Repair of UV lesions in nucleosomes--intrinsic properties and remodeling

Nucleotide excision repair and reversal of pyrimidine dimers by photolyase (photoreactivation) are two major pathways to remove UV-lesions from DNA. Here, it is discussed how lesions are recognized and removed when the DNA is condensed into nucleosomes. During the recent years it was shown that nucleosomes inhibit photolyase and excision repair in vitro and slow down repair in vivo. The correlation of DNA-repair rates with nucleosome positions in yeast suggests that intrinsic properties of nucleosomes such as mobility and transient unwrapping of nucleosomal DNA facilitate damage recognition. Moreover, it was shown that nucleosome remodeling activities can act on UV-damaged DNA in vitro and facilitate repair suggesting that random remodeling of chromatin might contribute to damage recognition in vivo. Recent work on nucleosome structure and mobility is included to evaluate how nucleosomes accommodate DNA lesions and how nucleosome mobility and remodeling can take place on damaged DNA.     

Quantifying the role of steric constraints in nucleosome positioning

Statistical positioning, the localization of nucleosomes packed against a fixed barrier, is conjectured to explain the array of well-positioned nucleosomes at the 5′ end of genes, but the extent and precise implications of statistical positioning in vivo are unclear. We examine this hypothesis quantitatively and generalize the idea to include moving barriers as well as nucleosomes actively packed against a barrier. Early experiments noted a similarity between the nucleosome profile aligned and averaged across genes and that predicted by statistical positioning; however, we demonstrate that aligning random nucleosomes also generates the same profile, calling the previous interpretation into question. New rigorous results reformulate statistical positioning as predictions on the variance structure of nucleosome locations in individual genes. In particular, a quantity termed the variance gradient, describing the change in variance between adjacent nucleosomes, is tested against recent high-throughput nucleosome sequencing data. Constant variance gradients provide support for generalized statistical positioning in ∼50% of long genes. Genes that deviate from predictions have high nucleosome turnover and cell-to-cell gene expression variability. The observed variance gradient suggests an effective nucleosome size of 158 bp, instead of the commonly perceived 147 bp. Our analyses thus clarify the role of statistical positioning in vivo.     

Nucleosome gaping supports a functional structure for the 30nm chromatin fiber

The biological functions played by the nucleus of eukaryotic cells and especially those involved in cellular differentiation not only depend on the genomic sequence but also on all the proteins which form the nucleo-protein complex named chromatin. The tridimensional organization of this huge polymer involves many structural levels, the most basic one being the nucleosome. Nucleosomes further organize into the so-called 30nm fiber, which, according to recent works, is likely to be the main functional level of chromatin. We wish here to propose a plausible structure for the 30nm chromatin fiber that could explain its functional role. In our model, silenced chromatin is locked by nucleosome stacking interactions. This is achieved by a conformational transition within the nucleosome core particle (NCP) which allows nucleosomes to stack along two helices without bending the DNA linkers. We used molecular modeling to check that this conformational transition was plausible. Then we proposed to modify the well-known two-angle model according to these atomic level results. The emerging picture is an allosteric behavior of the nucleosomes induced by their collective organization within the 30nm chromatin fiber.     

Functional role of extranucleosomal DNA and the entry site of the nucleosome in chromatin remodeling by ISW2

A minimal amount of extranucleosomal DNA was required for nucleosome mobilization by ISW2 as shown by using a photochemical histone mapping approach to analyze nucleosome movement on a set of nucleosomes with varied lengths of extranucleosomal DNA. ISW2 was ineffective in repositioning or mobilizing nucleosomes with 相似文献   

Activity of FEN1 endonuclease on nucleosome substrates is dependent upon DNA sequence but not flap orientation

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.     

The dynamics of the nucleosome: thermal effects, external forces and ATP

With nucleosomes being tightly associated with the majority of eukaryotic DNA, it is essential that mechanisms are in place that can move nucleosomes 'out of the way'. A focus of current research comprises chromatin remodeling complexes, which are ATP-consuming protein complexes that, for example, pull or push nucleosomes along DNA. The precise mechanisms used by those complexes are not yet understood. Hints for possible mechanisms might be found among the various spontaneous fluctuations that nucleosomes show in the absence of remodelers. Thermal fluctuations induce the partial unwrapping of DNA from the nucleosomes and introduce twist or loop defects in the wrapped DNA, leading to nucleosome sliding along DNA. In this minireview, we discuss nucleosome dynamics from two angles. First, we describe the dynamical modes of nucleosomes in the absence of remodelers that are experimentally fairly well characterized and theoretically understood. Then, we discuss remodelers and describe recent insights about the possible schemes that they might use.     

Sin mutations alter inherent nucleosome mobility

Previous studies have identified sin mutations that alleviate the requirement for the yeast SWI/SNF chromatin remodelling complex, which include point changes in the yeast genes encoding core histones. Here we characterise the biochemical properties of nucleosomes bearing these mutations. We find that sin mutant nucleosomes have a high inherent thermal mobility. As the SWI/SNF complex can alter nucleosome positioning, the higher mobility of sin mutant nucleosomes provides a means by which sin mutations may substitute for SWI/SNF function. The location of sin mutations also provides a new opportunity for insights into the mechanism for nucleosome mobilisation. We find that both mutations altering histone DNA contacts at the nucleosome dyad and mutations in the dimer-tetramer interface influence nucleosome mobility. Furthermore, incorporation of H2A.Z into nucleosomes, which also alters dimer-tetramer interactions, affects nucleosome mobility. Thus, variation of histone sequence or subtype provides a means by which eukaryotes may regulate access to chromatin through alterations to nucleosome mobility.