Double-stranded DNA breaks

Question 1

How do DSB’s occur?

Answer 1

Naturally, during:
• Meiosis
• Generation of immune diversity, both VDJ recombination and class switch recombination
• DNA replication, when the replication fork encounters a nick in one template strand or stalls at fragile sites, or sites of topoisomerase failure
• On insertion of retroviruses or retrotransposons into genomic DNA

Or from environment ionizing radiation:
• These include cosmic rays and radiation from soils. The damage from ionizing radiation can be both direct (deposition of energy) and indirect (clustered oxidative free radicals caused by the energy deposition in turn cause DNA breaks).
• Medical tests and treatments are another significant source of ionizing radiation. Many commonly used imaging techniques including X-rays and CT scans, as well as radiation treatment and some chemotherapeutic agents (e.g. bleomycin) cause DSBs.

Question 2

How to sense DSB’s

Answer 2

Two members of the phosphatidylinositol 3-kinase related kinases (PIKKs) family are principally used to sense DS breaks:

1) ataxia telangiectasia mutated (ATM) and
2) ataxia telangiectasia and RAD3-related (ATR).

These are protein kinases used to signal DNA damage more generally than just double-strand breaks. The disease ataxia telangiectasia causes genetic instability, a predisposition to cancer, and neurodegeneration.

Question 3

How to repair?

Answer 3

1) DNA non-homologous end joining (NHEJ) and 2) homologous recombination (HR); multiple proteins are involved in both.

Question 4

NHEJ

Answer 4

NHEJ proteins are required for resistance to ionizing radiation and for VDJ recombination and immunoglobulin class switching in the immune system. 
 NHEJ simply brings together the broken ends, and ligates them, typically (but not necessarily) losing a few nucleotides (nt) in the process.  
Thus fidelity of this system is imperfect, but its advantages include quick repair at any time in the cell cycle, and the process is not sequence-dependent. 
Recall that the human genome has a lot of space (introns, intragenic regions) where the loss of a few nucleotides here and there is not likely to cause a problem.  
The somatic mutations left behind by imperfections in the NHEJ process accumulate over time; a typical somatic cell in a 70 year old human has ~2000 little “footprints” from this imperfect repair system.

Question 5

HR

Answer 5

HR, on the other hand, occurs during and shortly after DNA replication, in S and G2 phases of the cell cycle.

It absolutely requires nearly identical DNA strands (homologous) of the unbroken sister chromatid.

Using this perfect template, HR repairs the break perfectly, with no gain or loss of nt. Multiple steps are required to repair double-strand breaks by homologous recombination.

1. ) broken ends are resected, i.e., processed to expose single-strands ending in 3’ OHs.
2. ) The single-strands then invade the homologous DNA, using it as a template for synthesis to bridge the gap caused by the break.
3. ) The crossover (Holliday junction) then has to be resolved back to two separate DNA double strands.
4. ) Typically in DNA repair using the identical sister chromatid perfectly restores the break with no exchange. 5.) Different resolutions of Holliday junctions can lead to exchange of genetic information between the two chromosomes undergoing HR.
5. ) This exchange is an essential feature of meiosis and can take two forms, gene conversion or crossing over.

In some cases of misregulated homologous recombination, using the other chromosomal homolog as a template for double-strand break repair instead of the sister chromatid in somatic cells can lead to loss of heterozygosity (LOH) by these same mechanisms.

Question 6

NHEJ vs HR?

Answer 6

Two tumor suppressors, BRCA1 and 53BP1, are pivotal regulators of the choice to repair a DSB using HR or NHEJ, respectively.
BRCA1 is one of the key protein targets of phosphorylation by activated ATM and ATR; phosphorylation recruits BRCA1 to a DSB. Proper control of normal BRCA1 activity is required for DSB repair by HR although the exact role remain a topic of active investigation.

Another important mediator of DS break signaling is 53BP1. 53BP1 plays a crucial role in DSB pathway choice, acting as a positive regulator of NHEJ by promoting the synapsis of distal broken ends and by antagonizing their resection.
Significantly, in BRCA1 mutant mice that exhibit embryonic lethality, tumorogenesis and chromosomal abnormalities, genetic instability can be phenotypically rescued by a 53BP1 deletion.
Importantly, the genetic instability caused by mutations in the core HR machinery, e.g., BRCA2 or XRCC2, are not rescued by a 53BP1 deletion. These findings indicate that, in the absence of HR mediated by BRCA1, 53BP1 promotes deleterious NHEJ; thus, the proteins encoded by BRCA1 and 53BP1 compete to determine the correct DNA DS break repair pathway.

Question 7

What happens if mis-repaired or not repaired?

Answer 7

Mis-repair is the source of genetic instability with translocations being particularly bad.
Loss of HR leads to gross genomic rearrangements and thus genome instability.
Translocations most often occur when DNA DSBs are healed between different chromosomes by NHEJ.

Failure to repair a DSB will lead to cell death, a fact exploited by cancer therapies (ie radiation and drugs that cause DSBs like bleomycin).
DSB repair deficits, which increase cancer susceptibility, can be caused by loss of proteins involved in the signaling (both at the level of sensors and transducers) of the DSBs and in the enzymes that mediate their actual repair.

Question 8

53BP1

Answer 8

Positive regulator of NHEJ

Question 9

BRCA1

Answer 9

leads to HR.

BRCA2 is a key target of Phosphorylation by ATMP/ATR.