F2023_BIS2A_Singer_DNA_Repair_and_Recombination

Cellular Stress and DNA Damage Response

In the previous section, we learn how the cell cycle checkpoint allows timely progression of the cell cycle in undamaged cells. As described in this section, the cell cycle checkpoint has another critical function in response to cellular stress and DNA damage.

Genetic damage produced by either exogenous or endogenous mechanisms represents an ongoing threat to the cell. To preserve genome integrity, eukaryotic cells have evolved repair mechanisms specific for different types of DNA Damage. However, regardless of the type of damage a sophisticated surveillance mechanism, that elicits DNA damage checkpoints, detects and signals its presence to the DNA repair machinery. DNA damage checkpoints have been functionally conserved throughout eukaryotic evolution, with most of the relevant players in the checkpoint response highly conserved from yeast to humans. Checkpoints are induced to delay cell cycle progression and to allow cells time to repair damaged DNA prior to DNA replication. Once the damaged DNA is repaired, the checkpoint machinery triggers signals that will resume cell cycle progression. Within cells, multiple pathways contribute to DNA repair, but independent of the specific repair pathway involved, three phases of checkpoint activation are traditionally identified: (1) Sensing of damage, (2) Activating the signaling cascade, and (3) Switching on downstream effectors. The sensor phase recognizes the damage and activates the signal transduction phase to block cell cycle progression and select the appropriate repair pathway.

In multicellular organisms, the response to DNA damage can result in two major physiological consequences: (1) Cells can undergo cell cycle arrest, repair the damage and re-enter the cell cycle, or (2) cells can be targeted for cell death (apoptosis) and removed from the population. The cell cycle process is highly conserved and precisely controlled to govern the genome duplication and separation into the daughter cells. The cell cycle consists of four distinct and ordered phases, termed G0/G1 (gap 1), S (DNA synthesis), G2 (gap 2), and M (mitosis). Multiple checkpoints exist within each stage of the cell cycle to ensure the faithful replication of DNA in the S phase and the precise separation of the chromosomes into daughter cells. The G1 and G2 phases are critical regulatory checkpoints, whereby the restriction point between the G1 and S phase determines whether the cells enter the S phase or exit the cell cycle to halt at the G0 phase.

 

Figure 1: The cell cycle is controlled at three checkpoints. Integrity of the DNA is assessed at the G₁ checkpoint. Proper chromosome duplication is assessed at the G₂ checkpoint. Attachment of each kinetochore to a spindle fiber is assessed at the M checkpoint.

Repair of Double-Stranded DNA Breaks

Cells have evolved two main mechanisms to repair double-strand breaks within the DNA: the non-homologous end-joining (NHEJ)(Figure 2), that ensures direct resealing of DNA ends; and the homologous recombination (HR) (Figure 3) that relies on the presence of homologous DNA sequences for DSB repair. NHEJ and HR are regulated based on the cell cycle.

Figure 2: Non-homologous end-joining (NHEJ)

NHEJ repair is the simplest and most widely utilized mechanism to repair DSB that occur in DNA. Repair by NHEJ involves direct resealing of the two broken ends independently of sequence homology. Although being active throughout the cell cycle, NHEJ is relatively more important during G1 phase.  NHEJ protects genetic integrity by rejoining broken strands of DNA that may otherwise be lost during DNA replication and cell regeneration. However, during the process of NHEJ, insertions or deletions within the joined regions may occur.

Figure 3: Homologous recombination (HR)

In contrast to NHEJ, homologous recombination (HR) requires a homologous DNA sequence to serve as a template for DNA-synthesis-dependent repair and involves extensive DNA-end processing. As expected, HR is extremely accurate, as it leads to precise repair of the damaged locus using DNA sequences homologous to the broken ends. In somatic cells, HR predominantly uses the sister chromatid as a template for DSB repair, rather than the homologous chromosome. Correspondingly, HR is largely inhibited while cells are in the G1 phase of the cell cycle, when the sister chromatid has not yet been replicated. HR repair mechanisms play a bigger role in DSB repair that occurs after S-phase DNA replication (S-phase, G2, and M).

Importantly, HR is not only employed to repair DNA lesions induced by DNA damaging agents but is also essential for proper genetic recombination and chromosome segregation during meiosis. In meiosis, HR uses homologous chromosomes as a template for programmed DSB repair, developmentally controlled at the onset of meiosis (we will learn meiosis in the next section).

CRISPR Genome editing

NHEJ and HR are the mechanisms we use for the CRISPR-genome editing. The development of tools that would allow scientists to make specific, targeted changes in the genome has been the Holy Grail of molecular biology. An ingenious new tool that is both simple and effective in making precise changes is poised to revolutionize the field, much as PCR did in the 1980s. Known as the CRISPR/Cas9 system, and often abbreviated simply as CRISPR, it is based on a sort of bacterial immune system that allows bacteria to recognize and inactivate viral invaders.

CRISPR stands for Clustered Regularly Interspaced Short Palindromic Repeats, short repeated sequences found in prokaryotic DNA, separated by spacer sequences derived from past encounters with, for example, a bacteriophage. Like the glass slipper left behind by Cinderella that was later used to identify her, the pieces of the invader's sequences are a way for the bacteria to identify the virus if it attacks again. Inserted into the bacterial genome, these sequences can later be transcribed into a guide RNA that matches, and base-pairs with, sections of the viral genome if it was encountered again. A nuclease associated with the guide RNA then cleaves the sequence base-paired with the guide RNA. (The nucleases are named Cas for CRISPR-associated.)

The essential elements of this system are a guide RNA that homes in on the target sequence and a nuclease that can make a cut in the sequence that is bound by the guide RNA. By engineering guide RNAs complementary to a target gene, it is possible to target the nuclease to cleave within that gene. In the CRISPR/Cas9 system, the Cas9 endonuclease cuts both strands of the gene sequence targeted by the guide RNA. This generates a double-strand break that the cell attempts to repair.

Figure 4: A guide RNA directs the Cas9 nuclease to its target gene. Image by Pehr Jacobsen

As mentioned above, double-strand breaks in DNA can be repaired by simple, NHEJ or by HR. When a break is fixed by NHEJ, there is good chance that there will be deletions or insertions that will inactivate the gene they are in. Thus, targeted cleavage of a site by CRISPR/Cas9 can easily and specifically inactivate a gene, making it easy to characterize the gene's function.

But, what if you wished to simply mutate the gene at a specific site to study the effect of the mutation? This, too, can be achieved. If a homologous sequence bearing the specific mutation is provided, homologous recombination can repair the break, and at the same time insert the exact mutation desired. It is obvious that if you can insert a mutation as just described, it should be possible to correct a mutation in the genome by cleaving at the appropriate spot and providing the correct sequence as a template for repair by homologous recombination. The simplicity of the system holds great promise for curing genetic diseases.

Link to external resources

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Cell cycle control | Cells | MCAT | Khan Academy

https://www.youtube.com/watch?v=542CMooowNY

p53 Tumour Suppressor and MDM2

https://www.youtube.com/watch?v=81NCnoRlbGI

What happens when your DNA is damaged? - Monica Menesini

https://www.youtube.com/watch?v=vP8-5Bhd2ag

Gene Editing Mechanism of CRISPR-Cas9 

https://wyss.harvard.edu/media-post/gene-editing-mechanism-of-crispr-cas9/

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