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DNA Repair: Rad52 — the Means to an End Kevin Hiom

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DNA Repair: Rad52 — the Means to an End Kevin Hiom CORE Metadata, citation and similar papers at core.ac.uk Provided by Elsevier - Publisher Connector R446 Dispatch DNA repair: Rad52 — the means to an end Kevin Hiom In eukaryotic organisms, double-strand breaks in in its damaged homologous partner. Our understanding of chromosomal DNA are repaired either by this process has been greatly enhanced by reconstitution non-homologous end-joining, or by homologous of the strand-exchange reaction in vitro using purified recombination. How do cells choose which pathway components [3]. In eukaryotic organisms, strand-exchange to use? is carried out by the Rad51 protein, a homologue of the bacterial RecA recombinase. In vitro, however, strand- Address: MRC Laboratory of Molecular Biology, Hills Road, Cambridge CB2 2QH, UK. exchange mediated by Rad51 alone is very inefficient [3]. Current Biology 1999, 9:R446–R448 More recently, experiments have shown that this in vitro http://biomednet.com/elecref/09609822009R0446 strand-exchange reaction can be stimulated in the © Elsevier Science Ltd ISSN 0960-9822 presence of another recombination protein, Rad52 [4–6]. Rad52 has been shown to stimulate annealing of comple- Double-strand breaks in chromosomal DNA can arise as a mentary regions of single-stranded DNA, and is therefore consequence of exposure to harmful exogenous agents, thought to assist Rad51 in the initial pairing of homolo- such as ionising radiation or cytotoxic molecules, or as a gous DNA molecules [4–6]. Genetic studies found that result of errors in cellular processes, such as DNA replica- yeast rad52 mutants exhibit extensive degradation of tion. Double-strand breaks are also generated as normal DNA ends compared with wild type [7], raising the possi- intermediates in V(D)J recombination, the process by bility that the Rad52 protein might also have a role in the which the genes encoding the antigen receptor molecules initial processing of DNA breaks. Now, van Dyck et al. [2] of lymphocytes are assembled from multiple coding have shown that Rad52 does indeed act at the earliest sequences [1]. Double-strand breaks pose a considerable stage of homologous DNA repair, playing a key part in the threat to genomic integrity and cell survival; if left unre- recognition and binding of double-strand breaks. paired, a single double-strand break is sufficient to cause cell death. Moreover, inefficient or inappropriate repair To investigate the interactions between human Rad52 can generate potentially oncogenic chromosomal aberra- (hRad52) and DNA, van Dyck et al. [2] used electron tions such as translocations. microscopy to directly visualize protein–DNA complexes. They observed that, in binding to the ends of linear DNA, To overcome the lethal potential of double-strand breaks, hRad52 exhibited a clear preference for DNA ends with eukaryotic organisms have evolved two major pathways for single-stranded tails. Tailed molecules of this kind are repairing these lesions. One pathway is known as non- likely to be common in cells that have been damaged by homologous end-joining and involves the direct joining of exposure to ionising radiation, as newly formed double- the broken DNA ends. Repair by this pathway is not nec- strand breaks are very quickly resected by exonucleases essarily error-free, however, as small deletions in the DNA such as the Mre11–Rad50–Nbs1 (Xrs2) complex. Once sequence are often introduced at the site of the double- bound to a DNA end, however, Rad52 protects the end strand break. The second pathway, in contrast, uses an from further degradation by exonucleases. van Dyck et al. undamaged copy of a chromosome as a template to repair [2] also found that hRad52 promoted the association of DNA breaks in a way that restores the genetic information DNA ends, mediated through hRad52–hRad52 intermole- lost at the break site. This high-fidelity repair mechanism cular interactions. They further determined that these is known as homologous recombination. What, then, deter- end-to-end interactions facilitated the joining of DNA mines whether a cell repairs a double-strand break by non- ends by T4 DNA ligase. While it is unlikely that the stim- homologous end-joining or by homologous recombination? ulation of end-ligation has any direct role in homologous New work from West and colleagues [2] suggests that the recombination, the preference of hRad52 for DNA ends choice between homologous recombination and non- with single-stranded tails, and the promotion of end-to- homologous end-joining might be determined by a compe- end interactions, are clearly consistent with a role in tition between different DNA-end-binding proteins, homologous pairing. which direct the repair of breaks into alternative pathways. The properties observed for Rad52 are strikingly similar to A key process in homologous recombination is the pairing those previously described for the Ku protein (reviewed in and strand-exchange between homologous DNA mole- [8]). Ku is a heterodimer of two proteins, Ku70 and Ku80, cules. It is this reaction which donates an undamaged and plays a critical part in non-homologous end-joining DNA template that is used to repair a double-strand break [8]. Mammalian cells that lack either Ku70 or Ku80 are Dispatch R447 Figure 1 A model for the initiation of double-strand break repair. DNA damage — by ionising radiation, cytotoxic drugs or errors of cellular processes — generates double-stranded DNA damage/ breaks in chromosomal DNA. Initially, these processing breaks may be resected by the action of cellular exonucleases, generating DNA ends Rad52 binding Ku binding with single-stranded tails. DNA ends can be bound either by Rad52 or by Ku. Binding of Rad52 (left) initiates repair of the break by homologous recombination; binding by Ku (right) directs repair by non-homologous end- Recruitment of Rad51 Recruitment of joining. It is proposed that Rad52 promotes DNA-PK homologous recombination by assisting in the pairing of homologous DNAs and also by Strand exchange recruiting Rad51 to the site of the DNA break, where it can initiate strand-exchange. Ku facilitates non-homologous end-joining by Trimming, ligation promoting the association of DNA ends and also by recruiting other repair factors such as DNA-dependent protein kinase (DNA-PK). In this diagram, Rad52 is represented by the red circles, Rad51 by the yellow ellipses, Ku by Homologous recombination Non-homologous end-joining the blue circles and DNA-dependent protein kinase by the orange ellipses. Current Biology deficient in non-homologous end-joining and exhibit synchronised human cells has shown that hRad52 is extreme sensitivity to ionising radiation. Like Rad52, Ku present only at very low levels during the G1 phase of the binds selectively to DNA ends, protects these ends from cell cycle, when recombinational repair is absent, but that digestion by exonucleases and promotes DNA end- its level rises steadily through S phase, reaching a joining. On the basis of these similarities, van Dyck et al. maximum in G2 phase when homologous repair is also at [2] have suggested a model for the initiation of double- its highest level [10]. strand break repair in which Ku and Rad52 have analogous roles in non-homologous end-joining and homologous By employing competing but overlapping repair pathways, recombination, respectively. In this model (Figure 1), eukaryotic cells ensure a level of redundancy for double- double-strand breaks are bound either by Ku or by Rad52. strand break repair, which has benefits to the cell. This While binding of Ku directs double-strand breaks into can be seen clearly by comparing repair of double-strand repair by non-homologous end-joining, binding of Rad52 breaks in G1/early S phase with that in late S/G2 phase of initiates repair by homologous recombination. Hence, the the mammalian cell cycle [9]. As previously mentioned, choice of repair pathway for double-strand breaks may G1 cells repair double-strand breaks by non-homologous well be determined by a competition between Rad52 and end-joining; loss of this pathway, for example by inactiva- Ku for binding DNA ends. tion of Ku, renders the G1 cell defenceless against double- strand breaks and extremely sensitive to DNA damage by The outcome of this competition is likely to be influ- ionising radiation. In S/G2-phase cells, where double- enced by a number of factors, such as the relative abun- strand breaks can be repaired either by non-homologous dance of hRad52 and Ku in a cell, and the different end-joining or by homologous recombination, the loss of affinities of these proteins for binding DNA ends. either repair pathway alone leads to only a mild sensitivity Although nothing is currently known about the relative to ionising radiation. This suggests that, in this phase of affinities of Rad52 and Ku for DNA ends, there is a corre- the cell cycle, one repair pathway is able to compensate lation between the levels of Rad52 protein and repair by for loss of the other. homologous recombination. In mammalian cells, repair of double-strand breaks during the G1 and early S phase of Although both non-homologous end-joining and homolo- the cell cycle occurs almost exclusively by non-homolo- gous recombination have been conserved in evolution from gous end-joining. In late S and early G2 phase, however, yeast to man, the relative contribution of each pathway to after cells have replicated an extra copy of the genome, the overall repair of breaks differs between ‘lower’ and homologous recombination operates in addition to non- ‘higher’ organisms. Whereas in yeast repair of double- homologous end-joining [9]. Analysis of protein levels in strand breaks occurs largely by homologous recombination, R448 Current Biology, Vol 9 No 12 mammalian cells predominantly use non-homologous end- 5. New JH, Sugiyama T, Zaitseva E, Kowalczykowski SC: Rad52 protein stimulates DNA strand exchange by Rad51 and replication joining.
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