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| 2. |
- Bacic, Luka, et al.
(författare)
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Asymmetric nucleosome PARylation at DNA breaks mediates directional nucleosome sliding by ALC1
- 2024
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Ingår i: Nature Communications. - : Springer Nature. - 2041-1723. ; 15:1
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Tidskriftsartikel (refereegranskat)abstract
- The chromatin remodeler ALC1 is activated by DNA damage-induced poly(ADP-ribose) deposited by PARP1/PARP2 and their co-factor HPF1. ALC1 has emerged as a cancer drug target, but how it is recruited to ADP-ribosylated nucleosomes to affect their positioning near DNA breaks is unknown. Here we find that PARP1/HPF1 preferentially initiates ADP-ribosylation on the histone H2B tail closest to the DNA break. To dissect the consequences of such asymmetry, we generate nucleosomes with a defined ADP-ribosylated H2B tail on one side only. The cryo-electron microscopy structure of ALC1 bound to such an asymmetric nucleosome indicates preferential engagement on one side. Using single-molecule FRET, we demonstrate that this asymmetric recruitment gives rise to directed sliding away from the DNA linker closest to the ADP-ribosylation site. Our data suggest a mechanism by which ALC1 slides nucleosomes away from a DNA break to render it more accessible to repair factors.
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| 3. |
- Bacic, Luka
(författare)
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Molecular mechanisms underlying the activation of ALC1 nucleosome remodeling
- 2022
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Packaging DNA into chromatin represses essential DNA-based processes, such as transcription, DNA replication, and repair. To change the accessibility of DNA, cells have evolved a set of enzymes referred to as chromatin remodelers that act on the basic repeat unit of chromatin, the nucleosome. Chromatin remodelers are critical for normal cell physiology and development. Dysfunction or aberrant regulation of chromatin remodelers can lead to multisystem developmental disorders and cancers. DNA damage represents a major threat to eukaryotic cells. When DNA damage persists, the cell can enter programmed cell death. To avoid such a dramatic outcome, cells must rapidly recognize the DNA damage and trigger DNA repair pathways. An early event following DNA damage is the relaxation of chromatin. Chromatin relaxation depends on ATP consumption and ADP-ribosylation, where the site of DNA damage is marked with ADP-ribose units. ADP-ribose, in turn, can be recognized by the macro domain of the remodeler ALC1 (Amplified in Liver Cancer 1). ALC1 has therefore been implicated in the DNA damage response. In the absence of DNA damage, the macro domain of ALC1 is placed against its ATPase motor to inhibit its activity. However, it is unclear how ALC1, in its active state, engages the nucleosome. Moreover, the mechanism by which ALC1 is fully activated upon recruitment is poorly understood, and the impact of ALC1-catalyzed nucleosome sliding in the vicinity of a DNA damage site is unknown. This thesis investigates how ALC1 engages its substrate, the nucleosome, and how histone modifications can regulate ALC1 activity. Structural and biophysical approaches revealed an ALC1 regulatory segment that binds to the acidic patch, a prominent feature on the nucleosome surface. Further analysis showed that the interaction between ALC1 and the acidic patch is required to fully activate ALC1. Moreover, in vitro ADP-ribosylation of nucleosomes enabled us to form a stable complex of nucleosome-bound ALC1 amenable to structural determination by cryogenic electron microscopy. Our structural models visualize nucleosomal epitopes that play an important role in stimulating productive remodeling by ALC1, as confirmed by various biochemical approaches. Taken together, our data suggested a possible mechanism by which ALC1 could render DNA breaks more accessible to downstream repair factors. Since recent studies defined ALC1 as an attractive anti-cancer target, this thesis provides insights into the molecular mechanisms that regulate ALC1 activity as a potential starting point for structure-based drug development.
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| 4. |
- Bacic, Luka, et al.
(författare)
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Recent advances in single-molecule fluorescence microscopy render structural biology dynamic
- 2020
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Ingår i: Current opinion in structural biology. - LONDON ENGLAND : Elsevier BV. - 0959-440X .- 1879-033X. ; 65, s. 61-68
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Tidskriftsartikel (refereegranskat)abstract
- Single-molecule fluorescence microscopy has long been appreciated as a powerful tool to study the structural dynamics that enable biological function of macromolecules. Recent years have witnessed the development of more complex single-molecule fluorescence techniques as well as powerful combinations with structural approaches to obtain mechanistic insights into the workings of various molecular machines and protein complexes. In this review, we highlight these developments that together bring us one step closer to a dynamic understanding of biological processes in atomic details.
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| 5. |
- Bacic, Luka, et al.
(författare)
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Structure and dynamics of the chromatin remodeler ALC1 bound to a PARylated nucleosome
- 2021
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Ingår i: eLIFE. - : eLife Sciences Publications Ltd. - 2050-084X. ; 10
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Tidskriftsartikel (refereegranskat)abstract
- The chromatin remodeler ALC1 is recruited to and activated by DNA damage-induced poly(ADP-ribose) (PAR) chains deposited by PARP1/PARP2/HPF1 upon detection of DNA lesions. ALC1 has emerged as a candidate drug target for cancer therapy as its loss confers synthetic lethality in homologous recombination-deficient cells. However, structure-based drug design and molecular analysis of ALC1 have been hindered by the requirement for PARylation and the highly heterogeneous nature of this post-translational modification. Here, we reconstituted an ALC1 and PARylated nucleosome complex modified in vitro using PARP2 and HPF1. This complex was amenable to cryo-EM structure determination without cross-linking, which enabled visualization of several intermediate states of ALC1 from the recognition of the PARylated nucleosome to the tight binding and activation of the remodeler. Functional biochemical assays with PARylated nucleosomes highlight the importance of nucleosomal epitopes for productive remodeling and suggest that ALC1 preferentially slides nucleosomes away from DNA breaks.
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| 7. |
- Douchkov, D., et al.
(författare)
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The barley (Hordeum vulgare) cellulose synthase-like D2 gene (HvCslD2) mediates penetration resistance to host-adapted and nonhost isolates of the powdery mildew fungus
- 2016
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Ingår i: New Phytologist. - : Blackwell Publishing. - 0028-646X .- 1469-8137. ; 212:2, s. 421-433
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Tidskriftsartikel (refereegranskat)abstract
- Cell walls and cellular turgor pressure shape and suspend the bodies of all vascular plants. In response to attack by fungal and oomycete pathogens, which usually breach their host's cell walls by mechanical force or by secreting lytic enzymes, plants often form local cell wall appositions (papillae) as an important first line of defence. The involvement of cell wall biosynthetic enzymes in the formation of these papillae is still poorly understood, especially in cereal crops. To investigate the role in plant defence of a candidate gene from barley (Hordeum vulgare) encoding cellulose synthase-like D2 (HvCslD2), we generated transgenic barley plants in which HvCslD2 was silenced through RNA interference (RNAi). The transgenic plants showed no growth defects but their papillae were more successfully penetrated by host-adapted, virulent as well as avirulent nonhost isolates of the powdery mildew fungus Blumeria graminis. Papilla penetration was associated with lower contents of cellulose in epidermal cell walls and increased digestion by fungal cell wall degrading enzymes. The results suggest that HvCslD2-mediated cell wall changes in the epidermal layer represent an important defence reaction both for nonhost and for quantitative host resistance against nonadapted wheat and host-adapted barley powdery mildew pathogens, respectively.
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| 8. |
- Ford, Kristina L., et al.
(författare)
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Comparative "Golgi" Proteome Study of Lolium multiflorum and Populus trichocarpa
- 2016
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Ingår i: PROTEOMES. - : MDPI AG. - 2227-7382. ; 4:3
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Tidskriftsartikel (refereegranskat)abstract
- The Golgi apparatus (GA) is a crucial organelle in the biosynthesis of non-cellulosic polysaccharides, glycoproteins and proteoglycans that are primarily destined for secretion to the cell surface (plasma membrane, cell wall and apoplast). Only a small proportion of the proteins involved in these processes have been identified in plants, with the majority of their functions still unknown. The availability of a GA proteome would greatly assist plant biochemists, cell and molecular biologists in determining the precise function of the cell wall-related proteins. There has been some progress towards defining the GA proteome in the model plant system Arabidopsis thaliana, yet in commercially important species, such as either the cereals or woody species there has been relatively less progress. In this study, we applied discontinuous sucrose gradient centrifugation to partially enrich GA from suspension cell cultures (SCCs) and combined this with stable isotope labelling (iTRAQ) to determine protein sub-cellular locations. Results from a representative grass species, Italian ryegrass (Lolium multiflorum) and a dicot species, black cottonwood (Populus trichocarpa) are compared. The results confirm that membrane fractionation approaches that provide effective GA-enriched fractions for proteomic analyses in Arabidopsis are much less effective in the species examined here and highlight the complexity of the GA, both within and between species.
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