DNA replication is an essential process in all domains of life. Replication must be precisely regulated, especially at the step of initiation. In bacteria, the replication initiator DnaA is regulated by multiple post-translational regulations to ensure timely replication. Caulobacter crescentus has the most strict replication regulation that DNA only replicates once per cell cycle, and proteolysis of DnaA identified in this species is the only irreversible way to inhibit DnaA, suggesting it might be pivotal to restricting DNA replication. However, the responsible protease(s) and mechanism for its degradation remain unclear since its first discovery in 2005. In this thesis, I describe the efforts to characterize the proteolysis regulation on C. crescentus DnaA. I identified and characterized DnaA degradation by two different proteases, Lon and ClpAP. Lon is the dominant protease for DnaA degradation, and my work on this degradation revealed a novel allosteric regulation mechanism by which Lon links unfolded substrate concentration with DnaA proteolysis, and provides a way for Lon to rapidly eliminate DnaA and arrest replication during proteotoxic stress. Mechanistic studies of Lon-dependent degradation shows that a complicated mechanism governs the recognition and degradation of DnaA, including the existence of multiple degradation determinants and the dependency of DnaA activity state. In contrast, ClpAP plays an auxiliary role on DnaA degradation, but this degradation is enhanced during nutrient starvation stress. Interestingly, Lon degrades DnaA more rapidly when it is in a complex with DnaA loaded on the replication origin DNA, but a specific structure of DNA, G-quadruplex, strongly inhibits general substrate degradation by Lon. Taken together, the studies in this thesis revealed the complex mechanisms on DnaA degradation in Caulobacter crescentus, and provided insights on how cells interrogate proliferation status in changing environments by modulating the levels of a replication factor.