Loss of Werner syndrome protein function promotes aberrant mitotic recombination - PubMed (original) (raw)

Loss of Werner syndrome protein function promotes aberrant mitotic recombination

P R Prince et al. Genes Dev. 2001.

Abstract

The chromosome 8p11-12 Werner syndrome (WRN ) locus encodes a RecQ helicase protein of unknown function that possesses both 3' --> 5' helicase and 3' --> 5' exonuclease activities. We show that WRN cell lines display a marked reduction in cell proliferation following mitotic recombination, and generate few viable gene conversion-type recombinants. These findings indicate that WRN plays a role in mitotic recombination, and that a loss of WRN function may promote genetic instability and disease via recombination-initiated mitotic arrest, cell death, or gene rearrangement.

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Figures

Figure 1

Reduced recovery of viable pNeoA recombinants in WRN cells. (a) pNeoA contains a direct repeat of bacterial neomycin phosphotransferase alleles (neo; open boxes) where each allele has been inactivated with an 8-bp _Bam_HI linker (filled boxes), separated by a hygromycin resistance gene (_hyg_r; hatched box). neo expression is controlled by a retroviral long terminal repeat (LTR, cross-hatched box; transcription start site, arrow). ΔLTR (cross-hatched box) is a partial LTR copy containing a 299-bp deletion (Meyn 1993). The location and extent of the 592-bp region of perfect sequence identity between the 5′ and 3′ neo alleles is shown by the black bars over the two alleles. (b) Reduced recombination-dependent G418-resistant colony formation in WRN cell lines. Dishes seeded with either 200 or 105 pNeoA-containing cells were grown in the absence or presence of G418, then crystal violet-stained to determine colony forming efficiency (left column) or pNeoA recombinant frequency (right column). Replicate cultures were grown from pools that contained 47 to 486 independent pNeoA clones/cell line. Results shown are from replicate cultures of GM639-500.1 (control) and AG11395-500.2 (Werner). Comparable results were obtained using replicate cultures derived from a second control line (GM-847) and from a WRN cell line (WV1) that lacks WRN protein (additional results not shown).

Figure 2

Mitotic recombination rate of control and WRN pLrec sublines. (a) pLrec contains a direct repeat of bacterial lacZ alleles (open boxes) that have been inactivated by an 8-bp _Xho_I linker (5′ allele, filled box) or by a 727-bp deletion (3′ allele), separated by the neomycin resistance gene (_neo_r; hatched box), lacZ expression is under SV40 early promoter control (P/E; cross-hatched box). The location and extent of the 693-bp region of perfect sequence identity between the 5′ and 3′ lacZ alleles are shown by the black bars over the two alleles. (b) Mitotic recombination rates for independent control and WRN pLrec sublines that lack (WV1-derived) or retain (AG07066-derived) WRN protein. A total of 273 independent G418-resistant colonies were screened by Southern blot hybridization to identify seven sublines that contained intact single copies of pLrec: control sublines 639L22 (a), 639rec75 (b) and 847L22 (a); and WRN sublines WVP45 (a), WVP46 (b), PSVL30 (a) and 811L45 (b). One additional GM847-derived pLrec subline, LNL1 (GM847b) was kindly provided by M.S. Meyn (Yale University, CT and Hospital for Sick Children, Toronto). Recombination rates were calculated from the fraction of colonies that contained no Lac+ cells (the P0 fraction) using data from 860 colonies/pLrec subline (range 226–1559) where mean colony size was 374 cells (range 229–530). The difference in recombination rate between WRN and control cells was not significant: P = 0.22, with 95% confidence intervals of −9.8, 30.2.

Figure 3

Reduced cell division of Lac+ WRN mitotic recombinants. (a) Control and WRN colonies were fixed and stained with X-gal to reveal Lac+ (dark, arrow) cells. A majority of WRN colonies had single Lac+ cells (arrow), in contrast to control Lac+ colonies. (b) Frequency distribution of the number of Lac+ WRN (filled bars) and control (open bars) colonies in which Lac+ recombinant cells had undergone no, 1–2 or >2 cell divisions. Lac+ cells were present in 127/2200 WRN and 151/4684 control colonies.

Figure 4

Model of WRN function. The replication of DNA containing strand breaks or damage can result in stalled replication/synthesis forks, ssDNA gaps or DNA double strand breaks (DSBs) that are substrates for homology- and/or recombination-dependent repair. WRN acts to promote error-free homology-dependent repair or restart to generate viable recombinant products while inhibiting illegitimate recombination or end-joining. In the absence of WRN, unresolved recombination products and DNA ends may lead to checkpoint activation and mitotic arrest or cell death, or to gene rearrangement or loss mediated by nonhomologous DNA end joining (NHEJ).

References

1. Birge EA, Low KB. Detection of transcribable recombination products following conjugation in Rec+, RecB− and RecC− strains of Escherichia coli K12. J Mol Biol. 1974;83:447–457. - PubMed
1. Chakraverty RK, Hickson ID. Defending genome integrity during DNA replication: A proposed role for RecQ family helicases. BioEssays. 1999;21:286–294. - PubMed
1. Chen C, Okayama H. High-efficiency transformation of mammalian cells by plasmid DNA. Mol Cell Biol. 1987;7:2745–2752. - PMC - PubMed
1. Constantinou A, Tarsounas M, Karow JK, Brosh RM, Jr, Bohr VA, Hickson ID, West SC. Werner's syndrome protein (WRN) migrates Holliday junctions and co-localizes with RPA upon replication arrest. EMBO Reports. 2000;1:80–84. - PMC - PubMed
1. Constantinou A, Davies AA, West SC. Branch migration and Holliday junction resolution catalyzed by activities from mammalian cells. Cell. 2001;104:259–269. - PubMed

Loss of Werner syndrome protein function promotes aberrant mitotic recombination - PubMed (original) (raw)