Investigating The Determinants Of Nucleosome Assembly And Positioning During Dna Replication In Saccharomyces Cerevisiae
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Eukaryotic nuclei are packaged into a higher-order, tightly regulated structure called chromatin. The repeating unit of chromatin, the nucleosome, is composed of 145-147bp of DNA wrapped twice around a histone octamer. Chromatin is highly dynamic and undergoes stepwise assembly and disassembly during transcription, replication, recombination and repair. During S phase, de novo chromatin assembly occurs rapidly on nascent DNA strands, behind the progressing replication fork. The process by which the de novo chromatin landscape arises behind the replication fork remains unclear. Numerous molecular factors have been implicated as determinants of nucleosome positioning and chromatin assembly. These include the intrinsic biophysical affinity of histone proteins towards different DNA sequences, histone chaperones (such as Asf1, CAF-1, Rtt106, Vps75), chromatin modifying enzymes (like the histone acetyltransferase, Rtt109), chromatin remodeling enzymes (Isw1, Chd1, Isw2) and General Regulatory Factors (Abf1, Reb1, Rap1). However, the contribution of various molecular players remains ambiguous. The goal of my doctoral work has been to dissect the intricate network of chromatin assembly factors in vivo, using Okazaki fragment synthesis as a tool. Work presented in this thesis confirms and extends upon the properties of various molecular players in nucleosome assembly and positioning during S-phase. In particular, I elucidate the contribution of ATP-dependent chromatin remodeling enzymes and histone chaperones in nucleosome assembly and positioning on nascent DNA. In addition, an important role for nucleosome abundance and histone structure is also described. This thesis provides new evidence for a concerted pathway involving the ‘loading and remodeling’ of nucleosomes on newly synthesized DNA during S-phase in vivo. These findings also shed light on the timing of chromatin maturation in budding yeast and the significance of various cis- & trans-determinants of nucleosome positioning in eukaryotes. Taken together, this thesis uncovers mechanistic insights into establishment and inheritance of chromatin structure that have far-reaching consequences for epigenetic regulation and genome maintenance.