Chromatin remodeling activities act on UV-damaged nucleosomes and modulate DNA damage accessibility to photolyase

Nucleosomes inhibit DNA repair in vitro, suggesting that chromatin remodeling activities might be required for efficient repair in vivo. To investigate how structural and dynamic properties of nucleosomes affect damage recognition and processing, we investigated repair of UV lesions by photolyase on a nucleosome positioned at one end of a 226-bp-long DNA fragment. Repair was slow in the nucleosome but efficient outside. No disruption or movement of the nucleosome was observed after UV irradiation and during repair. However, incubation with the nucleosome remodeling complex SWI/SNF and ATP altered the conformation of nucleosomal DNA as judged by UV photo-footprinting and promoted more homogeneous repair. Incubation with yISW2 and ATP moved the nucleosome to a more central position, thereby altering the repair pattern. This is the first demonstration that two different chromatin remodeling complexes can act on UV-damaged nucleosomes and modulate repair. Similar activities might relieve the inhibitory effect of nucleosomes on DNA repair processes in living cells.     

Chromatin remodeling: nucleosomes bulging at the seams

ATP-dependent chromatin remodeling enzymes, such as SWI/SNF, hydrolyze thousands of ATPs to regulate gene expression on chromatin fibers. Recent mechanistic studies suggest that these enzymes generate localized changes in DNA topology that drive formation of multiple, remodeled nucleosomal states.     

Chromatin remodeling and repair of DNA double-strand breaks

Eukaryotic cells have developed conserved mechanisms to efficiently sense and repair DNA damage that results from constant chromosomal lesions. DNA repair has to proceed in the context of chromatin, and both histone-modifiers and ATP-dependent chromatin remodelers have been implicated in this process. Here, we review the current understanding and new hypotheses on how different chromatin-modifying activities function in DNA repair in yeast and metazoan cells.     

Chromatin remodeling: RSC Motors along the DNA

Single-molecule experiments show that the chromatin-remodeling complex RSC, a member of the SNF2 ATPase family, induces formation of a negatively supercoiled DNA loop by active translocation.     

Chromatin remodeling in plants

In the past two years, a variety of forward genetic screens have revealed predicted plant chromatin remodeling components that are involved in either differential histone acetylation or ATP-dependent SWI2/SNF2-related complexes. Combined with the results of recent reverse genetic studies, these findings have begun to provide the groundwork for determining the function of chromatin-based control in plants.     

Chromatin reconstitution on small DNA rings. V. DNA thermal flexibility of single nucleosomes.

The thermal flexibility of DNA minicircles reconstituted with single nucleosomes was measured relative to the naked minicircles. The measurement used a new method based on the electrophoretic properties of these molecules, whose mobility strongly depended on the DNA writhe, either of the whole minicircle, when naked, or of the extranucleosomal loop, when reconstituted. The experiment was as follows. The DNA length was first increased by one base-pair (bp), and the correlative shift in mobility resulting from the altered DNA writhe was recorded. Second, the gel temperature was increased so that the former mobility was restored. Under these conditions, the untwisting of the thermally flexible DNA due to the temperature shift exactly compensates for the increase in the DNA mean twist number resulting from the one bp addition. The relative thermal flexibility was then calculated as the ratio between the increases in temperature measured for the naked and the reconstituted DNAs, respectively. The figure, 0.69 (+/- 0.07), was used to derive the length of DNA in interaction with the histones, 109 (+/- 25) bp. Such length was in good agreement with the mean value of 115 bp we have previously obtained from the distribution of the angles between DNAs at the entrance and exit of similar nucleosomes measured from high resolution electron microscopy. This consistency further reinforces our previous conclusion that minicircle-reconstituted nucleosomes, with 1.3(109/83) to 1.4(115/83) turns of superhelical DNA, show no crossing of entering and exiting DNAs when the loop is in its most probable configuration, and therefore, that these nucleosomes behave topologically as "single-turn" particles. The present data are also within the range of values, 50 to 100 bp of thermally rigid DNA per nucleosome, obtained by others for yeast plasmid chromatin, suggesting that the "single-turn" particle notion may be extended to this particular case of naturally-occurring H1-free chromatin. However, these data are quite different from the 230 bp figure derived from thermal measurements of reconstituted H1-free minichromosomes. It is proposed that nucleosome interactions occurring in this chromatin, but not in yeast chromatin, may be partly responsible for the discrepancy.     

Chromatin remodeling finds its place in the DNA double-strand break response

The accurate repair of chromosomal double-strand breaks (DSBs) arising from exposure to exogenous agents, such as ionizing radiation (IR) and radiomimetic drugs is crucial in maintaining genomic integrity, cellular viability and the prevention of tumorigenesis. Eukaryotic cells have evolved efficient mechanisms that sense and respond to DSBs. The DNA DSB response is facilitated by hierarchical signaling networks that orchestrate chromatin structural changes, cell-cycle checkpoints and multiple enzymatic activities to repair the broken DNA ends. Sensors and transducers signal to numerous downstream cellular effectors which function primarily by substrate posttranslational modifications including phosphorylation, acetylation, methylation and ubiquitylation. In particular, the past several years have provided important insight into the role of chromatin remodeling and histones-specific modifications to control DNA damage detection, signaling and repair. This review summarizes recently identified factors that influence this complex process and the repair of DNA DSBs in eukaryotic cells.     

TLK1B promotes repair of UV-damaged DNA through chromatin remodeling by Asf1

Background  

The mammalian protein kinase TLK1 is a homologue of Tousled, a gene involved in flower development in Arabidopsis thaliana. The function of TLK1 is not well known, although knockout of the gene in Drosophila, or expression of a dominant negative mutant in mouse mammary cells causes loss of nuclear divisions and chromosome mis-segregation. TLK1B is a splice variant of TLK1 and it confers radioresistance in a normal mammary mouse cell line possibly due to increased chromatin remodeling capacity, but the mechanism of resistance remains to be fully elucidated.     

Chromatin remodeling through directional DNA translocation from an internal nucleosomal site

The RSC chromatin remodeler contains Sth1, an ATP-dependent DNA translocase. On DNA substrates, RSC/Sth1 tracks along one strand of the duplex with a 3' --> 5' polarity and a tracking requirement of one base, properties that may enable directional DNA translocation on nucleosomes. The binding of RSC or Sth1 elicits a DNase I-hypersensitive site approximately two DNA turns from the nucleosomal dyad, and the binding of Sth1 requires intact DNA at this location. Results with various nucleosome substrates suggest that RSC/Sth1 remains at a fixed position on the histone octamer and that Sth1 conducts directional DNA translocation from a location about two turns from the nucleosomal dyad, drawing in DNA from one side of the nucleosome and pumping it toward the other. These studies suggest that nucleosome mobilization involves directional DNA translocation initiating from a fixed internal site on the nucleosome.     

Chromatin remodeling in mammalian zygotes.

With sperm-egg fusion at the time of fertilization the gamete nuclei are remodeled from genetically quiescent structures into pronuclei capable of DNA synthesis. Features of this process that are critical to insure the genetic integrity of the zygote and the success of subsequent embryonic development include: oocyte responses that prevent polyspermy; completion of the 2nd meiotic division by the oocyte; exchange of proteins in the sperm nucleus; and, remodelling of the oocyte chromosomes and sperm nucleus into functional pronuclei. Elucidation of the biological and molecular mechanisms underlying zygote formation and chromatin remodeling should enhance our understanding of the potential vulnerability of the zygote to toxicant-induced damage.     

Chromatin remodeling in development and differentiation

During development and differentiation, early inductive processes that influence cell fate at a later stage leave marks at distinct gene loci that are maintained through several rounds of mitosis. The structure of chromatin is part of this epigenetic memory that restricts or permits differential expression of genes in descendant cells. Establishing a cell-type-specific chromatin pattern thus predestines future cell differentiation and deters cell-lineage infidelity, as it often occurs during neoplastic transformation. As such, understanding the dynamics and mechanisms underlying chromatin remodeling has been a major focus of recent molecular genetic research that holds great promise for biomedical discoveries.     

Chromatin remodeling and genome stability

A report on the 12th Tenovus Scotland Symposium 'Stability and Regulation of Genes and Genomes', Glasgow UK, 6-7 April 2006.