To ensure accurate chromosome segregation, the yeast genome undergoes dramatic structural changes that include DNA replication, sister chromatid cohesion and chromosome condensation. Numerous studies reveal that DNA replication factors impact cohesion establishment, but the molecular mechanism through which this regulation occurs and the extent that replication factors similarly regulate chromosome condensation remains unknown. Here, we investigate two DNA replication factors that regulate temporally distinct steps in cohesion. Chl1 is a DEAH-box DNA helicase that functions early during DNA replication to promote sister chromatid cohesion. The extent to which Chl1 promotes condensation, however, remains unknown. To address this deficit, we tested for Chl1-dependent condensation at both the rDNA locus and along the chromosome arm. Using both Net1-GFP and a modified FISH procedure to visualize rDNA locus structure, the results show that chl1 deletion strain exhibit significant levels of decondensation, relative to wildtype cells. We also tested for condensation defects along the chromosome arm using a two GFP assay kindly provided by Dr. Frank Uhlmann. Compared to wildtype cells, chl1 mutant cells exhibited a significant increase in inter-GFP distances. These results reveal that Chl1 plays a critical role in condensation - extending the role of Chl1 beyond cohesion and identifying Chl1 as an early regulator of chromatin structure. PCNA is a sliding clamp that promotes processive DNA replication and interacts with the cohesion establishment factor Ctf7/Eco1. Previous results revealed that overexpression of PCNA rescues the conditional growth defects in ctf7/eco1 mutant cells. The molecular basis of this rescue, however, remains unknown. We thus tested whether PCNA overexpression rescues either the cohesion or condensation defects present in ctf7/eco1 mutant cells. The results reveal that PCNA overexpression rescues the cohesion defects, but not the condensation defects, of ctf7/eco1 mutants. These results support prior findings from our lab that PCNA rescues the cohesion, but not the condensation, defects in pds5 mutant cells. Thus, PCNA plays a critical role in regulating a unique and distinct aspect of cohesin biology. In combination with other findings from our lab, these results support a new model in which cohesin is deposited onto each sister chromatid early during DNA replication and that this deposition relies on Chl1 DNA helicase-dependent alterations of DNA structure. Later during DNA replication, and possibly signaled through completion of Okazaki maturation, PCNA promotes cohesin-dependent establishment of sister chromatin cohesion but not chromosome condensation. Thus, cohesin functions (and possibly structures) are separable and likely spatially regulated throughout the genome. .