DNA end-joining: from yeast to man

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Abstract

DNA non-homologous end-joining (NHEJ) is a crucial process that has been conserved highly throughout eukaryotic evolution. At its heart is a multiprotein complex containing the KU70–KU80 heterodimer. Recent work has identified additional proteins involved in this pathway, providing insights into the mechanism of NHEJ and revealing exciting links with the control of transcription, telomere length and chromatin structure.

Section snippets

Components of the DNA non-homologous-end-joining apparatus in mammalian cells

Essential to our current understanding of mammalian NHEJ was the generation and characterization of mutant mammalian cell lines that are hypersensitive to ionizing radiation (IR) and are defective in DSB rejoining and site-specific V(D)J recombination[1]. Characterization of these cell lines has revealed that they fall into four complementation groups: IR4, IR5, IR6 (Ref. [2]) and IR7. Cells of the IR5, IR6 and IR7 complementation groups lack components of the DNA-dependent protein kinase

Components of the DNA non-homologous-end-joining apparatus in yeast

To identify novel components of the NHEJ apparatus, many investigators have exploited the budding yeast, Saccharomyces cerevisiae, for which the complete genome sequence is known and in which it is relatively easy to perform gene-knockout experiments. In yeast, the predominant DSB-repair pathway is homologous recombination, which involves the exchange of genetic information between a damaged chromosome and its undamaged partner. Essential to homologous recombination in S. cerevisiae are the

Chromatin structure and double-strand-break repair

Recent work on yeast has provided exciting potential links between chromatin structure and NHEJ. A yeast two-hybrid screen revealed that the yeast silencing protein Sir4p interacts directly or indirectly with Yku70p (Ref. [40]). Sir4p, along with Sir2p and Sir3p, functions in transcriptional silencing at telomeres and also ensures that the genetic information in the yeast silent mating-type cassettes is not expressed inappropriately. Disruption of the SIR2, SIR3 or SIR4 genes leads to defective

Involvement of yeast non-homologous-end-joining proteins in telomere-length maintenance and transcriptional silencing

In addition to functioning in DNA repair, several yeast NHEJ proteins are involved in telomere-length maintenance and telomere-associated transcriptional silencing. Yeast telomeres are resistant to chemical and protein probes, which suggests that they exist in a protected location or condensed chromatin configuration[41]. Telomeric DNA consists of tandem copies of simple repeat sequences (C1–3A in yeast) that are maintained by the enzyme telomerase. Interestingly, strains defective in YKU70 or

Models for non-homologous end-joining

Much of what is known about the mechanisms of NHEJ has come from the analysis of rejoining of restriction-enzyme-cut plasmid DNA. Studies using S. cerevisiae have used a transformation-based in vivo plasmid-repair assay to assess both the relative efficiency and the accuracy of NHEJ20, 29. In this assay, restriction endonucleases are used to generate DSBs in a yeast–E. coli shuttle plasmid, within a region that has no homology to chromosomal sequences. Because only repaired circular plasmids

Mammalian V(D)J recombination

Given that components of the mammalian NHEJ apparatus, such as Ku, DNA-PKcs and XRCC4, are required for V(D)J recombination, NHEJ and V(D)J recombination are likely to be mechanistically very similar. Indeed, the DNA ends joined during V(D)J recombination are not generally homologous; this means that V(D)J recombination can be considered as an NHEJ process. Consistent with this idea, in mammalian cells in which KU80 is defective, signal join formation resulting from ligation of the

Concluding remarks

Since the discovery that Ku and DNA-PKcs play crucial roles in DSB repair, a number of new factors have emerged as essential or likely players in this process, including NBS1, XRCC4 and DNA ligase IV. Moreover, the important features of this pathway appear to have been conserved from yeast to man (Table 1). The yeast system has proved to be a powerful tool, not only for the identification of new components of the NHEJ apparatus, such as the Mre11p–Rad50p–Xrs2p complex and the Sir2p, Sir3p and

Acknowledgements

We apologize to those whose work has not been cited because of space restrictions. Thanks to Carol Featherstone and other members of the S. P. J. lab for their help and comments on this manuscript. S. P. J. thanks the CRC for support. S. E. C. is supported by a grant from the Association for International Cancer Research.

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