Mutation in the catalytic subunit of DNA polymerase α influences transcriptional gene silencing and homologous recombination in Arabidopsis

Abstract

SUMMARYREPRESSOR OF SILENCING 1 (ROS1) encodes a DNA demethylase that actively removes DNA methylation. Mutation in ROS1 leads to transcriptional gene silencing of a T-DNA locus that contains two genes, RD29A-LUC and 35S-NPTII, originally expressed in the C24 wild type. These units have different silencing regulation mechanisms: the former mechanism is dependent on small interfering RNA (siRNA)-directed DNA methylation, but the latter is not. We studied the latter gene silencing mechanism by screening the suppressors of the ros1 mutant using the silenced 35S-NPTII as a selection marker gene. The pola/incurvata2 (icu2) gene was isolated as one ros1 suppressor because its mutation leads to the reactivation of the silenced 35S-NPTII gene. POLa/ICU2 encodes a catalytic subunit of DNA polymerase a. Mutation of POLa/ICU2 did not affect DNA methylation, but reduced histone H3 Lys9 dimethylation (H3K9me2) modification in the 35S promoter. The pola mutation also influences the development of the shoot apical meristem, and delays the G2/M phase with high expression of a G2/M marker gene CycB1;1:GUS. Furthermore, the frequency of homologous recombination is greater in the pola/icu2 mutant than in the C24 wild type. Our results suggest that DNA polymerase a is involved in mediating epigenetic states and in DNA homologous recombination in Arabidopsis.

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

“…of the core DNA-replication proteins, including the DNA single-strand-binding protein RPA2A (Xia et al, 2006), Pola (Liu et al, 2010a), Pol « (Yin et al, 2009), POLD2 (this study), RFC1 (Liu et al, 2010b), and TOUSLED protein kinase (Wang et al, 2007). Other studies have identified other DNA replication-related proteins involved in mediating TGS, including BRU1 (Takeda et al, 2004), chromatin assembly factor (CAF-1) subunits FAS1 and FAS2 (Ono et al, 2006;Schonrock et al, 2006), and TEBICHI (Inagaki et al, 2009).…”

“…However, pold2-1 had little effect on the ProRD29A:LUC locus in dms3-4. Like mutants of DNA replication-related proteins (Elmayan et al, 2005;Xia et al, 2006;Yin et al, 2009;Liu et al, 2010aLiu et al, , 2010b, pold2-1 failed to suppress the silenced ProRD29A:LUC in the ros1-1 mutant (Fig. 1F).…”

“…1C). TSIs are endogenous TGS loci (Steimer et al, 2000), the expression of which is altered in some mutants involved in DNA methylation, histone modification, or DNA replication (Steimer et al, 2000;Xia et al, 2006;Yin et al, 2009;Liu et al, 2010aLiu et al, , 2010b. The expression of TSIs was increased in these isolated mutants (Fig.…”

“…In Arabidopsis (Arabidopsis thaliana), mutations in replication factor C1 (RFC1), Pol «, Pol a, and replication protein A2A (RPA2A) can suppress gene silencing in a DNA methylation-independent manner (Elmayan et al, 2005;Kapoor et al, 2005;Xia et al, 2006;Liu et al, 2010aLiu et al, , 2010b. These mutants are hypersensitive to DNA damage and exhibit reduced telomere length and increased genomic instability.…”

Regulating DNA Replication in Plants

“…Interestingly, the POLA1 gene, also known as INCURVATA2 (ICU2), was originally identified in a genetic screen for abnormal leaf morphology and has a role in transcriptional silencing (Barrero et al 2007;Liu et al 2010a). The sequence features of POLA1 are conserved with the exception of a YYRRLFP motif of unknown function present in yeast and animal POLA4 subunit but not in plants (Shultz et al 2007).…”