Figure 4.

The pold2-1 mutant has increased sensitivity to DNA damage, a delayed cell cycle, increased HR, and a reduced telomere length. A, pold2-1 is hypersensitive to MMS. Wild-type (WT), dms3-4, pold2-1 dms3-4, and pold2-1 seedlings that were grown for 15 d on MS medium alone or MS medium with 200 mg/L HU (200 HU), 50 ppm (65 μg mL⁻¹) MMS, or 25 µm cisplatin (25 CIS). P-values were calculated using Paired Student’s t test. B, Relative fresh weight of seedlings in D. The fresh weight relative to untreated controls after treatment with DNA-damaging reagents is shown. Values are means ± se, n = 3. C, Relative expression of DNA damage response genes in the wild type, dms3-4, pold2-1 dms3-4, and pold2-1 as determined by qRT-PCR. RNAs extracted from 7-d-old seedlings were used for qRT-PCR. Values were normalized to the expression level of the references gene (UBI). Two independent experiments (each with three technical replicates) were done with similar results. Values are means ± se, n = 3, from one experiment. D, pold2-1 increases the expression of ProCYCB1;1:GUS. ProCYCB1;1:GUS was introduced into the pold2-1 mutant by crossing the wild type carrying ProCYCB1;1:GUS with the pold2-1 mutant. a to d, GUS staining in a wild-type seedling (a), a wild-type root tip (b), a pold2-1 seedling (c), and a pold2-1 root tip (d). e, CYCB1;1 expression as determined by qRT-PCR. RNAs were extracted from 7-d-old seedlings and used for qRT-PCR. Values were normalized to the expression level of the references gene (UBI). Two independent experiments (each with three technical replicates) were done with similar results. Values are means ± se, n = 3, from one experiment. E, pold2-1 increases HR in 14-d-old seedlings. The wild-type 651 carrying a homologous reporter GUS gene was crossed with the dms3-4 pold2-1 mutant. Homozygous pold2-1 plants carrying the homozygous GUS reporter were isolated and used for GUS staining. F and G, pold2-1 exhibits a reduced telomere length. Genomic DNAs of the wild type, dms3-4, pold2-1, and pold2-1 dms3-4 digested by HinfI (DNA-methylation sensitive, F) or MseI (DNA-methylation insensitive, G) were subjected to Southern blot with a telomere probe.