Subunit Interaction Differences Between the Replication Factor C Complexes in Arabidopsis and Rice

Abstract

Replication factor C (RFC) is a multisubunit complex that opens the sliding clamp and loads it onto the DNA chain in an ATP-dependent manner and is thus critical for high-speed DNA synthesis. In yeast (Saccharomyces cerevisiae) and humans, biochemical studies and structural analysis revealed interaction patterns between the subunits and architectures of the clamp loaders. Mutations of ScRFC1/2/3/4/5 lead to loss of cell viability and defective replication. However, the functions of RFC subunits in higher plants are unclear, except for AtRFC1/3/4, and the interaction and arrangement of the subunits have not been studied. Here, we identified rfc2-1/+, rfc3-2/+, and rfc5-1/+ mutants in Arabidopsis, and found that embryos and endosperm arrested at the 2/4-celled embryo proper stage and 6-8 nuclei stages, respectively. Subcellular localization analysis revealed that AtRFC1 and OsRFC1/4/5 proteins were localized in the nucleus, while AtRFC2/3/4/5 and OsRFC2/3 proteins were present both in the nucleus and cytoplasm. By using yeast two-hybrid (Y2H) and bimolecular fluorescence complementation (BiFC) techniques, we demonstrated the interactions of Arabidopsis and rice (Oryza sativa) RFC subunits, and proposed arrangements of the five subunits within the RFC complex, which were AtRFC5-AtRFC4-AtRFC3/2-AtRFC2/3-AtRFC1 and OsRFC5-OsRFC2-OsRFC3-OsRFC4-OsRFC1, respectively. In addition, AtRFC1 could interact with AtRFC2/3/4/5 in the presence of other subunits, while OsRFC1 directly interacted with the other four subunits. To further characterize the regions required for complex formation, truncated RFC proteins of the subunits were created. The results showed that C-termini of the RFC subunits are required for complex formation. Our studies indicate that the localization and interactions of RFCs in Arabidopsis and rice are distinctly discrepant.

Arabidopsis mediator subunit 17 connects transcription with DNA repair after UV‐B exposure

“…In yeast, MED17 physically interacts with the Rad2/XPG subunit of TFIIH and participates in DNA repair after UV exposure, recruiting Rad2 to transcribed genes, whereas in human cells MED17 interacts with a DNA helicase XPB subunit of TFIIH, which is essential for both transcription and NER (Eyboulet et al., 2013; Kikuchi et al., 2015). In our experiments, MED17 also directly or indirectly interacted with proteins that participate in DNA repair in Arabidopsis, such as the histone chaperones NAP1;3, NRP1 and NRP2 (Casati & Gomez, 2021), a DNA repair ATPase‐related protein and a replication factor C subunit 4 (Chen et al., 2018). However, we did not find interactions with any TFIIH subunit, suggesting that the role of MED17 in DNA repair may be through a different interaction in Arabidopsis.…”

“…Interestingly, several proteins that were previously described to have a role during DNA repair were identified, such as the cohesin factor PDS5C (Pradillo et al., 2015), the histone chaperones NAP1;3, NRP1 and NRP2 (Casati & Gomez, 2021), two DEK domain‐containing chromatin‐associated proteins (Waidmann et al., 2014), a sister chromatid cohesion 1 protein 4 (SYN4; da Costa‐Nunes et al., 2006), a DNA repair ATPase‐related protein and a replication factor C subunit 4 (Chen et al., 2018; Tables 1 and S1). LC‐MS/MS data showed that MED17 is in the same complex as the transcription initiation factor TFIID subunit 9 and the transcription initiation factor IIE subunit alpha; therefore, MED17 together with these proteins may be required for correct transcription coupled dna repair (TCR), as previously described in other species.…”

Genome-wide identification of the restorer-of-fertility-like (RFL) gene family in Brassica napus and expression analysis in Shaan2A cytoplasmic male sterility

“…4 and Additional file 4 . Interestingly, AtRFL11, AtRFL12 and AtRFL13 and three HXKs, including HXK1, HXK2 and HXK3, shared a common interacting protein, namely replication factor C2 (RFC2), a multi-subunit complex critical for high-speed ATP-dependent DNA synthesis [ 33 ]. No homologs of AtRFL25 were identified in B. napus .…”

Comparative proteome analyses of rhizomania resistant transgenic sugar beets based on RNA silencing mechanism

“…Four proteins that function in DNA- and RNA-related processes and whose expressions were up-regulated in the transgenic plants include CST complex subunit STN1 which protects the end of chromosomes (telomere) from degradation, 34 replication factor C subunit 4 as a component of replication factor C (RFC) which causes high-speed DNA synthesis, 35 ribonuclease J that removes RNAs with inefficient transcription in chloroplasts by exoribonuclease activity. In this sense, it is involved in the development of the chloroplast by regulating gene expression, 36 and putative pentatricopeptide repeat-containing protein, which is involved in mitochondrial mRNA processing.…”