Strandbreak Repair

Repair of damaged DNA bases is a permanent process in living cells. It is only one of several processes, including chemical reactions and enzymatic actions, that generate single-strand breaks in DNA. These are therefore common, and it is estimated that 100,000 DNA single-breaks occur per cell each day. In the most simple case, single-strand breaks are repaired by DNA ligase, but components from the short- and long-patch base excision repair or nucleotide excision repair systems may be needed, when severe base damage or chemical modification of the sugar are associated with the loss of a base.

Like mismatch repair during DNA replication, repair of DNA strand-breaks goes on rather 'quietly'. However, this changes dramatically, when DNA double-strand breaks are generated. These can arise by physiological and non-physiological mechanisms, endogenous processes and exogenous mutagens. During DNA replication, a double-strand break can result from a single-strand break, if this is not repaired, before it is encountered by the replisome. Sometimes two single-strand breaks may by chance occur closely together leading to a double-strand break. Other double-strand breaks are caused by exogenous agents. Some viruses encode enzymes that cut DNA in a similar fashion as restriction enzymes, e.g. retroviral integrases. Ionizing radiation can generate single- as well as double-strand DNA breaks as do several chemical carcinogens and some drugs used in chemotherapy. Bleomycin, e.g., cuts DNA directly, and topoisomerase inhibitors generate strand breaks by inhibition of these enzymes (^22.2). Repair of DNA crosslinks also involves the generation of double-strand breaks.

Double-strand breaks are also created, in a controlled fashion, during physiological recombinations. Important processes of this kind are meiotic recombination and generation of functional T-cell receptor (TCR) and immunoglobulin (IG) genes in lymphocytes, which yield occasionally errors. Unequal recombination in germ cells is an important cause of inherited disease including cancer. Aberrant joining of genes encoding the T-cell receptor or immunoglobulins to other genes such as MYC is a frequent source of chromosomal translocations in lymphomas (^10.2). Other translocations and deletions in cancers of the lymphoid lineage can result when the lymphocyte-specific recombination system acts accidentially at sites outside the TCR and IG gene clusters.

Independent of how they arise, double-strand breaks are dangerous as long as they exist, especially to a proliferating cell. They separate a fragment of DNA from the centromere, predisposing it to loss during mitosis. Moreover, the open ends can recombine with other parts of the genome, starting a chain reaction of recombinations and chromosome alterations that can lead to cell death or transformation. Double-strand repair therefore involves blocking of the open DNA ends in addition to actually mending the break. In addition, activation of doublestrand DNA break repair is usually associated with the activation of cellular checkpoints that prevent the cell from entering or proceeding through S-phase and mitosis. Specifically, unrepaired DNA double-strand breaks in normal cells often elicit apoptosis. This mechanism provides another level of protection against carcinogenesis, in addition to DNA repair itself.

Several repair systems in human cells deal with double-strand breaks. They can be classified into non-homologous and homologous repair systems.

Non-homologous end-joining (NHEJ) is an imprecise mechanism which is nevertheless most often used in human cells (Figure 3.10). Double-strand breaks are protected by the KU70/KU80 protein heterodimer, and bound by the 'MRN' complex consisting of the MRE11, RAD50, and NBS1 (Nibrin) proteins. These proteins prevent them from illegitimate recombination and attempt to align them. Compatible ends may become ligated, but in many cases the ends are processed. Processing can involve filling in 5'-overhangs and degrading 3'-overhangs. MRE11 possesses nuclease activity. In addition the FEN1 nuclease may be involved as well as the WRN protein which may also supply helicase activity additional to that of the KU70/KU80 proteins. Apparently, processing, unwinding and alignment of the strands proceeds until short complementary base stretches are found which can be used to hybridize the two ends. Remaining overhangs are processed, gaps are filled in and the sugar-phosphate backbone is religated by DNA ligase IV/XRCC4. The end product of the repair process is a restored DNA double helix with a deletion, which is normally kept at a minimum. A distinct characteristic of sequences repaired by NHEJ are microhomologies, i.e. short stretches of 1-12 bp which were identical in the original sequences at both ends of the deletion. These stretches of homology

Figure 3.10 Repair of DNA double-strand breaks by non-homologous endjoining

See text for details.

Figure 3.10 Repair of DNA double-strand breaks by non-homologous endjoining

See text for details.

are much longer when deletions arise by illegitimate homologous recombination. In some cases, NHEJ repair leads to the insertion of a few additional nucleotides, as during V(D)J joining in lymphocytes. This may help to anneal sequences.

When NHEJ begins, it elicits signals that activate cellular checkpoints. The KU proteins constitute the regulatory subunits of DNA-dependent protein kinase (DNA-PK), which is essential for proper DNA repair. Its catalytic subunit phosphorylates not only itself and other proteins directly involved in repair, but also activates the TP53 protein, which is one of the most important regulators of cellular checkpoints. The phosphorylation of TP53 by DNA-PK and/or further enyzmes such as the ATM and ATR kinases elicits cell cycle arrest or even apoptosis (^5.3). The NHEJ protein complex itself is regulated by ATM and other proteins. Within the MRN complex, Nibrin seems to exert the major control. It is phosphorylated and activated by the ATM protein kinase and in turn interacts with BRCA proteins.

In contrast to NHEJ, homologous recombination repair (HRR) can be performed in an error-free fashion, at least in principle. In human cells, it is the mechanism of choice in the G2 phase of the cell cycle when a second sequence identical to the damaged one is available in the sister chromatid. NHEJ, in contrast, appears to be the predominant mechanism in G1 cells. HRR may also constitute the preferred method for the repair of double-strand breaks that arise when breaks in one DNA strand are extended into double-strand breaks during replication and the replisome has stalled. In this situation, the BLM helicase may be crucially involved. Demarcation of the double-strand lesion in all other cases is likely performed by the RAD52 protein (Figure 3.11). As a clear-cut difference towards NHEJ, the KU proteins are not involved. The double strand break is then processed to yield a 3'-overhanging single strand of several 100 bases. In this processing the MRE11/RAD50/NBS1 (MRN) complex is again involved together with additional, less well characterized components. With the help of the recombination protein RAD51, the single strands invade the intact homologous double-strand DNA forming D-loop structures ('D' for 'displacement'). The 3'-hydroxyls of the singlestrands are then extended and a structure with two Holliday junctions forms. This is resolved by endonuclease action. There are several possible outcomes, depending on how the Holliday junctions are resolved. In one alternative, both original sequences are restored, in the other a crossover takes places. This does not result in a change of sequence when the sister chromatid is used. However, if a homologous sequence from a different chromosome was involved, gene conversion can happen.

Not all parts of the HRR mechanism are well understood, as for NHEJ repair. However, some components have been identified with certainty, because they are mutated in human inherited diseases. Homozygous mutations in the NBS1 gene that compromise the function of Nibrin underlie the Nijmegen breakage syndrome. This very rare syndrome presents with mental retardation, immunodeficiency, and, tellingly, chromosomal instability and cancer susceptibility. Homozygous mutations in the WRN gene encoding a helicase/nuclease involved in double-strand break repair and telomere maintenance also increase the susceptibility to various types of cancer, particularly in soft tissues. However, the resulting Werner syndrome

Wrn Gene And Cancer

Figure 3.11 Mechanism of DNA double-strand break repair by homologous recombination

See text for details

Figure 3.11 Mechanism of DNA double-strand break repair by homologous recombination

See text for details impresses primarily as a premature aging disease (^7.4) manifesting typically around puberty.

The most prevalent syndrome in this context is the recessively inherited ataxia telangiectasia (AT). It is caused by homozygous mutations in the gene encoding the ATM protein kinase that regulates DNA double-strand break repair. Like NBS patients, AT patients are prone to infections and chromosomal aberrations. They have a «100-fold increased risk of cancers, mostly of leukemias and lymphomas. Both syndromes share, in particular, a hypersensitivity towards ionizing radiation. However, AT patients are not usually mentally retarded. Instead, they develop a gradual decline of the function of the cerebellum, which progressively impedes movements, speech and sight. This very specific ataxia led to the name along with the diagnostic telangiectasias which are aggregates of small dilated blood vessel appearing in unusual places such as the conjunctiva of the eye. They are thought to be caused by inappropriate angiogenesis. The chain of events leading to these lesions may involve lack of ATM function leading to incomplete function of TP53 alleviating suppression of angiogenesis induced by hypoxia (^9.4). Other aspects of the pleiotropic ATM phenotype are less understood, including an elevation of the fetal albumin homologue a-fetoprotein that is useful for the diagnosis of the disease.

In contrast, the chromosomal instability and hypersensitivity towards ionizing radiation in the syndrome fit well with the known function of ATM as a central coordinator of double-strand break repair. DNA double-strand breaks caused by physiological recombination, by viral or retrotransposon enzymes, by ionizing radiation or chemicals, or by oxidative stress all appear to activate ATM. Likely, this occurs by different routes. The protein may itself sense damage to some extent, but the MRN complex through NBS1 certainly plays a part. A variant histone, H2AX, accumulates within 1 min at double-strand breaks to become phosphorylated by ATM; this could well be another sensor protein. H2AX can alternatively be phosphorylated by DNA-PK. Further candidates for damage sensors are RAD9 and RAD17 which are also ATM substrates.

Following its activation, ATM goes on to phosphorylate further proteins involved in DNA repair such as FANCD2, BRCA1, and RPA. Significantly, it also activates checkpoints that block the cell from further proliferation. Phosphorylation by ATM activates the TP53 protein, whereas it prevents the TP53 inhibitor protein HDM2 from binding to TP53. Together these actions lead to cell cycle arrest at the G1/S checkpoint via induction ofthe p21CIP1 cell cycle inhibitor and at the G2/M checkpoint by other mediators (^6.6). DNA replication can be arrested via phosphorylation of CHK2 (checkpoint kinase 2) and Nibrin, while phosphorylation of TP53 and BRCA1 also activates the G2/M checkpoint.

Some aspects of ATM function can also be provided by other protein kinases such as CHK2, ABL, and ATR. Severe damage by UV radiation, e.g., is signaled by the ATR protein kinase in an otherwise quite similar fashion, including phosphorylation of TP53 and CHK1 (instead of CHK2). The somewhat complementary functions of ATM and ATR are the likely explanation why AT patients and their cells are sensitive to ionizing radiation, but not to UV.

A public debate has developed on the issue of whether heterozygosity for ATM mutations leads to an increased cancer risk. This is a particular concern, since several methods commonly used in cancer screening and diagnosis employ ionizing radiation. The results of different investigations vary. It is possible that the cancer risk of heterozygous carriers of the disease may depend on which mutation is present. Some mutations may completely inactivate the affected allele. Others may show some degree of a dominant-negative phenotype, i.e. an altered protein product is formed which does not function in repair, but inhibits the function of the protein produced by the normal allele. In the case of the ATM protein, this is conceivable, since the protein normally exists as a dimer and its activation involves cross-phosphorylation between the subunits. So, dysfunctional subunits may inactivate some of the functional subunits as well.

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