Abstract
Genetic screening identified a suppressor of ros1-1, a mutant of REPRESSOR OF SILENCING1 (ROS1; encoding a DNA demethylation protein). The suppressor is a mutation in the gene encoding the largest subunit of replication factor C (RFC1). This mutation of RFC1 reactivates the unlinked 35S-NPTII transgene, which is silenced in ros1 and also increases expression of the pericentromeric Athila retrotransposons named transcriptional silent information in a DNA methylation-independent manner. rfc1 is more sensitive than the wild type to the DNA-damaging agent methylmethane sulphonate and to the DNA inter- and intra- cross-linking agent cisplatin. The rfc1 mutant constitutively expresses the G2/M-specific cyclin CycB1;1 and other DNA repair-related genes. Treatment with DNA-damaging agents mimics the rfc1 mutation in releasing the silenced 35S-NPTII, suggesting that spontaneously induced genomic instability caused by the rfc1 mutation might partially contribute to the released transcriptional gene silencing (TGS). The frequency of somatic homologous recombination is significantly increased in the rfc1 mutant. Interestingly, ros1 mutants show increased telomere length, but rfc1 mutants show decreased telomere length and reduced expression of telomerase. Our results suggest that RFC1 helps mediate genomic stability and TGS in Arabidopsis thaliana.
Highlights
During DNA replication, DNA polymerase a interacts with DNA primase to form a complex for initiating synthesis of a 15-20 mer DNA primer using an RNA primer
This accession is referred to as the C24 wild type. ros1 mutations lead to the silencing of both RD29A-LUC and 35S-NPTII, and the ros1-1 mutant is sensitive to kanamycin (Gong et al, 2002a)
A kanamycin-resistant mutant, named rfc1-1, was isolated from ethyl methanesulfonate– mutagenized ros1-1 plants, for which the M2 generation was grown on Murashige and Skoog (MS) medium supplemented with 50 mg/L kanamycin
Summary
During DNA replication, DNA polymerase a interacts with DNA primase to form a complex for initiating synthesis of a 15-20 mer DNA primer using an RNA primer. Replication factor C (RFC), a clamp-loader complex consisting of five different subunits, binds DNA at the template–primer junctions and displaces polymerase a to terminate DNA primer synthesis. The binding of RFC to DNA creates a loading site for recruiting the DNA sliding clamp proliferating cell nuclear antigen (PCNA), a ringshaped homotrimer. RFC is an AAA+-type ATPase that requires ATP hydrolysis for opening and closing PCNA around DNA during DNA replication, repair, and recombination (Majka and Burgers, 2004). Telomeres can be elongated by some mutations, such as in Rfc and DNA polymerase a, suggesting that the replication machinery regulates telomere length (Adams and Holm, 1996).
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