Note: ATM, ATR, and DNA-PK: The Trinity at the Heart of the DNA Damage Response

Note for: ATM, ATR, and DNA-PK: The Trinity at the Heart of the DNA Damage Response

doi: 10.1016/j.molcel.2017.05.015

Freaking complicated and so many gaps in the research as well as the available tool to elaborate precise mechanism.

In general, a cell’s DNA damage response (DDR) involves DNA lesion recognition, followed by initiation of a cellular signaling cascade to promote DNA repair, which can be aided by a pause in cell-cycle progression (checkpoint activation).

In concert with such events, cells mediate other responses, including modulation of chromatin structure and transcription, both at sites of DNA damage and more globally.

DNA damage permanently exit the cell cycle (senescence) or undergo programmed cell death (apoptosis), presumably to mitigate the propagation of potentially mutated cells leading to cancer or other age-related pathologies.

DNA lesions also serve as intermediates in certain biological processes, such as DNA demethylation, meiotic recombination, and generation of antibody diversity in the immune system.

Notably, Ku had been shown to preferentially bind dsDNA ends.

DNA-PKcs was found to be absent in a radiosensitive human glioma cell line. and mutated in the radiosensitive, severe combined immunodeficient (SCID) mouse identified several years earlier. Taken together, these observations

established that DNA-PK was crucial for DSB repair, by a pathway now known as non-homologous end joining (NHEJ).

DNA-PK -- oligomeric protein which includes Ku70/Ku80+PRKDC (DNA-PKcs -- PI3K)

A-T is a rare inherited autosomal-recessive genetic condition, characterized by dilated blood vessels (telangiectasia) and progressive neurological decline, resulting in a lack of voluntary movement coordination, including gait abnormality (ataxia).

This radiosensitivity was also apparent at the chromosomal level, suggesting a connection between the gene product mutated in A-T and DNA repair.

An important subsequent observation was that, while replicative DNA synthesis is markedly inhibited when normal cells are exposed to IR, this does not occur in A-T cells, which instead display radio-resistant DNA synthesis. Later work showed that A-T cells are defective in establishing both the G1/S and G2/M cell-cycle checkpoints in response to IR.

Both reports suggested FRP1/ATR (hereafter ATR) to be the ortholog of Mec1 and Rad3, based on sequence similarity and the fact that the ATR cDNA complemented the radiation hypersensitivity of mec1 budding yeast mutants.

ATM, ATR, and DNA-PKcs are huge polypeptides with similar domain organizations and various common structural features.

Due to their large size, limited three-dimensional structural information is available on full-length ATM, ATR, and DNA-PKcs, with the highest resolution crystal structure for these currently being of DNA-PKcs, at 4.3A°.

DNA-PK revealed its preference for phosphorylating a serine or threonine residue followed by a glutamine (S/T-Q). This was later shown to be the case for ATM and ATR as well.

however, that DNA-PK has also been shown to target Ser/Thr residues within non-S/T-Q contexts in vitro.

Various S/T-Q motifs are present in ATM, ATR, and DNA-PKcs, and all three can autophosphorylate. For DNA-PKcs, multiple autophosphorylation sites have been identified, which mostly cluster in the HEAT-repeat region.

Neither S2056 nor T2609is required for DNA-PKcs kinase activity, but both are important for DNA repair, with current models suggesting that their phosphorylation causes conformational changes that promote DNA-PK disassembly from DSB sites to allow DNA-end ligation.

For ATM and ATR, roles for autophosphorylation are more controversial. Human ATM phosphorylates itself on S1981, an event proposed to transition the kinase from an inactive dimer into active monomers in response to DNA damage.

Further work is clearly needed to elucidate the functions of S1981 and other ATM autophosphorylation events.

One ATR autophosphorylation site is T1989, although there is disagreement about whether T1989 plays a major role in ATR function.

Indeed, each kinase requires a specific protein co-factor for stable recruitment to DNA damage sites, this is NBS1 for ATM, ATRIP for ATR, and Ku80 for DNA-PKcs. Although each kinase requires a distinct factor, a common principle applies to their recruitment because NBS1, ATRIP, and Ku80 all share a related C-terminal motif required for PIKK binding, probably via interactions with the PIKK HEAT repeats.

Only when classical NHEJ fails must cells use an alternative end-joining pathway such as that mediated by DNA polymerase q (POLQ), which can introduce extensive mutations.

NHEJ is initiated by Ku and DNA-PKcs binding to DSBs, with the ensuing DNA-PK holoenzyme promoting DNA-end tethering.

Later on,

- XRCC4

- XLF

- DNA ligase4

Stabilizing protein at damaged chromatin;

- PAXX

- XRCC4-like core NHEJ factor

Some DSBs repaired by NHEJ require additional accessory proteins such as DNA polymerases and nucleases for their repair. One such factor is the endonuclease Artemis, which interacts with DNA-PKcs to promote repair of a subset of DSBs.

However, it is not yet clear whether NHEJ requires DNA-PKcs to phosphorylate any proteins other than itself. However, it is not yet clear whether NHEJ requires DNA-PKcs to phosphorylate any proteins other than itself.

As such DSBs require NHEJ for efficient repair, defects in most core NHEJ factors cause severe immunodeficiency in mammals. PAXX and XLF are exceptional in this regard, as both must be simultaneously lost to produce substantial immune defects in mice due to their partial functional redundancy in

B cells.

While this suggests that NHEJ factors play important roles in repairing DSBs that arise during differentiation of neural progenitor cells, the nature and etiology of such DSBs remain to be determined.

ATM is the apical kinase responsible for global orchestration of cellular responses to DSBs.

ATM --> affect on the global cellular response;

- DNA repiar

- check point activation

- apoptosis

- senescence

- alteration chromatin structure

- alteration transcription

- pre-mRNA splicing

ATM phosphorylates hundreds of substrates in response to DNA damage.

Most ATM substrates are probably also phosphorylated by ATR in response to replication stress and a few, such as histone H2AX, by DNA-PKcs as well.

ATM-dependent signaling events are not just restricted to factors directly phosphorylated by ATM.

While CHK2-T68 phosphorylation is routinely used to indicate ATM activation, it is worth noting that this modification may not be solely ATM dependent in all circumstances.

The tumor suppressor p53 is stabilized in response to DNA damage to activate a transcriptional program that can lead to cell-cycle arrest in G1, senescence, or apoptosis. ATM can phosphorylate p53 on multiple sites.

S15 phosphorylation was proposed to inhibit interaction of p53 with the ubiquitin ligase MDM2, resulting in rapid p53 stabilization and activation in response to DSBs.

It is important to note, however, that ATM controls p53 stability via multiple mechanisms by phosphorylating not just p53 itself but also other proteins that directly or indirectly influence p53 stability.

ATM is recruited to chromatin in response to DSBs,  in a process that requires ATM binding to the C terminus of NBS1, a component of the MRE11-RAD50-NBS1 (MRN) complex.

MRN both recruits ATM to DNA lesions and stimulates ATM kinase activity once there. the exact mechanism whereby MRN activates ATM is still not understood. MRN/DSB-independent means of ATM activation also exist, for example in response to oxidative stress or chromatin changes.

Upon DNA damage, exposed nucleosomes bearing the H3K9me3 histone mark are recognized by the TIP60/KAT5 acetyltransferase, which is thus recruited to damaged chromatin and shielded from dephosphorylation.

While most (~80%) IR-induced DSBs outside of S phase are repaired by NHEJ independently of ATM, a minority is repaired by a pathway requiring ATM, DNA-PK, the MRN complex, and Artemis.

This subset may include DSBs in heterochromatic regions and those with blocked ends that require processing.

Mechanistically, MRN-dependent ATM activation may trigger phosphorylation of DNA-PKcs to promote Artemis recruitment and activation. a model supported by the demonstration that Ku and MRN can simultaneously localize to the same DSB

site in cells.

ATM plays at least one important additional role in NHEJ that is not yet clear but which is redundant with XLF and is therefore not normally apparent.

H2AX and 53BP1, also share redundant functions with XLF in NHEJ, it is possible that the role of ATM in NHEJ relates to DSB-end bridging.

This process, termed DNA-end resection, is a key determinant of DSB repair pathway choice.

ATM-mediated CtIP phosphorylation may also impact on later stages of HR, as it promotes the removal of Ku from single-ended DSB ends at collapsed replication forks to allow the strand invasion step of HR to proceed.

it is important to note that ATM is not essential for HR and that HR-driven processes can take place in its absence.

DSBs trigger rapid phosphorylation of S139 in the C-terminal tail of histone variant H2AX, This modification, termed gH2AX, is primarily mediated by ATM in response to DSBs and forms the foundation of a chromatin-based signaling cascade involving phosphorylation, ubiquitylation, and other post-translational modifications.

MRN recruitment by MDC1 leads to further recruitment of ATM via its interaction with NBS1, leading to additional gH2AX formation and MDC1-MRN-ATM recruitment. thus spreading the assembly along chromatin and amplifying DDR signaling.

that are recognized by the FHA domain of the ubiquitin ligase RNF8, thus promoting RNF8 retention on damaged chromatin. With the ubiquitin-conjugating enzyme UBC13, RNF8 stimulates ubiquitylation of the linker histone H1.

Ubiquitylated H1 is recognized by ubiquitin-binding domains in another ubiquitin ligase, RNF168, which ubiquitylates K15 of H2A-type histones to promote recruitment of the scaffold protein 53BP1 to channel DSB repair toward NHEJ. A second, RIF1, interacts with 53BP1 independently of PTIP but in a manner requiring phosphorylation of the 53BP1 N terminus by ATM; ensuing 53BP1 complexes containing PTIP, RIF1, REV7/MAD2L2, and other factors promote DSB repair by NHEJ through mechanisms that are not yet clear.

ATM, the loss of which also predisposes to breast cancer, functionally phosphorylates BRCA1 on multiple residues in response to DNA damage.

gH2AX-marked chromatin is transcriptionally inactive, possibly to help prevent collisions between DNA repair and transcription machineries.

Depending on its cellular context, transcriptional silencing can depend on ATM, ATR, or DNA-PKcs, with ATM being particularly important for nucleolar DNA silencing.

ATR is the apical DNA replication stress response kinase, phosphorylating many substrates in response to agents such as UV.

ATR is essential in proliferating cells.

ATR is activated by a much wider range of genotoxic stresses. This is because ATR is recruited, via its partner protein ATRIP, to extended tracts of ssDNA coated with the ssDNA binding protein complex, replication protein A (RPA).

The best-characterized ATR activator is TopBP1, which contains an ATR-activation domain that stimulates ATR kinase activity.

As with ATR itself, TopBP1 loss or an inactivating mutation in the TopBP1 ATR-activation domain is lethal in mammalian cells.

TopBP1 and ETAA1 may be recruited to different types of aberrant DNA structures to stimulate ATR toward discrete sets of substrates.

The importance of the ATR-CHK1-CDC25A axis for cell survival is highlighted by the lethality associated with ATR or CHK1 inhibition being circumvented by CDC25A inactivation.

ATR may prevent replication fork collapse primarily through the following mechanisms. First, by inhibiting CDKs, ATR signaling restrains replication origin firing. This is important to avoid exhaustion of replication and repair factor pools, particularly RPA.

Finally, ATR regulates deoxyribonucleotide availability in mammalian cells by promoting upregulation of the ribonucleotide reductase subunit RRM2 at the transcriptional and posttranslational levels in response to DNA damage.

While ATR phosphorylates many of the same substrates as ATM, it may also have unique targets—particularly those functioning in pathways connected to replication fork repair. One of these is the Fanconi anemia (FA) pathway, which promotes repair of DNA interstrand crosslinks (ICLs).

The G1/S, intra-S, and G2/M checkpoints induced by DNA damage signaling are similar in that the endpoint of all three is to inhibit CDK activity. DSBs are not resected to generate significant amounts of RPA-ssDNA in G1. so the G1/S checkpoint is believed to be controlled primarily by ATM rather than ATR.

both ATM and ATR contribute to establishment and maintenance of the intra-S and G2/M checkpoints, in part because ATM-dependent DNA-end resection provides the RPA-ssDNA signal for ATR recruitment and activation.

ATM- and ATR dependent phosphorylation of DNA-PKcs may be important for DNA repair, and recent evidence suggests that DNA-PK also phosphorylates ATM to restrain its activity. Furthermore, ATR has been reported to phosphorylate ATM in

response to UV exposure.

chromosome ends do not trigger DNA damage signaling, as telomeric DNA is bound by the shelterin complex, which inhibits ATM, ATR, and DNA-PK via multiple mechanisms.

Indeed, emerging evidence suggests the spliceosome is both a target and an effector for ATM in response to R-loop formation.

These findings raise the possibility that at least some neurological phenotypes associated with ATM deficiency may be linked to defective resolution of DNA-RNA hybrids.

Chromatin dynamics generate mechanical forces that can be transmitted to the nuclear envelope via lamin-associated chromatin domains.

many viruses encode proteins that specifically inhibit ATM, ATR, or DNA-PK activity, either completely or partially. A common strategy is to hijack cellular ubiquitylation pathways to target PIKKs, or their activator proteins such as TopBP1 and the MRN complex, for proteasomal degradation

As the DDR can be activated at early stages in tumorigenesis due to oncogene-induced replication stress or processes such as telomere shortening, this has suggested that cell-cycle arrest or cell death enforced by PIKK signaling may serve as a barrier to tumorigenesis.

tumor cells are inherently vulnerable to additional replication stress, exogenous DNA damage, or DDR inhibition. This probably in large part explains therapeutic successes with radiotherapy and various DNA-damaging chemotherapeutics and also contributes to the anti-cancer properties of emerging DDR-enzyme inhibitors.

Small molecule inhibitors are now available against all three PIKKs and have entered phase I or II clinical trials both as single agents and in combination with radiotherapy or traditional chemotherapeutics.

ATR is a particularly attractive target for anticancer therapy, as tumor cells are likely to rely heavily on ATR for survival, due to their high burden of replication stress.

Furthermore, genome instability in many tumors is caused by defects in certain DNA repair pathways, such as HR. This offers

the potential for targeted therapies to exploit the concept of synthetic lethality, whereby loss of one cellular pathway results in high reliance on another pathway that is not essential under normal settings.

highlights how loss of ATM and other DDR factors also leads to PARP-inhibitor sensitivity in cancers.

ATM inhibition has, for instance, been suggested as a potential anti-retroviral strategy. hyperactive DDR signaling is associated with various neurodegenerative diseases, as highlighted by recent work showing that ATM inhibition ameliorates pathologies in models of Huntington’s disease. (causing more apoptosis?)

Nevertheless, major questions still remain.

1. One key issue is the current lack of high-resolution DDR-PIKK structures.

2. Another is separating out functionally important phosphorylation events from the many residues that are modified simply because they by chance conform to DDR-PIKK consensus motifs.

New and emerging technologies,

such as

1. super-resolution microscopy,

2. cryo-electron microscopy,

3. CRISPR-Cas9, and

4. affordable high-throughput sequencing, will no doubt help address these and other issues.

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