DNA Breaks and Repair
Nucleic Acids Metabolism

Author: Gianpiero Pescarmona
Date: 18/04/2009


The maintenance of genome integrity and fidelity is essential for the proper function and survival of all organisms. This task is particularly daunting due to constant assault on the DNA by genotoxic agents (both endogenous and exogenous), nucleotide misincorporation during DNA replication, and the intrinsic biochemical instability of the DNA itself.

Failure to repair DNA lesions may result in blockages of transcription and replication, mutagenesis, and/or cellular cytotoxicity. In humans, DNA damage has been shown to be involved in a variety of :

  • genetically inherited disorders
  • aging
  • carcinogenesis.

DNA damage


Direct Reversal The simplest of the human DNA repair pathways involves the direct reversal of the highly mutagenic alkylation lesion O6-methylguanine (O6-mG) by the product of the MGMT gene (O6-methylguanine DNA methyltransferase).26 The O6-mG adduct is generated in low levels by the reaction of cellular catabolites with the guanine residues in the DNA.
Correction of the lesion occurs by direct transfer of the alkyl group on guanine to a cysteine residue in the active site of MGMT in a “suicide” reaction. The inactivated alkyl-MGMT protein is then degraded in an ATP-dependent ubiquitin proteolytic pathway.
This energetically expensive repair mechanism for the correction of a relatively simple alkyl-adduct implies O6-mG is extremely detrimental to the cell. Accordingly, a number of chemotherapeutic agents that attack the O6 position of guanine have been developed and are in clinical use.

BER Base excision repair (BER)

BER is a multi-step process that corrects non-bulky damage to bases resulting from oxidation, methylation, deamination, or spontaneous loss of the DNA base itself.30 These alterations, although simple in nature, are highly mutagenic and therefore represent a significant threat to genome fidelity and stability.

Nucleotide excision repair (NER)

NER is perhaps the most flexible of the DNA repair pathways considering the diversity of DNA lesions it acts upon. The most significant of these lesions are pyrimidine dimers (cyclobutane pyrimidine dimers and 6-4 photoproducts) caused by the UV component of sunlight. Other NER substrates include bulky chemical adducts, DNA intrastrand crosslinks, and some forms of oxidative damage. The common features of lesions recognized by the NER pathway are that they cause both a helical distortion of the DNA duplex and a modification of the DNA chemistry.


The DNA mismatch repair (MMR) pathway plays an essential role in the correction of replication errors such as base-base mismatches and insertion/deletion loops (IDLs) that result from DNA polymerase misincorporation of nucleotides and template slippage, respectively. Mispairs generated by the spontaneous deamination of 5-methylcytosine and heteroduplexes formed following genetic recombination are also corrected via MMR. Defects in this pathway result in a so-called mutator; cellular phenotype characterized by elevated frequencies in spontaneous mutations and increased microsatellite instability (MSI).

Mutations in several human MMR genes cause a predisposition to hereditary nonpolyposis colorectal carcinoma (HNPCC), as well as a variety of sporadic tumors that display MSI.

h4. DSB

Repair Double-strand breaks (DSBs) are perhaps the most serious form of DNA damage because they pose problems for transcription, replication, and chromosome segregation. Damage of this type is caused by a variety of sources including exogenous agents such as ionizing radiation and certain genotoxic chemicals, endogenously generated reactive oxygen species, replication of single-stranded DNA breaks, and mechanical stress on the chromosomes. DSBs differ from most other types of DNA lesions in that they affect both strands of the DNA duplex and therefore prevent use of the complementary strand as a template for repair (see BER, NER, and MMR). Failure to repair these defects can result in chromosomal instabilities leading to dysregulated gene expression and carcinogenesis. To counteract the detrimental effects of these potent lesions, cells have evolved two distinct pathways of DSB repair

  • homologous recombination (HR)
  • non-homologous end joining (NHEJ)

The cellular decision as to which pathway to utilize for DSB repair is unclear, however, it appears to be largely influenced by stage within the cell cycle at the time of damage acquisition.

HR-directed repair corrects DSB defects in an error-free manner using a mechanism that retrieves genetic information from a homologous, undamaged DNA molecule. The majority of HR-based repair takes place in late S- and G2-phases of the cell cycle when an undamaged sister chromatid is available for use as repair template. The RAD52 epistasis group of proteins, including RAD50, RAD51, RAD52, RAD54, and MRE11 mediate this process. The RAD52 protein itself is thought to be the initial sensor of the broken DNA ends. Processing of the damaged ends ensues resulting in the production of 3’ single-stranded DNA (ssDNA) overhangs. The newly generated ssDNA ends are bound by RAD51 to form a nucleoprotein filament. Other proteins including RPA,
RAD52, RAD54, BRCA1, BRCA2, and several additional RAD51-related proteins serve as accessory factors in filament assembly and subsequent RAD51 activities.The RAD51 nucleoprotein filament then searches the undamaged DNA on the sister chromatid for a homologous repair template. Once the homologous DNA has been identified, the damaged DNA strand invades the undamaged DNA duplex in a process referred to as DNA strand exchange. A DNA polymerase then extends the 3’ end of the invading strand and subsequent ligation by DNA Ligase I yields a heteroduplexed DNA structure. This recombination intermediate is resolved and the precise, error-free correction of the DSB is complete.

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DSB and repair


Homologous recombination (HR) takes place in late S–G2 phase and involves the generation of a single-stranded region of DNA, followed by strand invasion, formation of a Holliday junction, DNA synthesis using the intact strand as a template, branch migration and resolution. Non-homologous end-joining (NHEJ) takes place throughout the cell cycle and involves
binding of the KU heterodimer to double-stranded DNA ends, recruitment of DNA-PKcs (officially known as protein kinase, DNA-activated, catalytic polypeptide (PRKDC)), processing of ends (including Artemis-dependent processing) and recruitment of the DNA ligase IV (LIG4)–XRCC4 complex, which brings about ligation. The ataxia telangiectasia mutated (ATM) protein affects NHEJ as it is required for Artemis-dependent end processing. ATM signalling is the main signal-transduction process that responds to a DSB.

Ataxia telangiectasia and RAD3-related (ATR) signalling is activated later after irradiation, probably when radiation-induced lesions block replication. Both pathways lead to cell-cycle-checkpoint activation, which allows more time for repair or permanently prevents the proliferation of damaged cells. An important step in ATM- and ATR-dependent signalling is phosphorylation of H2A histone family, member X (H2AFX) and recruitment of the mediator proteins, mediator of DNA damage checkpoint 1 (MDC1), tumour protein 53 binding protein 1 (TP53BP1) and the MRN (MRE11–RAD50–NBS1) complex.

Additional proteins, such as structural maintenance of chromosomes 1 (SMC1), are also recruited to the site of damage. Other proteins that are recruited to the foci, such as replication protein A (RPA) and Bloom syndrome (BLM), have not been shown for clarity. ATR signalling is activated by single-stranded regions of DNA that arise, for example, at stalled replication forks.

RPA coats single-stranded DNA and recruits ATR and its partner ATRIP (ATR-interacting protein). H2AFX phosphorylation, recruitment of mediator proteins and downstream phosphorylation occur in a process that is similar to the ATM-dependent pathway, although with some distinctions (for example, CHEK1 and CHEK2 (checkpoint 1 and 2) might be differently
phosphorylated by ATM versus ATR). HR probably depends on ATR, either to stabilize stalled replication forks and/or to activate the process of HR. DNA crosslinks are repaired through a process that probably generates a DSB as an intermediate. b | DDR syndromes in which pathways that respond to DSBs are affected. AT, ataxia telangiectasia; ATLD, ataxia telangiectasia-like disorder; FA, Fanconi anaemia; FANCD2, Fanconi anaemia complementation group D2; NBS, Nijmegen breakage syndrome; PNK, polynucleotide kinase.

Pathways that respond to DSB


ATM is a nuclear protein kinase with a catalytic domain similar to the one of phosphatidylinositol 3-kinases (PI 3-kinases). This protein was discovered as a mutated proteins in patients with ataxia-telagiectasia (A-T), a sever genetic disorder characterized by cerebellar degeneration, neuromotor dysfunction, chromosomal instability, immune system defects, cancer
predisposition, and acute sensitivity to ionizing radiations.
In undamaged cells ATM is present as a dimer or ologomer molecule in which the kinase domain is silent because associated with the FAT region of another ATM monomer. Following DSB formation, ATM rapidly autophosphorylates on residue Serine 1981, and the inactive ATM dimers are converted (dissociated) into active ATM monomers. Active phosphorylated ATM molecules can now interact and phosphorylate downstream proteins that affect one or more of the cell cycle checkpoints. Some of the known substrates are the p53 protein and its ubiquitin ligase, MDM2; the Nbs1 protein; the Brca1 protein, which interacts with other repair proteins; the checkpoint kinase 2, Chk2; the Rad17 protein and the chromatin remodeling protein SMC1.

Because of the involvement of ATM in several downstream pathways, the phosphorylation of one substrate does not provide an accurate picture of the ATM-mediated cellular response to DNA damage. Moreover, a comparison between the signaling pathway of ATM and those of other PI-3-like kinase domain proteins * ATR and DNA-PK * has shown no clear differentiation between the signals.
Several downstream steps are commonly shared by these molecules, suggesting a common underlying mechanism for repairing damaged DNA.

fig.8 A model for activation of ATM by DSBs and other forms of DNA damage.


ATM resides predominantly in the nucleus in dividing cells, and responds swiftly and vigorously to DSBs by phosphorylating numerous substrates (see below). ATM-mediated phosphorylation either enhances or represses the activity of its targets, thereby affecting specific processes in which these proteins are involved. Similar to other active PIKKs (with the exception
of mTOR/FRAP), ATM targets serine or threonine residues followed by glutamine (the SQ/TQ; motif). The hallmark of ATMs response to DSBs is a rapid increase in its kinase activity immediately following DSB formation. Researchers have long been impressed by the rapid phosphorylation of the many ATM substrates, which converts them within minutes to phosphorylated derivatives. A marked change in the activity of ATM would account for this massive process. Initial evidence indicated that ATM activation might involve autophosphorylation. A breakthrough in our understanding of this process came in a landmark publication by Bakkenist and Kastan. They found that ATM molecules are inactive in undamaged cells, being held as dimers or higher-order multimers. In this configuration, the kinase domain of each molecule is blocked by the FAT domain of the other.
Following DNA damage, each ATM molecule phosphorylates the other on a serine residue at position 1981 within the FAT domain — a phosphorylation that releases the two molecules from each other’s grip, turning them into fully active monomers. Within minutes after the infliction of as few as several DSBs per genome, most ATM molecules become vigorously active.
Bakkenist and Kastan35 provide evidence that the signal for ATM activation might be chromatin alterations rather than direct contact of ATM with the broken DNA. However, it has previously been shown that, soon after damage infliction, a portion of the nuclear ATM is recruited to DSB sites and strongly adheres to them, possibly serving as a platform for further enzymatic reactions that take place at those sites. Both chromatin-bound and free ATM are autophosphorylated on serine 1981 (L. Moyal, unpublished observations). So, following DSB induction, activated ATM seems to divide between two fractions: one is chromatin bound and the other is free to move throughout the nucleus.


The list of published ATM substrates — more than a dozen at this time — is far from complete, and many ATM-dependent responses are likely to involve ATM targets that are unknown at present. However, the study of these pathways is gradually disclosing a remarkably broad cellular response to DSBs that is meticulously orchestrated by ATM.

A close look at the network of ATM-mediated pathways that is responsible for activation of the cell-cycle checkpoints explains how ATM precisely and decisively controls these pathways using several sophisticated strategies. The first strategy is to approach the same effector from several different directions. A main component in the G1–S cell-cycle checkpoint is mediated by activation and stabilization of p53, which, in turn, activates transcription of the gene that encodes the CDK2–cyclin-E inhibitor WAF1 (also known as p21 and CIP1). The main target of ATM in this pathway is p53, which is phosphorylated by ATM on Ser15 . This contributes primarily to enhancing the activity of p53 as a transcription factor. ATM also phosphorylates and activates CHK2, a checkpoint kinase41, 42 that phosphorylates p53 on Ser20. This interferes with the p53–MDM2 interaction. The oncogenic protein MDM2 is both a direct and indirect inhibitor of p53, as it serves as a ubiquitin ligase in p53 ubiquitylation, which mediates its proteasome-mediated degradation. ATM also directly phosphorylates MDM2 on Ser395, which interferes with nuclear export of the p53–MDM2 complex, and hence the degradation of p53 . Finally, it has been reported that phosphorylations of p53 on Ser9 and Ser46 , and dephosphorylation of Ser376 , are ATM dependent as well, although the function of these changes is unknown.

fig.9 ATM and related protein kinases: safeguarding genome integrity

2009-06-15T12:24:05 - Gianpiero Pescarmona

A field synopsis on low-penetrance variants in DNA repair genes and cancer susceptibility.
Vineis P, Manuguerra M, Kavvoura FK, Guarrera S, Allione A, Rosa F, Di Gregorio A, Polidoro S, Saletta F, Ioannidis JP, Matullo G.

Department of Epidemiology and Public Health, Imperial College, London, UK.

BACKGROUND: Several genes encoding for DNA repair molecules implicated in maintaining genomic integrity have been proposed as cancer-susceptibility genes. Although efforts have been made to create synopses for specific fields that summarize the data from genetic association studies, such an overview is not available for genes involved in DNA repair. METHODS: We have created a regularly updated database of studies addressing associations between DNA repair gene variants (excluding highly penetrant mutations) and different types of cancer. Using 1087 datasets and publicly available data from genome-wide association platforms, meta-analyses using dominant and recessive models were performed on 241 associations between individual variants and specific cancer types that had been tested in two or more independent studies. The epidemiological strength of each association was graded with Venice criteria that assess amount of evidence, replication, and protection from bias. All statistical tests were two-sided. RESULTS: Thirty-one nominally statistically significant (ie, P < .05 without adjustment for multiple comparisons) associations were recorded for 16 genes in dominant and/or recessive model analyses (BRCA2, CCND1, ERCC1, ERCC2, ERCC4, ERCC5, MGMT, NBN, PARP1, POLI, TP53, XPA, XRCC1, XRCC2, XRCC3, and XRCC4). XRCC1, XRCC2, TP53, and ERCC2 variants were each nominally associated with several types of cancer. Three associations were graded as having "strong" credibility, another four had modest credibility, and 24 had weak credibility based on Venice criteria. Requiring more stringent P values to account for multiplicity of comparisons, only the associations of ERCC2 codon 751 (recessive model) and of XRCC1 -77 T>C (dominant model) with lung cancer had P <or= .0001 and retained P <or= .001 even when the first published studies on the respective associations were excluded. CONCLUSIONS: We have conducted meta-analyses of 241 associations between variants in DNA repair genes and cancer and have found sparse association signals with strong epidemiological credibility. This synopsis offers a model to survey the current status and gaps in evidence in the field of DNA repair genes and cancer susceptibility, may indicate potential pleiotropic activity of genes and gene pathways, and may offer mechanistic insights in carcinogenesis.

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