Note: DNA double strand break repair pathway choice: a chromatin based decision?

Note for: DNA double-strand break repair pathway choice: a chromatin based decision?

(doi: 10.1080/19491034.2015.1010946)

The choice between these pathways is a critical, yet not completely understood, aspect of DSB repair. DSBs induced across the genome are not repaired by the same pathway. DSBs induced in active genes, naturally enriched in the trimethyl form of histone H3 lysine 36 (H3K36me3), are channeled to repair by HR, in a manner depending on SETD2, the major H3K36 trimethyltransferase. This “decision making” function of preexisting chromatin structure in DSB repair could connect the repair pathway used to the type and function of the damaged region, not only contributing to genome stability but also to its diversity.

Importantly failure or misuse of each of these DSB repair pathways can trigger very different consequences on the genome. NHEJ is the primary cause of translocations and dysfunctional telomeres fusion. HR pathways can be entirely conservative when the sister chromatid is used as a template, dramatic events such as repeat amplification/deletion or loss of heterozygosity (LOH) can occur when

HR operates on repeated sequences or homologous chromosomes.

the improper use of unequal homologous recombination to repair rDNA (rDNA) leads to the production of extrachromosomal rDNA circle (ERC), believed to be toxic for cells and associated with aging.

Clear evidence suggests that both HR and NHEJ can co-exist in the same cell. they compete to some extent for repair of a defined DSB. the choice between C-NHEJ, Alt-NHEJ/MMEJ, and the various HR-related pathways to repair a defined DSB is with no doubt a critical aspect of DSB repair.

However, how this choice is performed is far from understood. Mechanisms that have been proposed to participate in this decision include

1. cell type,

2. age of the cell and

3. cell cycle phase, as well as

4. the persistent nature

5. complexity of a break.

DNA associates with various proteins, mainly histones, to form chromatin, which tightly regulates its accessibility and therefore plays a key role in DNA metabolism. Chromatin is a highly dynamic structure, affected by multiple mechanisms, such as histone posttranslational modifications (i.e. methylation, acetylation, phosphorylation. . .), DNA methylation, incorporation of histone variants or local nucleosome density. chromatin protein occupancies have shown that the genome is divided into distinct functional chromatin “states”, broadening the classical distinction between euchromatin and heterochromatin. Effector proteins will be able to translate such “chromatin signatures” to functional outcomes based on their ability to “read” modified histones by means of a highly specialized protein domain. Such chromatin patterns are very tightly linked with cell identity and disease state -- connect chromatin signatures with specific patterns of gene expression. pre-established chromatin structure could also play an instructive, “decision making” role in addressing adequate DNA repair pathways depending on where a DSB occurs in the genome. Using a human cell line (called DIvA for DSB Inducible via AsiSI) in which multiple annotated DSBs can be induced in a controlled manner using a restriction enzyme -- not so sure whether they randomly integrated and whole genome sequencing to map the location where the DSB could occur once the enzymes has been introduced to the cell.

In agreement with the idea that repair pathway choice depends on the functional properties of the damaged locus, we found that HR-prone DSBs are located in actively transcribed genes and

repair at such DSBs can be switched to RAD51-independent repair pathway upon transcriptional inhibition. Supporting the “chromatin driven DSB repair choice” hypothesis, we found

that active genes are able to recruit the HR machinery thanks to the transcription-elongation associated H3K36me3 histone mark.

even in G2, the vast majority (roughly 85%) of irradiation or drugs induced DSBs are repaired by NHEJ, since active genes and H3K36me3 enriched loci represent only a minor fraction (few percent) of the genome.

all genomic loci are not repaired by the same pathway thanks to a chromatin dependent signaling,

part of which relies on the methylation state of H3K36. DSBs that occur in heterochromatin are repaired by a specific HR pathway (dependent on ATM kinase, Artemis exonuclease, 53BP1, and the RNF168 and RNF8 ubiquitin ligases). NHEJ might be inefficient to repair heterochromatic breaks due to the chromatin compaction observed in such regions. HP1 and H3K9me3 are mainly located in heterochromatin and HAT recruitment has been proposed to facilitate nucleosome removal and resection. Secondly, a fair amount of data describes the function of chromatin in the regulation of the 53BP1/BRCA1 axis. 53BP1 counteracts BRCA1 dependent resection, thus favoring NHEJ.

DSB-induced eviction of H4K20me2 binding proteins, such as L3MBTL1 and JMJD2A/KDM4A, would also permit unmasking of the preexisting H4K20me1/2 marks, allowing for damage-induced 53BP1 recruitment.

H4K16ac, H4K20me1 and H3K36me3 preferentially localize on genes, it will be of major interest now to investigate the interplay between these modifications and how they cooperate to regulate the 53BP1/BRCA1 balance and resection.

First of all, initial recruitment of repair/signaling proteins could be mediated by end detection (by the Ku heterodimer, MRN complex and other DNA end binding proteins). Histone modifications, already available at the site of a break, would next stabilize or destabilize these machineries, helping the cell to fine-tune the repair process at each DSB induced within the genome.

Finally, in the absence of breaks, specific DNA repair proteins might also already be settled on chromatin or scanning certain region in the genome, as instructed by the appropriate chromatin signatures

and be stabilized or activated upon the detection of a DSB.

Genomes do not evolve homogenously. while genes are highly conserved, some intergenic regions exhibit high mutation rates. differences were generally attributed to 2 driving forces: a stronger selective pressure for coding (or regulatory) regions and an increased sensibility of certain loci to damaging agents or oncogenic stresses, defined as “fragile sites”.

Even more importantly, chromatin can be directly modified in response to environmental inputs. opens the possibility that external signals may fine-tune repair accuracy at specific loci and therefore impact genome evolution and organism fitness. That higher eukaryotic genome evolution might also be under the control of the environment, thanks to a fine tuning of DNA repair by a chromatin interface,

represents an exciting area of future investigations.

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