Mutation in Rpa1 results in defective DNA double-strand break repair, chromosomal instability and cancer in mice

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Abstract

Most cancers have multiple chromosomal rearrangements; the molecular mechanisms that generate them remain largely unknown. Mice carrying a heterozygous missense change in one of the DNA-binding domains of Rpa1 develop lymphoid tumors, and their homozygous littermates succumb to early embryonic lethality. Array comparative genomic hybridization of the tumors identified large-scale chromosomal changes as well as segmental gains and losses. The Rpa1 mutation resulted in defects in DNA double-strand break repair and precipitated chromosomal breaks as well as aneuploidy in primary heterozygous mutant mouse embryonic fibroblasts. The equivalent mutation in yeast is hypomorphic and semidominant and enhanced the formation of gross chromosomal rearrangements in multiple genetic backgrounds. These results indicate that Rpa1 functions in DNA metabolism are essential for the maintenance of chromosomal stability and tumor suppression.

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Figure 1: Generation of Rpa1689C mutant mice.
Figure 2: Phenotypes of Rpa1 mutant mice.
Figure 3: Chromosome analyses of tumors and MEFs in Rpa1689C/+ mice.

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Acknowledgements

We thank H. Hou, Jr. and B. Jin for technical assistance and S. Brill for providing antibodies. This work was supported by grants from the US National Institutes of Health (to R.K., R.D.K. and W.E.).

Author information

Correspondence to Richard D Kolodner or Winfried Edelmann.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Fig. 1

Molecular dynamics suggests that the L221P mutation disrupts the DNA-binding interface of the oligonucleotide binding (OB) domain 1 of human RPA1. (PDF 5784 kb)

Supplementary Fig. 2

Laser capture microdissection (LCM) and loss of heterozygosity (LOH) analysis. (PDF 379 kb)

Supplementary Fig. 3

Partial dominance of plasmid borne rfa1-L221P alleles for sensitivity to constitutive expression of HO endonuclease. (PDF 7308 kb)

Supplementary Table 1

Summary of chromosomal aberrations in lymphomas of Rpa1L230P/+ mice. (PDF 60 kb)

Supplementary Table 2

Karyotypic aberrations in Rpa1L230P/+ MEFs. (PDF 51 kb)

Supplementary Table 3

Partial dominance of plasmid-borne rfa1-L221P alleles in MMS sensitivity. (PDF 64 kb)

Supplementary Table 4

Partial dominance of plasmid-borne rfa1-L221P alleles in HU sensitivity. (PDF 65 kb)

Supplementary Table 5

Partial dominance of plasmid-borne rfa1-L221P alleles in UV sensitivity. (PDF 719 kb)

Supplementary Methods (PDF 78 kb)

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