Shaping chromatin for repair

To counteract the adverse effects of various DNA lesions, cells have evolved an array of diverse repair pathways to restore DNA structure and to coordinate repair with cell cycle regulation. Chromatin changes are an integral part of the DNA damage response, particularly with regard to the types of repair that involve assembly of large multiprotein complexes such as those involved in double strand break (DSB) repair and nucleotide excision repair (NER). A number of phosphorylation, acetylation, methylation, ubiquitylation and chromatin remodeling events modulate chromatin structure at the lesion site. These changes demarcate chromatin neighboring the lesion, afford accessibility and binding surfaces to repair factors and provide on-the-spot means to coordinate repair and damage signaling. Thus, the hierarchical assembly of repair factors at a double strand break is mostly due to their regulated interactions with posttranslational modifications of histones. A large number of chromatin remodelers are required at different stages of DSB repair and NER. Remodelers physically interact with proteins involved in repair processes, suggesting that chromatin remodeling is a requisite for repair factors to access the damaged site. Together, recent findings define the roles of histone post-translational modifications and chromatin remodeling in the DNA damage response and underscore possible differences in the requirements for these events in relation to the chromatin context.     

Chromatin remodeling and the maintenance of genome integrity

DNA damage of any type is threatening for a cell. If lesions are left unrepaired, genomic instability can arise, faithful transmission of genetic information is greatly compromised eventually leading the cell to undergo apoptosis or carcinogenesis. In order to access/detect and repair these damages, repair factors must circumvent the natural repressive barrier of chromatin. This review will present recent progress showing the intricate link between chromatin, its remodeling and the DNA repair process. Several studies demonstrated that one of the first events following specific types of DNA damage is the phosphorylation of histone H2A. This mark or the damage itself are responsible for the association of chromatin-modifying complexes near damaged DNA. These complexes are able to change the chromatin structure around the wounded DNA in order to allow the repair machinery to gain access and repair the lesion. Chromatin modifiers include ATP-dependent remodelers such as SWI/SNF and Rad54 as well as histone acetyltransferases (HATs) like SAGA/NuA4-related complexes and p300/CBP, which have been shown to facilitate DNA accessibility and repair in different pathways leading to the maintenance of genome integrity.     

Recruitment of the type B histone acetyltransferase Hat1p to chromatin is linked to DNA double-strand breaks

Type B histone acetyltransferases are thought to catalyze the acetylation of the NH₂-terminal tails of newly synthesized histones. Although Hat1p has been implicated in cellular processes, such as telomeric silencing and DNA damage repair, the underlying molecular mechanisms by which it functions remain elusive. In an effort to understand how Hat1p is involved in the process of DNA double-strand break (DSB) repair, we examined whether Hat1p is directly recruited to sites of DNA damage. Following induction of the endonuclease HO, which generates a single DNA DSB at the MAT locus, we found that Hat1p becomes associated with chromatin near the site of DNA damage. The nuclear Hat1p-associated histone chaperone Hif1p is also recruited to an HO-induced DSB with a similar distribution. In addition, while the acetylation of all four histone H4 NH₂-terminal tail domain lysine residues is increased following DSB formation, only the acetylation of H4 lysine 12, the primary target of Hat1p activity, is dependent on the presence of Hat1p. Kinetic analysis of Hat1p localization indicates that it is recruited after the phosphorylation of histone H2A S129 and concomitant with the recombinational-repair factor Rad52p. Surprisingly, Hat1p is still recruited to chromatin in strains that cannot repair an HO-induced double-strand break. These results indicate that Hat1p plays a direct role in DNA damage repair and is responsible for specific changes in histone modification that occur during the course of recombinational DNA repair.     

Beyond Repair Foci: Subnuclear Domains and the Cellular Response to DNA Damage

In the mid to late 1990's several groups identified DNA damage-dependent focal accumulations in nuclei of both DNA repair factors and the phosphorylated form of the histone variant H2A.X. The term "repair foci" has since been used to describe these protein accumulations. As a molecular marker for DNA damage, they have been immensely useful in the study of signal transduction pathways triggered by DNA damage while aiding in the identification of new factors involved in DNA repair. In spite of their importance, many other changes in the nuclear landscape correlate with DNA damage and repair processes. These include dramatic changes in chromatin ultrastructure and epigenetic modifications, which occur at the site of DNA breaks as well as globally throughout the nucleus. Besides chromatin, DNA damage also affects the dynamic behaviour, morphology and biochemical composition of various subnuclear domains, including the nucleolus, promyelocytic leukemia (PML) nuclear bodies and Cajal bodies. These changes in the nuclear landscape, the topic of this review, appear to be intimately linked to the cellular response to DNA damage and may prove as useful as repair foci in elucidating mechanisms of DNA repair.     

High-resolution characterization of CPD hotspot formation in human fibroblasts

Repair of DNA lesions must occur within the chromatin landscape and is associated with alterations in histone modifications and nucleosome rearrangement. To directly associate these chromatin features with DNA damage and repair, it is necessary to be able to map DNA adducts. We have developed a cyclobutane pyrimidine dimer (CPD)-specific immunoprecipitation method and mapped ultraviolet damage hotspots across human chromosomes 1 and 6. CPD hotspots occur almost equally in genic and intergenic regions. However, these hotspots are significantly more prevalent adjacent to repeat elements, especially Alu repeats. Nucleosome mapping studies indicate that nucleosomes are consistently positioned at Alu elements where CPD hotspots form, but by 2 h post-irradiation, these same regions are significantly depleted of nucleosomes. These results indicate that nucleosomes associated with hotspots of CPD formation are readily rearranged, potentially making them accessible to DNA repair machinery. Our results represent the first chromosome scale map of ultraviolet-induced DNA lesions in the human genome, and reveal the sequence features and dynamic chromatin changes associated with CPD hotspots.     

Changes in chromatin structure and mobility in living cells at sites of DNA double-strand breaks

The repair of DNA double-strand breaks (DSBs) is facilitated by the phosphorylation of H2AX, which organizes DNA damage signaling and chromatin remodeling complexes in the vicinity of the lesion. The disruption of DNA integrity induces an alteration of chromatin architecture that has been proposed to activate the DNA damage transducing kinase ataxia telangiectasia mutated. However, little is known about the physical properties of damaged chromatin. In this study, we use a photoactivatable version of GFP-tagged histone H2B to examine the mobility and structure of chromatin containing DSBs in living cells. We find that chromatin containing DSBs exhibits limited mobility but undergoes an energy-dependent local expansion immediately after DNA damage. The localized expansion observed in real time corresponds to a 30-40% reduction in the density of chromatin fibers in the vicinity of DSBs, as measured by energy-filtering transmission electron microscopy. The observed opening of chromatin occurs independently of H2AX and ATM. We propose that localized adenosine triphosphate-dependent decondensation of chromatin at DSBs establishes an accessible subnuclear environment that facilitates DNA damage signaling and repair.     

HP1 proteins are essential for a dynamic nuclear response that rescues the function of perturbed heterochromatin in primary human cells

Cellular information is encoded genetically in the DNA nucleotide sequence and epigenetically by the "histone code," DNA methylation, and higher-order packaging of DNA into chromatin. Cells possess intricate mechanisms to sense and repair damage to DNA and the genetic code. However, nothing is known of the mechanisms, if any, that repair and/or compensate for damage to epigenetically encoded information, predicted to result from perturbation of DNA and histone modifications or other changes in chromatin structure. Here we show that primary human cells respond to a variety of small molecules that perturb DNA and histone modifications by recruiting HP1 proteins to sites of altered pericentromeric heterochromatin. This response is essential to maintain the HP1-binding kinetochore protein hMis12 at kinetochores and to suppress catastrophic mitotic defects. Recruitment of HP1 proteins to pericentromeres depends on histone H3.3 variant deposition, mediated by the HIRA histone chaperone. These data indicate that defects in pericentromeric epigenetic heterochromatin modifications initiate a dynamic HP1-dependent response that rescues pericentromeric heterochromatin function and is essential for viable progression through mitosis.     

Epigenetic regulation of genomic integrity

Inefficient and inaccurate repair of DNA damage is the principal cause of DNA mutations, chromosomal aberrations, and carcinogenesis. Numerous multiple-step DNA repair pathways exist whose deployment depends on the nature of the DNA lesion. Common to all eukaryotic DNA repair pathways is the need to unravel the compacted chromatin structure to facilitate access of the repair machinery to the DNA and restoration of the original chromatin state afterward. Accordingly, our cells utilize a plethora of coordinated mechanisms to locally open up the chromatin structure to reveal the underlying DNA sequence and to orchestrate the efficient and accurate repair of DNA lesions. Here we review changes to the chromatin structure that are intrinsic to the DNA damage response and the available mechanistic insight into how these chromatin changes facilitate distinct stages of the DNA damage repair pathways to maintain genomic stability.     

Chromatin Dynamics during DNA Repair Revealed by Pair Correlation Analysis of Molecular Flow in the Nucleus

Chromatin dynamics modulate DNA repair factor accessibility throughout the DNA damage response. The spatiotemporal scale upon which these dynamics occur render them invisible to live cell imaging. Here we present a believed novel assay to monitor the in vivo structural rearrangements of chromatin during DNA repair. By pair correlation analysis of EGFP molecular flow into chromatin before and after damage, this assay measures millisecond variations in chromatin compaction with submicron resolution. Combined with laser microirradiation we employ this assay to monitor the real-time accessibility of DNA at the damage site. We find from comparison of EGFP molecular flow with a molecule that has an affinity toward double-strand breaks (Ku-EGFP) that DNA damage induces a transient decrease in chromatin compaction at the damage site and an increase in compaction to adjacent regions, which together facilitate DNA repair factor recruitment to the lesion with high spatiotemporal control.     

DNA修复的表观遗传学调控

表观遗传学信息的改变是导致人类肿瘤形成的重要因素之一．基因组的稳定性经常会受到DNA损伤的威胁．然而,高度致密的染色质结构却极大地妨碍了DNA修复的进行．因此,真核生物细胞中必须有一个精确的机制来克服染色质这一天然的屏障．其中,组蛋白的共价修饰和ATP-依赖的染色质重塑通过改变染色质的结构,对DNA修复进程起着关键的调控作用．介绍了DNA修复过程中,发生在表观遗传学方面的主要调控过程,特别阐述了在DNA双链断裂损伤应答和修复过程中,组蛋白修饰和染色质重塑方面最新的研究进展,并对今后的发展方向进行了讨论．