Histone protein surface accessibility dictates direction of RSC-dependent nucleosome mobilization.

Chromatin remodeling enzymes use energy derived from ATP hydrolysis to mobilize nucleosomes and alter their structure to facilitate DNA access. The Remodels the Structure of Chromatin (RSC) complex has been extensively studied, yet aspects of how this complex functionally interacts with nucleosomes remain unclear. We introduce a steric mapping approach to determine how RSC activity depends on interaction with specific surfaces within the nucleosome. We find that blocking SHL + 4.5/-4.5 via streptavidin binding to the H2A N-terminal tail domains results in inhibition of RSC nucleosome mobilization. However, restriction enzyme assays indicate that remodeling-dependent exposure of an internal DNA site near the nucleosome dyad is not affected. In contrast, occlusion of both protein faces of the nucleosome by streptavidin attachment near the acidic patch completely blocks both remodeling-dependent nucleosome mobilization and internal DNA site exposure. However, we observed partial inhibition when only one protein surface is occluded, consistent with abrogation of one of two productive RSC binding orientations. Our results indicate that nucleosome mobilization requires RSC access to the trailing but not the leading protein surface, and reveals a mechanism by which RSC and related complexes may drive unidirectional movement of nucleosomes to regulate cis-acting DNA sequences in vivo.

HMGB1 Stimulates Activity of Polymerase ß on Nucleosome Substrates.

The process of base excision repair (BER) recognizes and repairs small lesions or inappropriate bases on DNA through either a short-patch or long-patch pathway. The enzymes involved in BER have been well-characterized on DNA substrates, and, somewhat surprisingly, many of these enzymes, including several DNA glycosylases, AP endonuclease (APE), FEN1 endonuclease, and DNA ligases, have been shown to have activity on DNA substrates within nucleosomes. DNA polymerase ß (Pol ß), however, exhibits drastically reduced or no activity on nucleosomal DNA. Interestingly, acetylation of Pol ß, by the acetyltransferase p300, inhibits its 5' dRP-lyase activity and presumably pushes repair of DNA substrates through the long-patch base excision repair (LP-BER) pathway. In addition to the major enzymes involved in BER, a chromatin architectural factor, HMGB1, was found to directly interact with and enhance the activity of APE1 and FEN1, and thus may aid in altering the structure of the nucleosome to be more accessible to BER factors. In this work, we investigated whether acetylation of Pol ß, either alone or in conjunction with HMGB1, facilitates its activity on nucleosome substrates. We find acetylated Pol ß exhibits enhanced strand displacement synthesis activity on DNA substrates, but, similar to the unmodified enzyme, has little or no activity on nucleosomes. Preincubation of DNA templates with HMGB1 has little or no stimulatory effect on Pol ß and even is inhibitory at higher concentrations. In contrast, preincubation of nucleosomes with HMGB1 rescues Pol ß gap-filling activity in nucleosomes, suggesting that this factor may help overcome the repressive effects of chromatin.

Base excision repair in chromatin: Insights from reconstituted systems.

The process of base excision repair has been completely reconstituted in vitro and structural and biochemical properties of the component enzymes thoroughly studied on naked DNA templates. More recent work in this field aims to understand how BER operates on the natural substrate, chromatin [1,2]. Toward this end, a number of researchers, including the Smerdon group, have focused attention to understand how individual enzymes and reconstituted BER operate on nucleosome substrates. While nucleosomes were once thought to completely restrict access of DNA-dependent factors, the surprising finding from these studies suggests that at least some BER components can utilize target DNA bound within nucleosomes as substrates for their enzymatic processes. This data correlates well with both structural studies of these enzymes and our developing understanding of nucleosome conformation and dynamics. While more needs to be learned, these studies highlight the utility of reconstituted BER and chromatin systems to inform our understanding of in vivo biological processes.

Mechanism(s) of SWI/SNF-induced nucleosome mobilization.

Impediments to DNA access due to assembly of the eukaryotic genome into chromatin are in part overcome by the activity of ATP-dependent chromatin-remodeling complexes. These complexes employ energy derived from ATP hydrolysis to destabilize histone-DNA interactions and alter nucleosome positions, thereby increasing the accessibility of DNA-binding factors to their targets. However, the mechanism by which theses complexes accomplish this task remains unresolved. We review aspects of nucleosome alteration by the SWI/SNF complex, the archetypal remodeling enzyme. We focus on experiments that provide insights into how SWI/SNF induces nucleosome movement along DNA. Numerous biochemical activities have been characterized for this complex, all likely providing clues as to the molecular mechanism of translocation.