AtRAD5A is a DNA translocase harboring a HIRAN domain which confers binding to branched DNA structures and is required for DNA repair in vivo

Abstract

SUMMARYDNA lesions such as crosslinks represent obstacles for the replication machinery. Nonetheless, replication can proceed via the DNA damage tolerance pathway also known as postreplicative repair pathway. SNF2 ATPase Rad5 homologs, such as RAD5A of the model plant Arabidopsis thaliana, are important for the error-free mode of this pathway. We able to demonstrate before, that RAD5A is a key factor in the repair of DNA crosslinks in Arabidopsis. Here, we show by in vitro analysis that AtRAD5A protein is a DNA translocase able to catalyse fork regression. Interestingly, replication forks with a gap in the leading strand are processed best, in line with its suggested function. Furthermore AtRAD5A catalyses branch migration of a Holliday junction and is furthermore not impaired by the DNA binding of a model protein, which is indicative of its ability to displace other proteins. Rad5 homologs possess HIRAN (Hip116, Rad5; domains. By biochemical analysis we were able to demonstrate that the HIRAN domain variant from Arabidopsis RAD5A mediates structure selective DNA binding without the necessity for a free 3 0 OH group as has been shown to be required for binding of HIRAN domains in a mammalian RAD5 homolog. The biological importance of the HIRAN domain in AtRAD5A is demonstrated by our result that it is required for its function in DNA crosslink repair in vivo.

The RecQ‐like helicase HRQ1 is involved in DNA crosslink repair in Arabidopsis in a common pathway with the Fanconi anemia‐associated nuclease FAN1 and the postreplicative repair ATPase RAD5A

“…We previously demonstrated that the RecQ helicase AtRECQ4A acts independently of AtRAD5A in CL repair (Mannuss et al ., ). The ATPase AtRAD5A is involved both regulatorily and mechanistically in the DNA damage tolerance pathway and thereby enables the error‐free repair of blocked replication forks (Chen et al ., ; Mannuss et al ., ; Kobbe et al ., ; Klemm et al ., ). To elucidate the role of AtHRQ1 in relation to AtRAD5A, we generated the hrq1‐1 rad5A‐2 double mutant.…”

The DNA translocase RAD5A acts independently of the other main DNA repair pathways, and requires both its ATPase and RING domain for activity in Arabidopsis thaliana

“…We identified the respective conserved amino acids in AtRAD5A and produced the constructs RAD5A‐HD (KT425/426AA) and RAD5A‐RD (C794S) with the respective amino acid exchanges via mutagenesis (Figure ). In vitro analysis of RAD5A expressed in Escherichia coli demonstrated that the KT425/426AA mutated protein ( RAD5A‐HD ), in contrast to the wild‐type protein, is indeed devoid of ATPase activity (Kobbe et al ., ).…”

“…To verify the genuine expression of the RAD5A transgene in the transformed rad5a‐2 lines, real‐time PCR expression analysis was performed and revealed similar or slightly higher levels of expression compared with the WT (Col‐0) (Figure S2). The presence of the different protein variants was furthermore confirmed via western blotting using a RAD5A antibody (Kobbe et al ., ) (Figure S3). This analysis also confirmed the absence of RAD5A protein in rad5a‐2 mutants.…”

“…In a previous study, we were already able to show that the conserved HIRAN domain of Arabidopsis RAD5A, which is involved in binding DNA repair intermediates, is essential for DNA repair in planta (Kobbe et al ., ). In the present study, we addressed the question as to whether both the RING and the helicase domain of Arabidopsis RAD5A are also essential for DNA repair, in general, or whether a specific domain is especially important for the repair of a particular kind of damage.…”

“…More recently, we could demonstrate in vitro that RAD5A is a DNA translocase that is able to catalyze fork regression and branch migration of Holliday junctions. Interestingly, the protein harbors a conserved HIP116 Rad5p N‐terminal (HIRAN) domain, the presence of which is essential for DNA repair in vivo and is required for DNA repair intermediate binding in vitro (Kobbe et al ., ). Besides the HIRAN domain, AtRAD5A harbors two additional conserved domains, the functions of which have not been tested in DNA repair in planta : the C‐terminal half of the protein contains a helicase domain, subdivided into seven conserved motifs.…”