RAD5A, RECQ4A, and MUS81 Have Specific Functions in Homologous Recombination and Define Different Pathways of DNA Repair inArabidopsis thaliana 

Abstract

Complex DNA structures, such as double Holliday junctions and stalled replication forks, arise during DNA replication and DNA repair. Factors processing these intermediates include the endonuclease MUS81, helicases of the RecQ family, and the yeast SNF2 ATPase RAD5 and its Arabidopsis thaliana homolog RAD5A. By testing sensitivity of mutant plants to DNAdamaging agents, we defined the roles of these factors in Arabidopsis. rad5A recq4A and rad5A mus81 double mutants are more sensitive to cross-linking and methylating agents, showing that RAD5A is required for damage-induced DNA repair, independent of MUS81 and RECQ4A. The lethality of the recq4A mus81 double mutant indicates that MUS81 and RECQ4A also define parallel DNA repair pathways. The recq4A/mus81 lethality is suppressed by blocking homologous recombination (HR) through disruption of RAD51C, showing that RECQ4A and MUS81 are required for processing recombination-induced aberrant intermediates during replication. Thus, plants possess at least three different pathways to process DNA repair intermediates. We also examined HR-mediated double-strand break (DSB) repair using recombination substrates with inducible site-specific DSBs: MUS81 and RECQ4A are required for efficient synthesis-dependent strand annealing (SDSA) but only to a small extent for single-strand annealing (SSA). Interestingly, RAD5A plays a significant role in SDSA but not in SSA.

The translesion polymerase ζ has roles dependent and independent of the nuclease MUS81 and the helicase RECQ4A in DNA damage repair in Arabidopsis

“…Thus, REV3 is not active in intrastrand cross-link repair pathways in which either RECQ4A or MUS81 is involved. Considering the previous results regarding RAD5A (Mannuss et al, 2010;Wang et al, 2011), we now have evidence for the existence of all four different subpathways of intrastrand cross-link repair in plants.…”

“…In contrast to the situation in animals and fungi, the DNA helicases RECQ4A and RAD5A as well as the endonuclease MUS81 define three parallel pathways of DNA cross-link repair in Arabidopsis (Mannuss et al, 2010). Interestingly, RAD5A and REV3 act independently in the repair of interstrand and intrastrand cross links and base alkylation (Wang, 2007).…”

“…We previously identified AtRAD5A as a functional Arabidopsis homolog of Rad5 (Chen et al, 2008). Interestingly, AtRAD5A is required for efficient repair by homologous recombination via the synthesis-dependent strand-annealing mechanism, a pathway that in some steps is related to the invasion model of PRR (Mannuss et al, 2010).…”

“…However, as plants, animals, and yeast differ in their DNA cross-link repair machinery (e.g. Mannuss et al, 2010;Knoll et al, 2012;Dangel et al, 2014;Herrmann et al, 2015), it is of particular importance to define the role of REV3 in relation to MUS81 and RECQ4A.…”

“…These independent pathways are defined by the ATPase RAD5A, the helicase RECQ4A, and MMS and UV-sensitive protein81 (MUS81; Mannuss et al, 2010). The structure-specific endonuclease MUS81 together with its noncatalytic subunit (Mms4 in yeast, Eme1 in Schizosaccharomyces pombe, and MMS4 or Crossover Junction Endonuclease (EME1) in humans and plants) functions in the rescue of stalled replication forks.…”