The integrity of the genome is constantly challenged by genotoxic factors and also DNA duplication during every cell cycle. Resolving replication stress and DNA lesions critically involves the kinases ATR and ATM as part of the DNA damage response (DDR). Until now, several clinical studies revealed that histone deacetylase inhibitors (HDACi) favorably combine with chemotherapeutic treatments. The underlying molecular mechanisms, however, remained to be defined. The present study shows for the first time that class I HDAC activity is necessary to keep up a sustained activation of ATR and ATM following replication stress. Specifically, simultaneous loss of HDAC1 and HDAC2 attenuated signaling of ATR/ATM, downstream checkpoint kinases, and p53. Despite a higher incidence of DNA damage, stressed cells were unable to establish a stable cell cycle arrest in S phase in the presence of HDACi and also showed increased levels of apoptosis. This work proposes a suppressed mRNA and protein expression of the PP2A regulatory subunit PR130 as the underlying molecular mechanism governed by HDAC1 and HDAC2. HDAC inhibition elevated the amount of PR130 protein, which was shown to associate with phosphorylated ATM after replication stress. Conversely, elimination of PR130 or inhibition of PP2A re-established ATM phosphorylation. Taken together, this study describes a novel molecular link between the epigenetic regulators HDAC1/2 and a phosphatase-dependent modulation of DDR signaling intensity in cells suffering from replication stress. PR130 was identified as a potential modulator of the DDR to terminate kinase activity and restore normal cellular physiology.