Localization of a DNA repair gene (XRCC5) involved in double-strand-break rejoining to human chromosome 2.

Jeggo, Penny A.; Hafezparast, Majid; Thompson, A.; Broughton, Bernard C.; Kaur, Gagandeep; Zdzienicka, Małgorzata Z.; Athwal, Raghbir S.

doi:10.1073/pnas.89.14.6423

Cited by 51 publications

(25 citation statements)

References 30 publications

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“…Importantly, human p86Ku colocalizes with xrs, another XRCC5 mutant (23). xrs cells are restored for IRS and V(D)J recombination defects by microcell-mediated transfer of human chromosome 2 (15,33). During the publication of this work, the repair and recombination defects of xrs-6 cells were shown to be complemented by p86Ku (34).…”

Section: Discussionmentioning

confidence: 86%

Complementation of the ionizing radiation sensitivity, DNA end binding, and V(D)J recombination defects of double-strand break repair mutants by the p86 Ku autoantigen.

Boubnov

Hall

Wills

et al. 1995

Proc. Natl. Acad. Sci. U.S.A.

124

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Two ionizing radiation-sensitive (IRS) and DNA double-strand break (DSB) mutants, sxi-3 and sxi-2, were shown to be severely deficient in a DNA end binding activity, similar to a previously described activity of the Ku autoantigen, correlating with thexrs (XRCC5) mutations. Cell fusions with xrs-6, another IRS, DSB repair-deficient cell line, defined these sxi mutants in the XRCC5 group. sr-3 cells have low expression levels of the p86Ku mRNA. Here we show the sxi-3 mutant of DSB repair is defective for both product formation steps of V(D)J recombination. sxi-3 cells were found to be in the XRCC5 IRS group and were complemented by the p86Ku gene for DNA repair and recombination functions. Thus, Ku must coordinate DSB repair and V(D)J recombination. MATERIALS AND METHODSTransfection and IR Cell Survival. Expression plasmids for human p70Ku and p86Ku cDNAs were constructed in SRa, a derivative of pcDL-SRa296 (24). SRa-p7OKu or ug) was transfected into sxi-3 cells grown in 10:1 excess over pPGKhygromycin by standard calcium phosphate precipitation and hygromycin selection (Sigma, 200 ,ug/ml). One hundred to 200 colonies were pooled and replated for IRS tests at 200 cells per 60-mm plate (13).DNA End Binding. A 32P-labeled Pvu II-Xma I fragment of pJH290 was prepared. Nuclear extracts, DNA end binding, and mobility shift gels were by standard methods (18,19). Antibodies (1 ,ul of ascites fluid) for supershifting were directly added to the DNA end binding mixture.Cell Fusion. DSB mutant and control cell lines were scid (scSV3, V-3) (13, 25); XRCC5 (sxi-2, sxi-3), xrs-6 (26); XR-1 (27); V79-4, and CHO. Cells were tagged with pPGKhygromycin or pPGKpuromycin by calcium phosphate transfection. Cell fusions were generated between puromycin-resistant and hygromycin-resistant cells. Cells (1 x 106) of each fusion partner were plated into a 60-mm plate for 24 hr, washed four times with serum-free F12 medium (SF12), then 0.5 ml of PEG 1500 (Boehringer Mannheim) added for 1 min, washed four times with SF12, incubated for 60-90 min at 37°C, and then incubated in F12 plus 10% fetal bovine serum (FBS), puromycin (2 ,ug/ml), and hygromycin (350 ,ug/ml). After 24 hr, fusions were replated at 103 cells per plate in duplicate. Twenty-four colonies of each fusion were tested for IRr (13). Methionine Labeling. sxi-3, sxi-3/p86Ku, and LAZ388 (5 x 106 cells) were preincubated with 2 ml of methionine-free medium plus 10% dialyzed FBS for 1 hr. Three hundred microcuries of [35S]methionine (1 Ci = 37 GBq) was added for 5 hr. Following a phosphate-buffered saline wash, cells were lysed on ice in 200 ,ul of 40 mM Tris-HCl, pH 8.0/10 mM EDTA/0.5 M NaCl/0.5% Nonidet P-40 for 30 min; 100 ,ul of 18% PEG 8000 and 0.5 M NaCl were added, and cells were spun to remove debris. For immunoprecipitations, GE2-9.5

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Section: Discussionmentioning

confidence: 86%

Complementation of the ionizing radiation sensitivity, DNA end binding, and V(D)J recombination defects of double-strand break repair mutants by the p86 Ku autoantigen.

Boubnov

Hall

Wills

et al. 1995

Proc. Natl. Acad. Sci. U.S.A.

124

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“…However, poly(ADP-ribose) polymerase has a much higher affinity for heat-denatured calf thymus DNA than for untreated DNA (32), making it clearly distinct from DEB factor. Furthermore, the gene maps to human chromosome 1 (9), while chromosome 2 restores X-ray resistance and V(D)J recombination to group 5 cells (19,43).…”

Section: Resultsmentioning

confidence: 99%

A DNA end-binding factor involved in double-strand break repair and V(D)J recombination.

1994

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We have identified a nuclear factor that binds to double-stranded DNA ends, independently of the structure of the ends. It had equivalent affinities for DNA ends created by sonication or by restriction enzymes leaving 5', 3', or blunt ends but had no detectable affinity for single-stranded DNA ends. Since X rays induce DNA double-strand breaks, extracts from several complementation groups of X-ray-sensitive mammalian cells were tested for this DNA end-binding (DEB) activity. DEB activity was deficient in three independently derived cell lines from complementation group 5. Furthermore, when the cell lines reverted to X-ray resistance, expression of the DEB factor was restored to normal levels. Previous studies had shown that group 5 cells are defective for both double-strand break repair and V(D)J recombination. The residual V(D)J recombination activity in these cells produces abnormally large deletions at the sites of DNA joining (F. Pergola, M. Z. Zdzienicka, and M. R. Lieber, Mol. Cell. Biol. 13:3464-3471, 1993, and G. Taccioli, G. Rathbun, E. Oltz, T. Stamato, P. Jeggo, and F. Alt, Science 260:207-210, 1993), consistent with deficiency of a factor that protects DNA ends from degradation. Therefore, DEB factor may be involved in a biochemical pathway common to both double-strand break repair and V(D)J recombination.X rays and oxidative metabolism induce a number of different DNA lesions, including base damage, single-strand breaks, and double-strand breaks. In response to this onslaught, cells have evolved specific biochemical pathways for repairing X-ray damage. The search for the genetic basis of these pathways has been greatly aided by the development of a number of easily cultured rodent cell lines that are hypersensitive to the lethal effects of X rays. Cell fusion experiments have shown that these cell lines fall into at least nine genetic complementation groups (10). Three of these groups are defective in the repair of DNA double-strand breaks (DSBs). Presumably, one or more of these genes encode a protein involved in protecting and resolving the free DNA ends generated by X rays.Another process that requires the resolution of free DNA ends is V(D)J recombination, a process that rearranges germ line DNA to assemble exons encoding immunoglobulin and T-cell receptor proteins (44). Early steps in the process involve the lymphocyte-specific recombination-activating genes RAG-1 and RAG-2. Cotransfection of both genes confers V(D)J recombination activity to nonlymphoid cells; therefore, RAG-1 and RAG-2 initiate V(D)J recombination in some still-undefined way (31, 39). Endonucleases recognize and cleave the DNA at sites defined by conserved signal sequences. Other proteins must resolve the free DNA ends generated by cleavage and rejoin the ends, forming a coding join and a signal join. The result is a rearranged immunoglobulin or T-cell receptor gene.A mutant mouse model provided the first indication that DSB repair and V(D)J recombination share common gene products. Mice homozygous for the scid mutation suffe...

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“…The XRCC5 gene has been mapped to 2q35, the same region as that of the Ku86 gene (6,16,21). In addition, group 5 cells lack a DNA-end-binding (DEB) activity with an abundance, nuclear localization, DNA substrate specificities, and antigenic determinants similar to those of Ku (13,36,37).…”

mentioning

confidence: 99%

“…First, human Ku86 cDNA did not completely rescue group 5 mutants. Second, chromosome mapping experiments sampled only a fraction of the human genome (6,21) and therefore failed to rule out the possibility that complementing activity was present on another chromosome in addition to chromosome 2. Therefore, we in-troduced a wild-type hamster cDNA into group 5 mutant cells and demonstrated that this could completely restore DEB activity, Ku70 protein levels, X-ray resistance, and V(D)J recombination.…”

mentioning

confidence: 99%

Ku86 Defines the Genetic Defect and Restores X-Ray Resistance and V(D)J Recombination to Complementation Group 5 Hamster Cell Mutants

Errami

Smider

Rathmell

et al. 1996

Molecular and Cellular Biology

Self Cite

152

117

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X-ray-sensitive hamster cells in complementation groups 4, 5, 6, and 7 are impaired for both double-strand break repair and V(D)J recombination. Here we show that in two mutant cell lines (XR-V15B and XR-V9B) from group 5, the genetic defects are in the gene encoding the 86-kDa subunit of the Ku autoantigen, a nuclear protein that binds to double-stranded DNA ends. These mutants express Ku86 mRNA containing deletions of 138 and 252 bp, respectively, and the encoded proteins contain internal, in-frame deletions of 46 and 84 amino acids. Two X-ray-resistant revertants of XR-V15B expressed two Ku86 transcripts, one with and one without the deletion, suggesting that reversion occurred by activation of a silent wild-type allele. Transfection of fulllength cDNA encoding hamster Ku86 into XR-V15B cells resulted in a complete rescue of DNA-end-binding (DEB) activity and Ku70 levels, suggesting that Ku86 stabilizes the Ku70 polypeptide. In addition, cells expressing wild-type levels of DEB activity were fully rescued for X-ray resistance and V(D)J recombination, whereas cells expressing lower levels of DEB activity were only partially rescued. Thus, Ku is an essential component of the pathway(s) utilized for the resolution of DNA double-strand breaks induced by either X rays or V(D)J recombination, and mutations in the Ku86 gene are responsible for the phenotype of group 5 cells.All cells possess a mechanism for repairing DNA doublestrand breaks (DSBs) produced by ionizing radiation. Cells of the immune system must also resolve DNA DSBs produced by V(D)J recombination of the immunoglobulin and T-cell receptor genes during development of B and T cells (reviewed in reference 28). In fact, the biochemical pathways for DSB repair and V(D)J recombination have a number of common factors. Evidence of this was first described for the severe combined immune deficient (scid) mouse, which is hypersensitive to ionizing radiation because of defective DSB repair (1, 12, 18) and immune deficient because of defective V(D)J recombination (29). Subsequently, other X-ray-sensitive rodent cell lines defective in DSB repair were also found to have impaired V(D)J recombination (17,27,34,45,47). These cell lines fall into four complementation groups, 4, 5, 6, and 7, with group 7 corresponding to the scid defect (43, 52). The human genes capable of rescuing these mutants are designated XRCC4, XRCC5, XRCC6, and XRCC7, respectively (XRCC denotes X ray cross-complementing) (for a review, see reference 51).Recently, it has been shown that the Ku protein is involved in both DSB repair and V(D)J recombination. Ku is an abundant nuclear protein identified originally as an autoantigen recognized by sera from patients with autoimmune diseases, including scleroderma-polymyositis overlap syndrome and systemic lupus erythematosus (31

show abstract

Localization of a DNA repair gene (XRCC5) involved in double-strand-break rejoining to human chromosome 2.

Cited by 51 publications

References 30 publications

Complementation of the ionizing radiation sensitivity, DNA end binding, and V(D)J recombination defects of double-strand break repair mutants by the p86 Ku autoantigen.

Complementation of the ionizing radiation sensitivity, DNA end binding, and V(D)J recombination defects of double-strand break repair mutants by the p86 Ku autoantigen.

A DNA end-binding factor involved in double-strand break repair and V(D)J recombination.

Ku86 Defines the Genetic Defect and Restores X-Ray Resistance and V(D)J Recombination to Complementation Group 5 Hamster Cell Mutants

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