Accelerated publication
XRCC3 is required for efficient repair of chromosome breaks by homologous recombination

https://doi.org/10.1016/S0921-8777(00)00002-1Get rights and content

Abstract

XRCC3 was originally identified as a human gene able to complement the DNA damage sensitivity, chromosomal instability and impaired growth of the mutant hamster cell line irs1SF. More recently, it has been cloned, sequenced and found to bear sequence homology to the highly conserved eukaryotic repair and recombination gene RAD51. The phenotype of irs1SF and the identification of XRCC3 as a member of the RAD51 gene family have suggested a role for XRCC3 in repair of DNA damage by homologous recombination. Homologous recombinational repair (HRR) of a specifically induced chromosomal double-strand break (DSB) was assayed in irs1SF cells with and without transient complementation by human XRCC3. Complementation with XRCC3 increased the frequencies of repair by 34- to 260-fold. The results confirm a role for XRCC3 in HRR of DNA DSB, and the importance of this repair pathway for the maintenance of chromosomal integrity in mammalian cells.

Introduction

XRCC3 is one of a group of genes originally identified by their ability to complement certain mutated rodent cell lines for hypersensitivity to ionizing radiation (and other DNA damaging agents), and hence designated “X-ray repair cross-complementing”. Four of these genes are now known to function in the repair of DNA double-strand breaks (DSB) through the process of nonhomologous end-joining (NHEJ). The product of the XRCC4 gene functions as an accessory to DNA ligase IV 1, 2. XRCC5, XRCC6 and XRCC7 encode, respectively, the Ku80 subunit, the Ku70 subunit and the catalytic subunit of DNA-dependent protein kinase (DNA-PK) (reviewed in Ref. [3]).

XRCC2 and XRCC3 constitute a subset apart from the XRCC mutants linked to defects in NHEJ, as distinguished by the phenotypes of the mutant hamster cell lines in which they were first defined 4, 5, 6, 7, 8, 9(reviewed in Ref. [10]). XRCC2 and XRCC3 mutant cells show only moderate hypersensitivity to ionizing radiation (two- to three-fold), in contrast to the more severe sensitivities of XRCC4, XRCC5, XRCC6 and XRCC7 mutants 11, 12, 13. XRCC2 and XRCC3 mutant cells also display moderate hypersensitivity to ultraviolet radiation and the monofunctional alkylating agent ethyl methanesulfonate 4, 5, and extreme sensitivity to DNA cross-linking drugs such as mitomycin C or cisplatin 4, 7. XRCC4, XRCC5 and XRCC7 mutants display little or no hypersensitivity to these classes of DNA damaging agents 11, 14. While XRCC4, XRCC5, XRCC6 and XRCC7 mutants exhibit delayed or incomplete rejoining of DSB after gamma irradiation 11, 12, there is no demonstrable deficit in DSB rejoining in XRCC3 or XRCC2 mutant cells after irradiation. Cells mutated for XRCC4, XRCC5, XRCC6 and XRCC7 all have reduced ability to complete V(D)J recombination 12, 13, 15, indicating that V(D)J recombination and repair of DSB by NHEJ employ overlapping sets of proteins. No deficiency in V(D)J recombination has been reported for XRCC2 or XRCC3 mutants.

XRCC2 and XRCC3 have recently been cloned and sequenced 16, 17. Both genes show limited sequence homology to the highly conserved eukaryotic repair and recombination gene RAD51, and are now considered members of the RAD51 gene family. Because of the particular hypersensitivity of XRCC2 and XRCC3 mutants to DNA cross-linking drugs, both genes have been candidates for involvement in homologous recombinational repair (HRR). The identification of XRCC2 and XRCC3 as RAD51-related genes has reinforced this expectation. Here, we have assayed the ability of the XRCC3 mutant cell line irs1SF to repair a specifically induced chromosomal break by homologous recombination, with and without complementation for XRCC3.

Section snippets

Cell lines and plasmid vectors

The XRCC3 mutant cell line irs1SF was grown in minimal essential meduim-alpha (α-MEM, from BRL-Gibco) with 10% fetal calf serum (HyClone), in a humidified atmosphere with 6% CO2. Construction of a plasmid vector, pMSGneo2S12HIS, which contains a complete reporter locus for recombinational repair of a site-specific DSB, has been described previously [18]. pMSGneo2S12HIS was transfected into irs1SF cells by electroporation with 0.04–1.0 μg of plasmid vector, using a BioRad GenePulser set to 250

Results

A recombination substrate that can be used to detect repair of a site-specific chromosome break by homologous recombination was integrated into irs1SF cells. This substrate, and its use in a wild-type hamster cell line (CHO-K1), has been described previously [18]. It consists of two disabled neo (neomycin resistance) genes configured as direct repeats (Fig. 1). The upstream or left neo gene is equipped with a viral enhancer/promoter and a polyadenylation signal, but is nonfunctional because a

Discussion

The sharply elevated frequencies of homologous DSB repair measured in irs1SF cells complemented with human XRCC3 show that this repair pathway requires the XRCC3 protein to function at full efficiency. The frequencies of repair seen here in transiently complemented irs1SF cells are similar to that measured previously in a wild-type hamster cell line carrying the same recombination substrate [18]. In a recent work using a recombination substrate of similar design, Pierce et al. [21]have also

Acknowledgements

We thank Brant M. Wagener for expert technical support; Chris P. Allen for technical advice and critical discussions; Greg Donoho (Lexicon Genetics, The Woodlands, TX) for providing the I–SceI expression vector pCMV(3×NLS)I–SceI, and Nan Liu (Lawrence Livermore National Laboratory) for providing the human XRCC3 expression vector pREP–XRCC3. We also thank Dr. Shunichi Takeda (Faculty of Medicine, Kyoto University, Japan) for discussing data prior to publication. This study was supported by the

References (39)

Cited by (159)

  • Association of buccal micronucleus cytome assay (BMNCyt) biomarkers with inorganic element concentration and genetic polymorphisms in welders

    2023, Environmental Toxicology and Pharmacology
    Citation Excerpt :

    These findings may indicate that double breaks caused by oxidative damage are not being repaired, thereby leading to cell death. The X-Ray Repair Cross Complementing 3 gene (XRCC3) encodes the protein XRCC3 which is involved in homologous recombination of double-strand breaks and cross-link repair in mammalian cells (Brenneman et al., 2000). XRCC3 and other paralogous proteins are required by RAD51 for the formation of stable and active repair complexes (Forget et al., 2004; Nowacka-Zawisza et al., 2019).

  • Cytogenetic status of interventional radiology unit workers occupationally exposed to low-dose ionising radiation: A pilot study

    2019, Mutation Research - Genetic Toxicology and Environmental Mutagenesis
    Citation Excerpt :

    The occupationally absorbed dose of 1.7 ± 2.3 mSv over the previous 6 months was reflected in a higher MNi frequency. Moreover, the volunteers carrying the XRCC3 Met 241 allele had more MNi than homozygotes, possibly due to a lower capacity to repair double-stranded DNA breaks [9,45]. Our results showed a higher frequency of NBs, which are biomarkers of nuclear elimination of amplified DNA and/or DNA repair complexes [27].

  • Paths from DNA damage and signaling to genome rearrangements via homologous recombination

    2017, Mutation Research - Fundamental and Molecular Mechanisms of Mutagenesis
View all citing articles on Scopus
1

Present address: Department of Molecular Genetics and Microbiology, University of New Mexico School of Medicine, 915 Camino de Salud, Albuquerque, NM 87131, USA.

View full text