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

Abstract

Multiple pathways exist to repair DNA damage induced by methylating and crosslinking agents in Arabidopsis thaliana. The SWI2/SNF2 translocase RAD5A, the functional homolog of budding yeast Rad5 that is required for the error-free branch of post-replicative repair, plays a surprisingly prominent role in the repair of both kinds of lesions in Arabidopsis. Here we show that both the ATPase domain and the ubiquitination function of the RING domain of the Arabidopsis protein are essential for the cellular response to different forms of DNA damage. To define the exact role of RAD5A within the complex network of DNA repair pathways, we crossed the rad5a mutant line with mutants of different known repair factors of Arabidopsis. We had previously shown that RAD5A acts independently of two main pathways of replication-associated DNA repair defined by the helicase RECQ4A and the endonuclease MUS81. The enhanced sensitivity of all double mutants tested in this study indicates that the repair of damaged DNA by RAD5A also occurs independently of nucleotide excision repair (AtRAD1), single-strand break repair (AtPARP1), as well as microhomology-mediated double-strand break repair (AtTEB). Moreover, RAD5A can partially complement for a deficient AtATM-mediated DNA damage response in plants, as the double mutant shows phenotypic growth defects.

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.…”

“…AtRAD5A defines a pathway in the repair of DNA CLs separate from the helicase RECQ4A and the nuclease MUS81 (Mannuss et al ., ). Furthermore, we recently demonstrated that AtRAD5A acts independently of NER, single‐strand break repair, microhomology‐mediated end‐joining and the ATM‐mediated DNA damage response (Klemm et al ., ). Therefore, it was surprising to find a common involvement of AtHRQ1 and AtRAD5A in DNA CL repair.…”

“…In this pathway, the excision of the affected single strand occurs 5′ and 3′ of the lesion mediated by endonucleolytic incisions through HsXPF‐HsERCC1 (Staresincic et al ., ; Fagbemi et al ., ). In Arabidopsis, mutants of RAD1 were shown to be highly sensitive to CL agents (Klemm et al ., ). NER is a main repair mechanism for intrastrand CLs.…”

“…Mutants of At RAD5A exhibit hypersensitivity against CL agents and base methylations (Chen et al ., ). Recently, it was shown that AtRAD5A acts in a pathway independent of the main DNA repair pathways, NER, single‐strand break repair, as well as microhomology‐mediated double‐strand break repair (Klemm et al ., ). As AtFAN1 was found to genetically interact with both AtRECQ4A and AtRAD5A, it was classified into the CL repair pathway above both factors, uniting them into a FAN1‐dependent branch independently of AtMUS81 (Herrmann et al ., ).…”

Poly(ADP-Ribose) Polymerases in Plants and Their Human Counterparts: Parallels and Peculiarities

“…In agreement with increased gene expression and activity, the genetic inhibition of AtPARPs in parp1 and parp2 mutant plants enhanced the sensitivity of plant growth to methyl methane sulfonate (MMS, a DNA alkylation agent that induces N-alkyl lesions and single strand breaks (SSB)) and to bleomycin [97,100,108,117]. Similarly, formation of true leaves was reduced in parp2 seedlings grown on bleomycin and mitomycin C (a DNA cross-linking agent) [92].…”