Eukaryotic chromatin is a hierarchical collection of nucleoprotein structures that package DNA to form chromosomes. The initial levels of packaging include folding of long strings of nucleosomes into secondary structures and array-array association into higher-order tertiary chromatin structures. The core histone tail domains are required for the assembly of higher-order structures and mediate short- and long-range intra- and inter-nucleosome interactions with both DNA and protein targets to direct their assembly. However, important details of these interactions remain unclear and are a subject of much interest and recent investigations. Here, we review work defining the interactions of the histone N-terminal tails with DNA and protein targets relevant to chromatin higher-order structures, with a specific emphasis on the contributions of H3 and H4 tails to oligonucleosome folding and stabilization. We evaluate both classic and recent experiments determining tail structures, affect of tail cleavage/loss, and posttranslational modifications of the tails on nucleosomes and nucleosome arrays, as well as internucleosomal and inter-array interactions of the H3 and H4 N-terminal tails.

The activation of a silent gene locus is thought to involve pioneering transcription factors that initiate changes in the local chromatin structure to increase promoter accessibility and binding of downstream effectors. To better understand the molecular requirements for the first steps of locus activation, we investigated whether acetylation of a single nucleosome is sufficient to alter DNA accessibility within a condensed 25-nucleosome array. We found that acetylation mimics within the histone H4 tail domain increased accessibility of the surrounding linker DNA, with the increased accessibility localized to the immediate vicinity of the modified nucleosome. In contrast, acetylation mimics within the H3 tail had little effect, but were able to synergize with H4 tail acetylation mimics to further increase accessibility. Moreover, replacement of the central nucleosome with a nucleosome free region also resulted in increased local, but not global DNA accessibility. Our results indicate that modification or disruption of only a single target nucleosome results in significant changes in local chromatin architecture and suggest that very localized chromatin modifications imparted by pioneer transcription factors are sufficient to initiate a cascade of events leading to promoter activation.

Background:The core histone tail domains are essential for formation of native chromatin structures, but interactions are poorly understood. Results: H4 tail-DNA intra-nucleosome contacts are lost upon nucleosome array condensation, but intra-array, inter-nucleosome DNA contacts are not detected. Conclusion:The multiple functions of the H4 tail require targeted distinct interactions within condensed chromatin. Significance: These results illuminate how the H4 tail stabilizes higher-order chromatin structures.

We demonstrated previously that human FEN1 endonuclease, an enzyme involved in excising single-stranded DNA flaps that arise during Okazaki fragment processing and base excision repair, cleaves model flap substrates assembled into nucleosomes. Here we explore the effect of flap orientation with respect to the surface of the histone octamer on nucleosome structure and FEN1 activity in vitro. We find that orienting the flap substrate toward the histone octamer does not significantly alter the rotational orientation of two different nucleosome positioning sequences on the surface of the histone octamer but does cause minor perturbation of nucleosome structure. Surprisingly, flaps oriented toward the nucleosome surface are accessible to FEN1 cleavage in nucleosomes containing the Xenopus 5S positioning sequence. In contrast, neither flaps oriented toward nor away from the nucleosome surface are cleaved by the enzyme in nucleosomes containing the high-affinity 601 nucleosome positioning sequence. The data are consistent with a model in which sequence-dependent motility of DNA on the nucleosome is a major determinant of FEN1 activity. The implications of these findings for the activity of FEN1 in vivo are discussed. Flap endonuclease 1 (FEN1)2 plays critical roles in both DNA replication and repair. During processing of Okazaki fragments, FEN1 specifically recognizes a 5Ј-unannealed flap formed by the displacement synthesis activity of DNA polymerase ␦. FEN1 binds and cleaves the phosphodiester backbone at the base of the flap, leaving a ligatable nick (1, 2). The significant role of FEN1 in DNA synthesis is highlighted by genetic studies demonstrating that RNase H is not essential for eliminating the RNA initiator (3) and that homozygous deletion of FEN1 results in a complete inactivation of DNA synthesis in mouse blastocysts (4).FEN1 is also involved in the penultimate step of a long patch base excision repair pathway where the enzyme removes a flap containing the damaged base that was generated by DNA polymerase ␤ displacement synthesis. In vivo studies have shown that deletion of both copies of the FEN1 gene results in hypersensitivity of cells to methylating agents and hydrogen peroxide (5), and a FEN1 null mutation has been found to render Saccharomyces cerevisiae and Schizosaccharomyces pombe sensitive to UV radiation (2). Further, mice with FEN1 haploinsufficiency show a mild predisposition phenotype which, in combination with a mutation in the adenomatous polyposis coli (Apc) gene, leads to rapid tumor progression and genomic instability. In addition, deletion of both copies of the FEN1 gene results in early embryonic lethality (6). Finally, blastocysts with both copies of the FEN1 gene deleted and exposed to ␥ radiation have been observed to undergo extensive apoptosis (4), likely because of the essential role of FEN1 in the repair of radiation-induced DNA damage in vivo.A number of in vitro studies have addressed the kinetics and mechanism of FEN1 activity on DNA substrates. FEN1 specifically recognizes sin...

DNA polymerase delta is the primary polymerase that is involved in undamaged nuclear lagging strand DNA replication. Our mass-spectroscopic analysis has revealed that the human DNA polymerase δ is acetylated on subunits p125, p68, and p12. Using substrates that simulate Okazaki fragment intermediates, we studied alterations in the catalytic properties of acetylated polymerase and compared it to the unmodified form. The current data show that the acetylated form of human pol δ displays a higher polymerization activity compared to the unmodified form of the enzyme. Additionally, acetylation enhances the ability of the polymerase to resolve complex structures such as G-quadruplexes and other secondary structures that might be present on the template strand. More importantly, the ability of pol δ to displace a downstream DNA fragment is enhanced upon acetylation. Our current results suggest that acetylation has a profound effect on the activity of pol δ and supports the hypothesis that acetylation may promote higher-fidelity DNA replication.