Histone acetylation and nucleosome remodeling regulate DNA damage repair, 
replication and transcription. Rtt109, a recently discovered histone 
acetyltransferase (HAT) from Saccharomyces cerevisiae, functions with the 
histone chaperone Asf1 to acetylate lysine K56 on histone H3 (H3K56), a 
modification associated with newly synthesized histones. In vitro analysis of 
Rtt109 revealed that Vps75, a Nap1 family histone chaperone, could also 
stimulate Rtt109-dependent acetylation of H3K56. However, the molecular function 
of the Rtt109-Vps75 complex remains elusive. Here we have probed the molecular 
functions of Vps75 and the Rtt109-Vps75 complex through biochemical, structural 
and genetic means. We find that Vps75 stimulates the kcat of histone acetylation 
by approximately 100-fold relative to Rtt109 alone and enhances acetylation of 
K9 in the H3 histone tail. Consistent with the in vitro evidence, cells lacking 
Vps75 showed a substantial reduction (60%) in H3K9 acetylation during S phase. 
X-ray structural, biochemical and genetic analyses of Vps75 indicate a unique, 
structurally dynamic Nap1-like fold that suggests a potential mechanism of 
Vps75-dependent activation of Rttl09. Together, these data provide evidence for 
a multifunctional HAT-chaperone complex that acetylates histone H3 and deposits 
H3-H4 onto DNA, linking histone modification and nucleosome assembly.