Similar to CPT, UV-B and MMC above, therpa1c rpa1edouble mutant displays supra-additive hypersensitivity to both HU (0.5 Gfap mM) and APH in comparison to either singlerpa1mutants. conditions. We show these defects in therpa1b rpa1dmutant are likely the result of defective DNA replication leading to reduction in cell division. == INTRODUCTION == Replication Protein A (RPA) is a eukaryotic, single-stranded DNA (ssDNA)-binding protein composed of three associated subunits, RPA1 (70 kDa), RPA2 Cimetidine (32 kDa) and RPA3 (14 kDa). The primary biochemical function of the heterotrimeric RPA complex (referred to hereafter as RPA) is to protect and preserve ssDNA from nucleolytic degradation and hairpin formation, similar to SSB (single-stranded binding protein) in prokaryotes (1,2). Consistent with this function, RPA plays essential roles in almost all DNA metabolic pathways including S-phase genome replication, DNA recombination and DNA excision repair. Importantly, RPA plays a key role in the activation and maintenance of cellular responses to DNA damage. Downstream cellular responses to detected DNA damage include regulation of cell-cycle transitions Cimetidine (checkpoints), induction of DNA repair, changes in gene transcription and in some cases apoptosis (programmed cell death). These responses are ultimately mediated through the two closely related protein kinases, ATM (Ataxia Telangiectasia mutated) and ATR (ATM and Rad3-related) (35). While ATM is activated primarily by double-strand breaks, ATR is activated by a wide variety of lesions that result in stalled replication forks, such as DNA breaks, UV photoproducts and DNA crosslinks. These stalled replication forks, as well as DNA excision activities involved in repairing the lesions, induce functional uncoupling (physical disassociation) of helicase and polymerase activities resulting in the persistence of abnormally long stretches of ssDNA (68). Studies in yeasts and animal cells suggest that RPA-coating of these ssDNA stretches act as a molecular signal to activate ATR-dependent downstream phosphorylation, primarily through an associated Cimetidine protein called ATRIP (911). An ATRIP ortholog has recently been described in plants, and mutants inATRIPdisplay a nearly identical phenotype toatrmutants when challenged with replication blocking agents (12,13). This suggests plants encode a similar system of RPA-dependent activation of ATR in the DNA-damage response. Interestingly, RPA itself is a target of phosphorylation by ATM, ATR, and the related kinase DNA-PK (found only in animals) in response to DNA damage. During the unperturbed cell cycle, RPA activity is regulated through cyclin-dependent kinase phosphorylation of the RPA2 subunit during DNA replication and mitosis, and dephosphorylation as cells progress into G1 (14,15). Cyclin-dependent kinase phosphorylation can prime RPA2 for additional phosphorylation by ATR, ATM and DNA-PK in response to DNA damage (1). These phosphorylation changes to RPA2 can have effects on RPA activity during DNA repair and replication (1621). For example, RPA hyper-phosphorylation mimetic mutants, engineered with multiple negative amino acids at known phosphorylation sites within RPA32 are unable to interact with replication centers (20). Thus, models have been proposed whereby phosphorylation of RPA2 by ATR/ATM/DNA-PK acts as a switch to modulate active DNA replication if DNA damage persists within the cell. Although plant and animal DNA metabolism and DNA-repair responses are highly conserved in most aspects, RPA regulation in plants appears surprisingly different. In contrast to the single RPA1, RPA2 and RPA3 subunits found in yeasts and mammals [excluding humans where there are two RPA2-like genes (22)], plants encode multiple RPA1, RPA2 and RPA3 subunits. Rice contains three RPA1 paralogs, three RPA2 paralogs and one RPA3 homolog (23,24). Interaction studies Cimetidine suggest the subunits form at least three heterotrimeric complexes (25). Although studies in pea suggest that an RPA32 subunit is phosphorylated at certain developmental stages (26),in.