucleases cleave DNA into smaller fragments, ligases join DNA strands, and polymerases essentially build the DNA through nucleotide assembly. This non-specific and generalized mechanism is advantageous due to its flexibility and efficiency. Since it requires no additional genetic material, it is always an available option for the cell regardless of cell cycle stage and can therefore be thought of as a backup plan if HR fails (Lieber).
However, a disadvantage of NHEJ is that the outcome may differ from the original DNA genome. There is a greater chance of heterogeneity due to the variable number of nucleotides that could have been removed from the DNA and therefore variable amounts of nucleotide addition to the DNA ends. This can lead to inaccuracy during repair with an increased number of mutations or DNA losses (Lieber).
Figure 1: NHEJ mechanism with potential outcomes, underscoring its variability (Lieber).
The other repair mechanism, HR, is used when sister chromatids are available to copy. This process can ensure a more accurate DNA repair as ideally an identical piece of DNA is inserted into the area of breakage. First, the MRN complex binds to each loose end of the broken DNA to initiate a signaling sequence that will result in the repairing of the DNA. The three proteins that make up the complex are Rad50, Mre11 (Meiotic Recombination Protein), and NBS-1 (NiJmegan Breakage Syndrome-1). This enables the activation of ATM, a serine-threonine kinase enzyme, to phosphorylate a histone protein, H2AX. The becomes γH2AX causing a change in the conformation of the DNA complex which can be amplified across the DNA. Additionally, γH2AX can recruit many different types of proteins including CHK2 (Checkpoint Kinase 2 protein) which can activate p53- a protein which can increase the expression of genes involved with DNA repair ([Ben1994]).
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Meanwhile, on each side of the breakage, the protein Ctlp binds to the MRN complex and creates an endonuclease cut about 100-200 base pairs from the breakage. This cut is very specific as it only happens on one strand of the double stranded DNA and on the end that specifically has its 5’ end facing into the broken edge. As the MRN complex travels back to the original breakage site, it will cut off the remaining nucleotides ranging from the endonuclease cut to the break. By the end of the MRN complex action, there will be an unpaired DNA strand on each side of the breakage known as the 3’overhang that is about 1,000 base pairs long. As soon as the DNA is unpaired, a protein called RPA will bind as protection until another protein, Rad51 can replace it. The proteins that assist in this switch are BRCA1 and BRCA2. Once Rad51 replaces RPA, the structure becomes a Rad51-single stranded DNA nucleofilament, and it is officially ready to take part in homologous recombination. To do this, the sister chromatid that has been made in the S phase of the cell cycle will be implemented. In a process known as Strand Invasion, the Rad51 protein will catalyze the opening of the sister chromatid and the binding of the 3’ overhang to the identical genomic portion. This is an extremely important step in the mechanism as Rad51 is responsible for finding the correct genetic sequence on the sister chromatid using the free end of the 3’ overhang. Once it has been found, it is possible to use the sister chromatids as a template for extension. The structure that is created as a result of this binding is called the D-loop. Once the D-loop has been created, the original 3’ overhang will be extended, by the means of complementary base pairing with the sister chromatid D-loop. This is known as Strand Extension, and it is executed by DNA polymerase enzymes. As the nucleotides are added to the 3’ overhang, the chromatid will peel itself off the newly extended DNA to rejoin with its original DNA strand. Ideally, enough of the 3’ overhang will be synthesized to rejoin with the 3’ overhang on the other side of the DNA break without damaging the other chromosome. The final step is for DNA polymerases and ligases to fill in the final gaps surrounding the joined newly made DNA and other 3’ overhang ([Ben1994]).
This is advantageous for the cell because of its accuracy as the original genome could be synthesized despite the double stranded break and potential loss of nucleotides. However, it is only possible if a sister chromatid is in proximity, so it is fairly limited in terms of its commonality (Group 6 Discussion).
BRCA1 and BRCA2 were only briefly mentioned above, but they play a critical role in this process by regulating the binding of Rad51 to the single stranded 3’ overhang. Without their contributions, the cell would not be able to properly undergo homologous recombination thereby sentencing the cell to make repairs using only NHEJ, the quick but sloppy approach. Repairing DNA with only NHEJ leads to increased likelihood of cancer as mutations are more probable. This outcome is more likely to occur in people who are heterozygous for the mutated version of BRCA1 or BRCA2, otherwise known as a germline mutation. It only takes one somatic mutation, acquired through life experience, for their only working copies of the proteins to be disabled (Group 6 Discussion).
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