Poly(ADP-ribose)1 and PARP2 are critical DNA damage sensors, involved in nearly every known pathway of DNA repair. While unbound, PARP1 and PARP2 have a “beads-on-a-string” conformation, which maintains low basal activity. Upon sensing a DNA break, these domains collapse around the damage, triggering a complex network of domain intercommunications that open the active site to substrate NAD+ binding, and enable >1000-fold increase in catalytic rate. The structural and backbone dynamic changes that are driven by these domain intercommunications during DNA damage engagement have been extensively characterized in PARP1. In comparison, there is limited structural information, and no backbone dynamic information, on PARP2. Additionally, current studies have yet to sufficiently addressed the importance of these complex networks of autoinhibition and subsequent activation in cells. In this thesis, I first utilized hydrogen-deuterium exchange mass spectrometry (HXMS) and biochemical methods to investigate the backbone dynamic changes that maintain PARP2 autoinhibition, and subsequently relieve it upon binding to its preferred DNA damage substrate, a 5’ phosphorylated nick, and how these changes are further modified in the presence of cofactor Histone PARylation factor 1 (HPF1). Like PARP1, I observed that PARP2 similarly relies upon rapid HD unfolding to regulate its activity. However, I found that PARP2 will not fully activate until the additional melting of a small 310-helix adjacent to the NAD+ binding site. In response to these findings, I utilized a robust ectopic expression system to inducibly express a spectrum of hyperactive PARP1 or PARP2 mutants in our DLD-1Flp-In Trex Tir1 cell line, allowing me to study the cellular consequences of disrupting PARP1 or PARP2 autoinhibition. Using this system, I reported that disruptions to PARP1 or PARP2 autoinhibition, in the absence of DNA damage, lead to acute cellular toxicity and increased PARGi sensitivity. Put together, these findings have advanced our understanding of the key similarities and differences between PARP1 and PARP2 autoinhibition and activation, and the importance of the complex allosteric network that maintains both of these states.