Replication Stress Leads to Genome Instabilities inArabidopsisDNA Polymerase Δ Mutants

Abstract

Impeded DNA replication or a deficiency of its control may critically threaten the genetic information of cells, possibly resulting in genome alterations, such as gross chromosomal translocations, microsatellite instabilities, or increased rates of homologous recombination (HR). We examined an Arabidopsis thaliana line derived from a forward genetic screen, which exhibits an elevated frequency of somatic HR. These HR events originate from replication stress in endoreduplicating cells caused by reduced expression of the gene coding for the catalytic subunit of the DNA polymerase d (POLd1). The analysis of recombination types induced by diverse alleles of pold1 and by replication inhibitors allows the conclusion that two not mutually exclusive mechanisms lead to the generation of recombinogenic breaks at replication forks. In plants with weak pold1 alleles, we observe genome instabilities predominantly at sites with inverted repeats, suggesting the formation and processing of aberrant secondary DNA structures as a result of the accumulation of unreplicated DNA. Stalled and collapsed replication forks account for the more drastic enhancement of HR in plants with strong pold1 mutant alleles. Our data suggest that efficient progression of DNA replication, foremost on the lagging strand, relies on the physiological level of the polymerase d complex and that even a minor disturbance of the replication process critically threatens genomic integrity of Arabidopsis cells.

The second subunit of DNA-polymerase delta is required for genomic stability and epigenetic regulation

“…DNA Pol d is conserved in eukaryotes and is important in regulating genomic stability and development in Arabidopsis (Schuermann et al, 2009;Iglesias et al, 2015). To our knowledge, the biological functions of Arabidopsis POLD2 have not been studied yet.…”

“…As shown in Figure 4E, many more GUS staining sectors were observed in the cotyledons in pold2-1 than in the wild type, suggesting that HR is negatively modulated by POLD2. A previous study indicated that mutations in the catalytic subunit of Pol d (POLD1) result in genome instability and enhance the frequency of somatic intramolecular HR (Schuermann et al, 2009). We also found that the telomere was shorter in pold2-1 or pold2-1 dms3-4 than in the wild type or dms3-4 (Fig.…”

NAP1 Family Histone Chaperones Are Required for Somatic Homologous Recombination in Arabidopsis

“…We next focused on nrp1-1 nrp2-1 for more detailed analyses. To address whether the observed somatic HR defects are reproducible with different recombination substrates, the line 1445 containing a similar GUS reporter construct as in 1406 but at a different genome location (Fritsch et al, 2004;Schuermann et al, 2009) was introgressed into nrp1-1 nrp2-1 by crossing. Similar to 1406, 1445 also showed a reduction in somatic HR in the nrp1-1 nrp2-1 mutant background compared with Col under either standard growth conditions or UV treatment (Figure 2A).…”

“…The two fragments contain an overlapping region and are spatially arranged as inverted repeats that restore a functional GUS after an HR event ( Figure 1A; Schuermann et al, 2009). The restored GUS activity can be visualized in planta as a blue spot/sector by histochemical staining ( Figure 1B).…”

“…To analyze the role of NAP1s and NRPs in somatic HR, we used the recombination substrate line 1406, which contains a nonfunctional reporter transgene consisting of two separated fragments of the b-glucuronidase (GUS) gene (Gherbi et al, 2001;Schuermann et al, 2009). The two fragments contain an overlapping region and are spatially arranged as inverted repeats that restore a functional GUS after an HR event ( Figure 1A; Schuermann et al, 2009).…”