Note: DNA Damage Foci: Meaning and Significance

Note: DNA Damage Foci: Meaning and Significance

(doi: 10.1002/em.21944)

Basically, it can reflect DNA damage response but it cannot be solely related to DSB.

(ionizing) radiation-induced foci (IRIF or RIF) or DNA repair foci. the histone variant H2AX which gets phosphorylated at its C-terminal Ser-139 residue by the DNA damage-activated kinases ATM, ATR, and DNA-PK, to form gH2AX. gH2AX then acts as a docking station for other DNA damage signaling factors such as MDC1 and 53BP1 which accumulate to form foci in a histone-modification-dependent manner.

Foci can also be analysed using fluorescent protein fusion constructs, enabling foci formation and loss to be monitored in live cells. As scoring is severely influenced by staining quality and imaging characteristics, it is good practice to include positive and negative reference samples which help confirm the validity and reproducibility of the results obtained in a particular experiment. Intensity-based approaches such as flow cytometry or Western blotting are also commonly used to study foci-forming DNA damage response proteins. total abundance of the protein or modification, are typically less sensitive than imaging approaches, and blind to the intranuclear spatial distribution of the proteins of interest.

DSBs occur when the two complementary strands of the DNA are broken within a distance of a few base pairs. DSBs can be induced by exogenous agents such as ionizing radiation, chemicals, anti-cancer drugs and environmental stress or endogenously as a result of reactive oxygen species (ROS) produced during normal cell metabolism or when DNA replication forks collapse.

As radiation-induced gH2AX foci tend to colocalise very reliably with 53BP1 and ATM-pS1981, these other DNA damage response proteins can be used as alternative or, in situations where accuracy is of crucial importance, as additional markers of double-strand breaks through co-immunostaining. quantitative and spatio-temporal inconsistencies in the relationship between foci and double-strand breaks and heterogeneous foci dynamics within the nucleus have been reported and some of the underlying issues have been explored at least for ionizing radiation.

Foci formation issue;

1.heterogeneous distribution of the H2AX histone in the nucleus 

2.the delay between DSB induction and the formation of microscopically visible foci

3.pan-nuclear H2AX phosphorylation and

4.MDC1 recruitment following localised induction of complex DNA damage

5.expulsion of DNA damage foci from heterochromatin

6.the possible coalescence of multiple foci in close proximity into one

The situation is even less clear for “spontaneous” foci and those triggered by other stimuli, whether intrinsic (e.g. replication stress, aging, oxidative damage, DNA metabolism), or extrinsic (e.g. ultraviolet radiation, chemical exposures).

Most genotoxins induce foci only as secondary events, when unrepaired DNA lesions cause replication forks to stall and/or collapse in cells passing through S phase following exposure. Also it is not absolutely clear whether such secondary foci do in fact always reflect the presence of DSB. For example, ageing haematopoietic stem cells have been reported to harbor replication stress-induced nucleolar gH2AX foci which persist owing to ineffective H2AX dephosphorylation by mislocalized PP4c phosphatase

rather than ongoing DNA damage.

However, there is still no proof that these persistent foci do indeed reflect unrepaired DSB. The lack

of a reliable, alternative assay for the sensitive DSB detection means that this ambiguity is likely to remain unresolved for the foreseeable future, although promising complementary DSB detection methods have been reported recently.

DSB repair deficiency has been associated with chromosomal breaks and translocations resulting in cell death, cell transformation and tumorigenesis, developmental defects, neurodegeneration, immunodeficiency, radiosensitivity, sterility, and cancer disposition. NHEJ involves three steps which result in the ligation of two DNA ends in close proximity: (a) recognition of break ends and their binding by the Ku subunit of the DNA-dependent protein kinase (DNA-PK), (b) removal of nonligatable termini, and (c) joining of the ends by DNA ligase IV, supported by the scaffold proteins XLF and XRCC4. These pathways are evolutionary conserved in eukaryotes, but their significance differs between species and changes during the cell cycle. For example, HR is favored in simple eukaryotes, such as yeast and is generally more active during or after DNA replication. On the other hand, NHEJ is the dominant pathway in mammals and is active throughout the cell cycle, whereas HR is active only in the S- and G2-phases when a sister chromatid is available.

PARP-1, DNA ligases 1 and 3 as well as XRCC1 have been found to contribute to backup end joining.

These alternative DSB repair processes are thought to gain special importance in tumour cells, in which the canonical DNA damage signaling and repair pathways are often disturbed.

Apart from the cell cycle position, discussed above, DSB repair pathway choice and the composition of DNA damage foci may also be influenced by the nature of DSB ends as well as by the localisation of the DSB within the nucleus and its chromatin context.

The chemical nature of DSB ends can have a major impact on their processing with NHEJ being inhibited by modified/damaged DNA ends, but also by those with extended single-stranded DNA overhangs.

A range of specialised enzymes act to process DNA ends to restore them to a ligatable state and at the same time determine the choice of repair pathway to be utilized.

The position of a DSB within the nucleus also affects how it is repaired. DSBs located at the nuclear membrane, but not at nuclear pores or in the centre of the nucleus, were shown not to activate the canonical DNA damage response and to be repaired by alternative end-joining.

DNA damage signaling and foci dynamics differ significantly for DSBs located in different chromatin environments such as hetero- and euchromatin, and are also affected by the transcriptional status.

some of the key proteins used in DNA damage foci assays, i.e. gH2AX, 53BP1, and ATM, are expendable for the bulk repair of most DSBs induced by ionizing radiation, with only a small fraction of repair events appearing to require these factors.

This surprising finding illustrates the high level of redundancy and wide range of back-up options available to the cell. Therefore, the induction of gH2AX may serve as a surrogate marker of DNA damage in general but may not always be associated with DSBs. however, gH2AX seems to contribute much more critically to the response to DSBs than to other DNA lesions, and specifically to a certain subset of DSBs that also require ATM, MRE11, NBS1, 53BP1, and Artemis for their repair.

It should always be kept in mind that gamma- H2AX foci represent dynamic events of continued phosphorylation by the DNA damage kinases ATM, DNA-PK, and ATR and dephosphorylation by a range of phosphatases. Just because foci persist this does not necessarily mean that the underlying DSBs are not repaired, just that the foci have not yet been dephosphorylated.

a deficiency in RAD51 focus formation, such as that observed in rad51d null mutants, is not necessarily associated with increased cellular sensitivity to agents that block replication. a highly complex picture emerges, in which the choice of DSB repair pathway is made separately for each DSB, depending on a combination of criteria and regulated by a functional network of protein phosphorylation and ubiquitilation in a chromatin context.

DNA damage foci, and especially gH2AX foci in peripheral white blood cells, are promising biomarkers in biological dosimetry where radiation exposures need to be estimated retrospectively. The growing

interest in combined treatment modalities and personalized therapies will create an ever increasing demand for reliable markers of individual exposure and effect, which DNA damage foci assays will help to address. Additional effort on standardisation and regular performance testing will be required to fully establish DNA damage foci assays as routine biodosimetric tools. gH2AX is increasingly employed as a biomarker for DSB in environmental, occupational and clinical toxicology.

However, it is important to keep in mind that, whilst DSBs tend to be closely associated with DNA damage foci in most situations, there are cases where one may be present without the other. In cells undergoing apoptosis H2AX phosphorylation occurs in an intranuclear shell. This response, which microscopic analysis can easily distinguish from foci formation, may serve as an additional pharmacodynamics biomarker for anticancer therapies.

DSB repair measured using DNA damage foci in ex vivo or in vivo-irradiated peripheral blood lymphocytes has also been proposed as a predictive marker of individual risk of oral mucositis in head and neck cancer radiotherapy patients.

The specific utility of gH2AX as a prognostic biomarker in lung cancer has recently been proposed. DNA damage foci assays have been used to study the relationship between DNA repair and radiotherapy fraction size sensitivity. RAD51 foci based functional assays are being developed to profile HR repair pathway activity in tissue biopsies and enable the selection of patients with HR-deficient tumours for specific treatments such as PARP inhibitors.

A radiation-induced bystander effect (RIBE) was first described as a radiation-induced DNA damage response in cells adjacent to directly targeted cells, manifesting as increased yields of micronuclei, sister chromatid exchanges, apoptosis, mutations, genomic instability and neoplastic transformation.

gH2AX foci induction in distant proliferating tissue was reported as manifestation of a systemic tumour-induced bystander effect caused by the presence of a malignant tumour. DSBs caused by persistent genotoxic stress. In pre-cancerous tissues it was shown that increased numbers of DNA double-strand breaks demonstrated by 53BP1 foci accumulation were associated with DNA replication stress.

Beyond the technical issues, there are still a number of fundamental gaps in our understanding of the meaning and significance of DNA damage foci, especially in situations where foci form as secondary events as a consequence of the cellular response to non-DSB damage.

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