Cohesion between sister chromatids, mediated by the chromosomal cohesin complex, 
is a prerequisite for their alignment on the spindle apparatus and segregation 
in mitosis. Budding yeast cohesin first associates with chromosomes in G1. Then, 
during DNA replication in S-phase, the replication fork-associated 
acetyltransferase Eco1 acetylates the cohesin subunit Smc3 to make cohesin's DNA 
binding resistant to destabilization by the Wapl protein. Whether stabilization 
of cohesin molecules that happen to link sister chromatids is sufficient to 
build sister chromatid cohesion, or whether additional reactions are required to 
establish these links, is not known. In addition to Eco1, several other factors 
contribute to cohesion establishment, including Ctf4, Ctf18, Tof1, Csm3, Chl1 
and Mrc1, but little is known about their roles. Here, we show that each of 
these factors facilitates cohesin acetylation. Moreover, the absence of Ctf4 and 
Chl1, but not of the other factors, causes a synthetic growth defect in cells 
lacking Eco1. Distinct from acetylation defects, sister chromatid cohesion in 
ctf4Δ and chl1Δ cells is not improved by removing Wapl. Unlike previously 
thought, we do not find evidence for a role of Ctf4 and Chl1 in Okazaki fragment 
processing, or of Okazaki fragment processing in sister chromatid cohesion. 
Thus, Ctf4 and Chl1 delineate an additional acetylation-independent pathway that 
might hold important clues as to the mechanism of sister chromatid cohesion 
establishment.