Cell cycle progression is orchestrated by a complex interplay between post-translational modifications (PTMs) controlling activation (phosphorylation) and degradation (ubiquitination) of key cell cycle regulators. To comprehensively identify ubiquitin signalling components critical for cell proliferation, we employed a chemical-genetic CRISPR knock-out screen, exploiting the first-in-class ubiquitin E1 inhibitor TAK-243. Our findings provide a systems view of key protein networks underpinning cell cycle progression. We identified 239 genes, including C16orf72/HAPSTR1, whose ablation rendered cells sensitive to a ubiquitin signalling blockade, and 55 genes that conferred resistance under the same conditions. We reveal broad transcriptional changes accompanied with proteome-wide alterations in the ubiquitin signalling landscape upon loss of HAPSTR1 expression, revealing that HAPSTR1 is involved in homeostasis of cell cycle regulators such as CDK6 and CDC6. Mechanistically, we found that HAPSTR1 orchestrates the timing of the G1/S-transition via RB-E2F1 pathway activation by CDK6. Under diminished ubiquitin signalling, compensatory mechanisms fail to respond to accelerated S phase entry and replication stress upon loss of HAPSTR1, eventually compromising cell cycle integrity. Altogether, we provide a valuable resource on the interplay between the ubiquitin system and cell proliferation and uncover HAPSTR1 as a transcriptional regulator orchestrating the G1/S transition.