Anastas Gospodinov

RAD51 foci formation in response to DNA damage is modulated by TIP49

Chromatin modification plays an important role in modulating the access of homologous recombination proteins to the sites of DNA damage. TIP49 is highly conserved component of chromatin modification/remodeling complexes, but its involvement in homologous recombination repair in mammalian cells has not been examined in details. In the present communication we studied the role of TIP49 in recruitment of the key homologous recombination protein RAD51 to sites of DNA damage. RAD51 redistribution to chromatin and nuclear foci formation induced by double-strand breaks and interstrand crosslinks were followed under conditions of TIP49 depletion by RNA interference. TIP49 silencing reduced RAD51 recruitment to chromatin and nuclear foci formation to about 50% of that of the control. Silencing of TIP48, which is closely related to TIP49, induced a similar reduction in RAD51 foci formation. RAD51 foci reduction in TIP49-silenced cells was not a result of defective DNA damage checkpoint signaling as judged by the normal histone H2AX phosphorylation and cell cycle distribution. Treatment with the histone deacetylase inhibitor sodium butyrate restored RAD51 foci formation in the TIP49-depleted cells. The results suggest that as a constituent of chromatin modification complexes TIP49 may facilitate the access of the repair machinery to the sites of DNA damage.

Chromatin structure in double strand break repair.

Cells are under constant assault by endogenous and environmental DNA damaging agents. DNA double strand breaks (DSBs) sever entire chromosomes and pose a major threat to genome integrity as a result of chromosomal fragment loss or chromosomal rearrangements. Exogenous factors such as ionizing radiation, crosslinking agents, and topoisomerase poisons, contribute to break formation. DSBs are associated with oxidative metabolism, form during the normal S phase, when replication forks collapse and are generated during physiological processes such as V(D)J recombination, yeast mating type switching and meiosis. It is estimated that in mammalian cells ∼10 DSBs per cell are formed daily. If left unrepaired DSBs can lead to cell death or deregulated growth, and cancer development. Cellular response to DSB damage includes mechanisms to halt the progression of the cell cycle and to restore the structure of the broken chromosome. Changes in chromatin adjacent to DNA break sites are instrumental to the DNA damage response (DDR) with two apparent ends: to control compaction and to bind repair and signaling molecules to the lesion. Here, we review the key findings related to each of these functions and examine their cross-talk.

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.

The mammalian INO80 chromatin remodeling complex is required for replication stress recovery

A number of studies have implicated the yeast INO80 chromatin remodeling complex in DNA replication, but the function of the human INO80 complex during S phase remains poorly understood. Here, we have systematically investigated the involvement of the catalytic subunit of the human INO80 complex during unchallenged replication and under replication stress by following the effects of its depletion on cell survival, S-phase checkpoint activation, the fate of individual replication forks, and the consequences of fork collapse. We report that INO80 was specifically needed for efficient replication elongation, while it was not required for initiation of replication. In the absence of the Ino80 protein, cells became hypersensitive to hydroxyurea and displayed hyperactive ATR-Chk1 signaling. Using bulk and fiber labeling of DNA, we found that cells deficient for Ino80 and Arp8 had impaired replication restart after treatment with replication inhibitors and accumulated double-strand breaks as evidenced by the formation of γ-H2AX and Rad51 foci. These data indicate that under conditions of replication stress mammalian INO80 protects stalled forks from collapsing and allows their subsequent restart.

Repair of DNA interstrand crosslinks may take place at the nuclear matrix

Host cell reactivation assay using Trioxsalen‐crosslinked plasmid pEGFP‐N1 showed that human cells were able to repair Trioxsalen interstrand crosslinks (ICL). To study the mechanism of this repair pathway, cells were transfected with the plasmids pEGFP‐1, which did not contain the promoter of the egfp gene, and with pEGFP‐G−, which did not contain the egfp gene. Neither of these plasmids alone was able to express the green fluorescent protein. After cotransfection with the two plasmids, 1%–2% of the cells developed fluorescent signal, which showed that recombination events had taken place in these cells to create DNA constructs containing the promoter and the gene properly aligned. When one or both of the plasmids were crosslinked with Trioxsalen, the recombination rate increased several fold. To identify the nuclear compartment where recombination takes place, cells were transfected with crosslinked pEGFP‐N1 and the amount of plasmid DNA in the different nuclear fractions was determined. The results showed that Trioxsalen crosslinking increased the percentage of matrix attached plasmid DNA in a dose‐dependant way. Immunoblotting experiments showed that after transfection with Trioxsalen crosslinked plasmids the homologous recombination protein Rad51 also associated with the nuclear matrix fraction. These studies provide a model system for investigating the precise molecular mechanisms that appear to couple repair of DNA ICL with nuclear matrix attachment.

The Inhibitor of Histone Deacetylases Sodium Butyrate Enhances the Cytotoxicity of Mitomycin C

The use of histone deacetylase inhibitors has been proposed as a promising approach to increase the cell killing effect of DNA damage–inducing drugs in chemotherapy. However, the molecular mechanism of their action remains understudied. In the present article, we have assessed the effect of the histone deacetylase inhibitor sodium butyrate on the DNA damage response induced by the crosslinking agent mitomycin C. Sodium butyrate increased mitomycin C cytotoxicity, but did not impair the repair pathways required to remove mitomycin C-induced lesions as neither the rate of nucleotide excision repair nor the homologous recombination repair rate were diminished. Sodium butyrate treatment abrogated the S-phase cell-cycle checkpoint in mitomycin C-treated cells and induced the G2–M checkpoint. However, sodium butyrate treatment alone resulted in accumulation of reactive oxygen species, double-strand breaks in DNA, and apoptosis. These results imply that the accumulation of reactive oxygen species–mediated increase in DNA lesion burden may be the major mechanism by which sodium butyrate enhances the cytotoxicity of mitomycin C. Mol Cancer Ther; 11(10); 2116–26. ©2012 AACR.

The repair capacity of lung cancer cell lines A549 and H1299 depends on HMGB1 expression level and the p53 status

Elucidation of the cellular components responsive to chemotherapeutic agents as cisplatin rationalizes the strategy for anticancer chemotherapy. The removal of the cisplatin/DNA lesions gives the chance to the cancer cells to survive and compromises the chemotherapeutical treatment. Therefore, the cell repair efficiency is substantial for the clinical outcome. High mobility group box 1 (HMGB1) protein is considered to be involved in the removal of the lesions as it binds with high affinity to cisplatin/DNA adducts. We demonstrated that overexpression of HMGB1 protein inhibited cis-platinated DNA repair in vivo and the effect strongly depended on its C-terminus. We registered increased levels of DNA repair after HMGB1 silencing only in p53 defective H1299 lung cancer cells. Next, introduction of functional p53 resulted in DNA repair inhibition. H1299 cells overexpressing HMGB1 were significantly sensitized to treatment with cisplatin demonstrating the close relation between the role of HMGB1 in repair of cis-platinated DNA and the efficiency of the anticancer drug, the process being modulated by the C-terminus. In A549 cells with functional p53, the repair of cisplatin/DNA adducts is determined by а complex action of HMGB1 and p53 as an increase of DNA repair capacity was registered only after silencing of both proteins.

Cells synchronized in S phase show increased rate of repair of UV damaged plasmids.

The capacity for nucleotide excision repair of cells synchronized in S phase and unsynchronized cells was compared by the host cell reactivation assay and the cell‐free repair system. HeLa cells were transfected with in vitro damaged by UV irradiation pEGFP and the repair capacity was determined by the number of fluorescent cells. In the cell‐free repair system, the repair capacity of protein extracts isolated from K562 cells was determined by measuring the transformation efficiency of UV irradiated pBlueScript incubated in the extracts. In both cases, the repair capacity of the cells synchronized in S phase cells was 30–50% higher than the repair capacity of unsynchronized cells.

Possible Emergence of Sequence Specific RNA Aminoacylation via Peptide Intermediary to Initiate Darwinian Evolution and Code Through Origin of Life

One of the most intriguing questions in biological science is how life originated on Earth. A large number of hypotheses have been proposed to explain it, each putting an emphasis on different events leading to functional translation and self-sustained system. Here, we propose a set of interactions that could have taken place in the prebiotic environment. According to our hypothesis, hybridization-induced proximity of short aminoacylated RNAs led to the synthesis of peptides of random sequence. We postulate that among these emerged a type of peptide(s) capable of stimulating the interaction between specific RNAs and specific amino acids, which we call “bridge peptide” (BP). We conclude that translation should have emerged at the same time when the standard genetic code begun to evolve due to the stabilizing effect on RNA-peptide complexes with the help of BPs. Ribosomes, ribozymes, and the enzyme-directed RNA replication could co-evolve within the same period, as logical outcome of RNA-peptide world without the need of RNA only self-sustained step.

Structural Aspects of Double Strand Break Repair

ABSTRACT Double strand breaks are extremely deleterious to the stability of the genome as they lead to loss of whole chromosome fragments or chromosomal rearrangements with the consequent cell death or tumor development. The principal players in the cellular response to DNA damage have been rigorously studied. At the same time, knowledge about the organization of the repair process in the context of local chromatin and the nucleus as a whole has started to emerge only recently. Here, we summerize the most pertinent results regarding the spatial organization of the double strand break repair. On the basis of the data obtained by others and us, we propose a possible chain of events that may take place in chromatin in the vicinity of the breaks to balance the needs for spatial stability and accessibility of the damaged DNA.