The maintenance of genomic stability relies on the concerted action of DNA repair and DNA damage signaling pathways. The PIAS (protein inhibitor of activated STAT) family of SUMO (small ubiquitin-like modifier) ligases has been implicated in DNA repair, but whether it plays a role in DNA damage signaling is still unclear. Here, we show that the PIAS3 SUMO ligase is important for activation of the ATR (ataxia telangiectasia and Rad3 related)-regulated DNA damage signaling pathway. PIAS3 is the only member of the PIAS family that is indispensable for ATR activation. In response to different types of DNA damage and replication stress, PIAS3 plays multiple roles in ATR activation. In cells treated with camptothecin (CPT), PIAS3 contributes to formation of DNA double-stranded breaks. In UV (ultraviolet light)-or HU (hydroxyurea)-treated cells, PIAS3 is required for efficient ATR autophosphorylation, one of the earliest events during ATR activation. Although PIAS3 is dispensable for ATRIP (ATR-interacting protein) SUMOylation and the ATR-ATRIP interaction, it is required for maintaining the basal kinase activity of ATR prior to DNA damage. In the absence of PIAS3, ATR fails to display normal kinase activity after DNA damage, which accompanies with reduced phosphorylation of ATR substrates. Together, these results suggest that PIAS3 primes ATR for checkpoint activation by sustaining its basal kinase activity, revealing a new function of the PIAS family in DNA damage signaling.Multiple types of post-translational modifications (PTMs), 2 including protein SUMOylation, are important for regulation of the DNA damage response (DDR) from yeast to human (1, 2). Several SUMO E3 ligases, particularly the members of the PIAS family, are shown to modify DDR proteins. In the budding yeast, the PIAS family member Siz1 SUMOylates PCNA during S phase, promoting the recruitment of Srs2 to replication forks to suppress unwanted recombination (3-6). Siz2, the second PIAS ligase in yeast, SUMOylates a large number of DNA replication and repair proteins in response to DNA damage, which may enhance the efficiency of DNA repair by stabilizing multiple protein interactions in repair complexes (7,8). A recent study showed that Siz2 is recruited to sites of DNA damage through its interaction with RPA, a sensor of single-stranded DNA (ssDNA), explaining the regulation of protein SUMOylation by DNA damage (9). Together these findings in yeast clearly show that the PIAS family SUMO ligases are critical regulators of the DDR.In human cells, four members of the PIAS family SUMO ligases have been identified: PIAS1, PIAS2 (PIASx␣/), PIAS3, and PIAS4 (PIASy) (10). Two of these SUMO ligases, PIAS1 and PIAS4, are recruited to sites of DNA damage (11). Both PIAS1 and PIAS4 contribute to the efficient recruitment of 53BP1, BRCA1, and RNF168 (11). A number of DDR proteins, such as 53BP1, BRCA1, MDC1, HERC2, RNF168, FNACI, and FANCD2, are SUMOylated by PIAS1 and/or PIAS4. The SUMOylation of these DDR proteins regulate their functions in several different w...