Double strand break repair

DSB repair in humans

Two major mechanisms exist for the repair of DSBs, non-homologous end joining (NHEJ), and homologous recombination (or recombinational repair (RR)) [2]. NHEJ involves a direct rejoining of the separated DNA ends. However, if there are multiple DSBs in a cell, this process is inefficient at determing which pieces should be joined back together and can thus lead to large scale alterations of DNA structure (insertions, deletions, transversions etc.). Furthermore damage or degradation of the DNA ends is not repaired by this process, which can lead to the generation of mutations. In contrast to NHEJ, RR uses homologous DNA as a template in rejoining DSBs. This allows damaged DNA ends to be both repaired and appropriately re-joined. However, higher eukaryotes (such as humans) contain high numbers of repetitive DNA sequences, which may be (incorrectly) used by RR as homologous sequences in repairing DSBs. Consequently, RR is only active at times in the cell cycle after the cell has duplicated its DNA, but before it has divided (late S through early M phases) (reviewed in [3]). This provides RR with nearby identical DNA sequences to use in repairing DSBs.

Thus, NHEJ dominates in the G1/early S phases, while homologous recombination accounts for most DSB repair in the late S/G2 phases (reviewed in [3]).

Although many of the proteins involved in the two DSB repair pathways have been identified, the precise mechanisms involved remain poorly understood. Furthermore, it appears that at least one protein complex (consisting of rad50, Mre11 and Xrs2), may contribute to repair by both of these mechanisms.

Non-homologous end-joining (NHEJ)

NHEJ accounts for most DSB repair in the G1/early S phases (reviewed in [3]) and involves the direct rejoining of DSBs. This requires little or no sequence homology between the DNA ends (reviewed in [4, 5]). According to the current model, each DNA end is recognised by a heterodimer of KU80 and KU70, which probably protects the ends from degradation [6] and may play a role in repositioning them together as it can transfer between DNA with complementary overhangs [7]. Upon binding a DNA end, the KU heterodimer attracts the catalytic subunit (DNA-PKcs) and stimulates its protein kinase activity [8, 9]. Next the Rad50, Mre11 and Xrs2 complex is believed to join the complex, which may play a role in processing the ends. The products of the SIR2, SIR3 and SIR4 genes, which function in chromatin remodeling, have also been implicated in the NHEJ complex [10, 11]. Finally a complex of XRCC4 and DNA ligase IV rejoins the strands.

Although only a minor contributor to the repair of DSB in S. cerevisiae, this pathway has been proprosed to be the major DSB repair pathway in mammals. However, the process is inherently error-prone as nucleotides at the break could be added or lost, and incorrect ends might be joined. Indeed, the immune systen exploits this inaccuracy to create additional diversity in antibodies and T-cell receptors. In contrast to NHEJ, homologous recombination accurately repairs DSBs using information on the undamaged sister chromatid or homologous chromosome.

Recombinational repair

Although homologous recombination is the major DSB repair pathway in S. cerevisiae, in mammals it is restricted to certain phases of the cell cycle (reviewed in [3]). It has been suggested that the inaccurate NHEJ repair pathway is tolerated by differentiated somatic cells, where a large fraction of the genome is no longer functional, because misalignment of repetitive sequences may complicate homologous recombination in mammals [4]. As such misalignments could lead to deletions and translocations, it would be advantageous to restrict homologous recombination of DSBs to the late S/G2 phases of the cell cycle where recombination would preferentially occur between replicating chromatids.

In response to ionising radiation the BRCA1 (breast cancer gene1) protein is phosphorylated by the ATM (mutated in ataxia telangiectasia) gene product [12], and relocalises to distinct foci with the recombination protein Rad51 [13]. The second "breast cancer gene", BRCA2, plays an essential role in formation of the Rad51 complex in response to IR [14]. BRCA1 can bind DNA strand breaks [15], and both BRCA1 [16] and BRCA2 [17, 18] have been implicated in the repair of DSB. A specific role for BRCA1 in recombinational repair of DSB has recently been demonstrated [19].

In homologous recombination, the DNA ends resulting from a DSB are believed to be processed by nucleases and/or helicases to a single stranded 3' overhang (reviewed in [4, 5]). Yeast and human Rad51 (which has homology to E. coli RecA) polymerises onto the single stranded DNA overhang to form a nucleoprotein filament that searches for homologous duplex DNA. Recognition and invasion of the homologous duplex requires a number of additional genes (Rad52, Rad54, Rad55/57 and RPA, as well as Mre11, Xrs2 and Rad50, have all been implicated in yeast), although the exact roles of most remain poorly defined. Strand exchange generates a joint molecule between the damaged and undamaged DNAs, where the invading strand is extended by a DNA polymerase restoring the missing information. Ligation and resolution of the Holliday junction by a resolvase restores two intact duplex DNAs.

Ataxia telangiectasia

Ataxia-telangiectasia is an autosomal recessive disorder, occurring in 1/40 000 live births. It is clinically characterised by cerebellar ataxia (results in staggering gait, severe muscle discoordination, and progressive mental retardation), dilation of small blood vessels (telengiectasia), immune dysfunction and sensitivity to ionizing radiation (reviewed in [1]). The molecular defect appears to be an aberrant response to DNA damage induced by ionizing radiation, and may involve a DNA repair deficiency per se, or a defect in other signalling pathways which are affected by DNA damage (ie. cell cycle arrest or induction of stress response genes).

AT heterozygotes (i.e. carry one mutant copy and one normal copy of the ataxia telangiectasia gene, ATM) are believed to comprise approximately 1.4% of the general population [28], and have been shown to exhibit an intermediate increase in cellular sensitivity to ionising radiation [29, 30]. Furthermore, AT heterozygotes are at increased risk for the development of breast cancer [25, 27, 31-35]. Approximately 7% of all breast cancer patients have been estimated to be heterozygous for a defect in the AT gene [25, 33]. Modest radiation doses result in a 6-fold increase in the risk of breast cancer for AT heterozygotes [34], and it has been suggested that low exposures to ionising radiation (below 20 mGy) may induce breast cancer in AT hetrozygotes while higher exposures (100-200 mGy) are required to induce a similar effect in other populations [33].

For more information see: ATM

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