A helicase-tethered ORC flip enables bidirectional helicase loading

Replication origins are licensed by loading two Mcm2‑7 helicases around DNA in a head-to-head conformation poised to initiate bidirectional replication. This process requires ORC, Cdc6, and Cdt1. Although different Cdc6 and Cdt1 molecules load each helicase, whether two ORC proteins are required is unclear. Using colocalization single-molecule spectroscopy combined with FRET, we investigated interactions between ORC and Mcm2‑7 during helicase loading. In the large majority of events, we observed a single ORC molecule recruiting both Mcm2‑7/Cdt1 complexes via similar interactions that end upon Cdt1 release. Between first and second helicase recruitment, a rapid change in interactions between ORC and the first Mcm2-7 occurs. Within seconds, ORC breaks the interactions mediating first Mcm2-7 recruitment, releases from its initial DNA-binding site, and forms a new interaction with the opposite face of the first Mcm2-7. This rearrangement requires release of the first Cdt1 and tethers ORC as it flips over the first Mcm2-7 to form an inverted Mcm2‑7-ORC-DNA complex required for second-helicase recruitment. To ensure correct licensing, this complex is maintained until head-to-head interactions between the two helicases are formed. Our findings reconcile previous observations and reveal a highly-coordinated series of events through which a single ORC molecule can load two oppositely-oriented helicases.

A Helicase-tethered ORC Flip Enables Bidirectional Helicase Loading

Replication origins are licensed by loading two Mcm2-7 helicases around DNA in a head-to-head conformation poised to initiate bidirectional replication. This process requires ORC, Cdc6, and Cdt1. Although different Cdc6 and Cdt1 molecules load each helicase, whether two ORC proteins are required is unclear. Using colocalization single-molecule spectroscopy combined with FRET, we investigated interactions between ORC and Mcm2-7 during helicase loading. We demonstrate that a single ORC molecule can recruit both Mcm2-7/Cdt1 complexes via similar interactions that end upon Cdt1 release. Between the first and second helicase recruitment, we observe a rapid change in interactions between ORC and the first Mcm2-7. In quick succession ORC breaks the interactions mediating first Mcm2-7 recruitment, releases from its initial DNA-binding site, and forms a new interaction with the opposite face of the first Mcm2-7. This rearrangement requires release of the first Cdt1 and tethers ORC as it flips over the first Mcm2-7 to form an inverted Mcm2-7-ORC-DNA complex required for second-helicase recruitment. To ensure correct licensing, this complex is maintained until head-to-head interactions between the two helicases are formed. Our findings reconcile previous observations and reveal a highly-coordinated series of events through which a single ORC molecule can load two oppositely-oriented helicases.

Rad24-RFC loads the 9-1-1 clamp by inserting DNA from the top of a wide-open ring, opposite the mechanism of RFC/PCNA

ABSTRACTIn response to DNA damage, the ring-shaped 9-1-1 clamp is loaded onto 5’ recessed DNA to arrest the cell cycle and activate the DNA damage checkpoint. The 9-1-1 clamp is a heterotrimeric ring that is loaded in S. cerevisiae by Rad24-RFC, an alternative clamp loader in which Rad24 replaces the Rfc1 subunit in the RFC1-5 clamp loader of PCNA. Unlike RFC that loads the PCNA ring onto a 3’-ss/ds DNA junction, Rad24-RFC loads the 9-1-1 ring onto a 5’-ss/ds DNA junction, a consequence of DNA damage. The underlying 9-1-1 clamp loading mechanism has been a mystery. Here we report two 3.2-Å cryo-EM structures of Rad24-RFC bound to DNA and either a closed or 27 Å open 9-1-1 clamp. The structures reveal a completely unexpected mechanism by which a clamp can be loaded onto DNA. The Rad24 subunit specifically recognizes the 5’-DNA junction and holds ds DNA outside the clamp loader and above the plane of the 9-1-1 ring, rather than holding DNA inside and below the clamp as in RFC. The 3’ ssDNA overhang is required to obtain the structure, and thus confers a second DNA binding site. The bipartite DNA binding by Rad24-RFC suggests that ssDNA may be flipped into the open 9-1-1 ring, similar to ORC-Cdc6 that loads the Mcm2-7 ring on DNA. We propose that entry of ssDNA through the 9-1-1 ring triggers the ATP hydrolysis and release of the Rad24-RFC. The key DNA binding residues are conserved in higher eukaryotes, and thus the 9-1-1 clamp loading mechanism likely generalizes.

Three ParA Dimers Cooperatively Assemble on Type Ia Partition Promoters

Accurate DNA segregation is essential for faithful inheritance of genetic material. In bacteria, this process is mainly ensured by partition systems composed of two proteins, ParA and ParB, and a centromere site. Auto-regulation of Par operon expression is important for efficient partitioning and is primarily mediated by ParA for type Ia plasmid partition systems. For the F-plasmid, four ParAF monomers were proposed to bind to four repeated sequences in the promoter region. By contrast, using quantitative surface-plasmon-resonance, we showed that three ParAF dimers bind to this region. We uncovered that one perfect inverted repeat (IR) motif, consisting of two hexamer sequences spaced by 28-bp, constitutes the primary ParAF DNA binding site. A similar but degenerated motif overlaps the former. ParAF binding to these motifs is well supported by biochemical and modeling analyses. Molecular dynamics simulations predict that the winged-HTH domain displays high flexibility, which may favor the cooperative ParA binding to the promoter. We propose that three ParAF dimers bind cooperatively to overlapping motifs, thus covering the promoter region. A similar organization is found on closely related and distant plasmid partition systems, suggesting that such promoter organization for auto-regulated Par operons is widespread and may have evolved from a common ancestor.

Abstract P-28: Structural Dynamics of DNA-Associated Chaperon Facilitates Chromatine Transcription

Background: Histone chaperon FACT (“FAcilitates Chromatin Transcription”) is a multifunctional and conserved eukaryotic protein involved in DNA transcription, replication and repair; which can reversibly unfold nucleosomes in presence of ATP. FACT is necessary for the viability and growth of breast tumor cells meanwhile in normal cells it can be knocked out without loss of vitality. Human FACT (hFACT) is a target for promising anticancer drug curaxins, which causes FACT trapping in chromatin of cancer cells and destabilizes the nucleosome. The nucleosome-unfolding activity is an important function of hFACT in vivo; however, the mechanism of FACT-dependent nucleosome unfolding remains unknown. Methods: Here, we studied negative stained hFACT structure using single particle electron microscopy using JEOL 2100 TEM. Micrographs were captured with 25k magnification, and 4.1 Å pixel size. EM images pre-processing and single particles collection were performed in EMAN2.3, followed by 2D-particles analysis in RELION2.0. Final 2D-classes included ~70 000 single particles images. Results: Based on 2D-classess data analysis we evaluated several states of hFACT reflecting its conformational flexibility: the “closed” complex is characterized by four domains localized close to each other and forming a compact structure; “intermediate” state represented by classes with identified three domains having compact structure and more disordered fourth domain, and the “open” complex, represented by three domains forming almost linear structure. The “closed” and “open” states are present in comparable amounts and significantly outnumber the “intermediate” state. It has been shown that hFACT domains are connected through flexible linkers and SPT16 and SSRP1 dimerization domains (DDs) form the “joint”-like connection between the two subunits. In the “сlosed” conformation, the DNA-binding surface of FACT is covered by its two C-terminal and middle domains (MDs). The N-terminal domain (NTD) of SPT16 was not resolved previously, but it is the best candidate for the forth domain that is clearly visible only in the “closed” conformation of hFACT, based on its dimensions and the longest linker length. Conclusion: We propose that during conversion to the “open” complexes SPT16 NTD is moving away from the other subunits leading to formation of the first intermediate state with the NTD domain poorly resolved or not resolved, while less mobile DDs and MDs maintain more compact structure and the DNA-binding site is still protected by the CTDs. In the “open” state SPT16/SSRP1 visible MDs and DDs form almost linear structure, unmasking the DNA-binding sites and making them accessible for the interaction with a nucleosome.

DeepDISE: DNA Binding Site Prediction Using a Deep Learning Method

It is essential for future research to develop a new, reliable prediction method of DNA binding sites because DNA binding sites on DNA-binding proteins provide critical clues about protein function and drug discovery. However, the current prediction methods of DNA binding sites have relatively poor accuracy. Using 3D coordinates and the atom-type of surface protein atom as the input, we trained and tested a deep learning model to predict how likely a voxel on the protein surface is to be a DNA-binding site. Based on three different evaluation datasets, the results show that our model not only outperforms several previous methods on two commonly used datasets, but also demonstrates its robust performance to be consistent among the three datasets. The visualized prediction outcomes show that the binding sites are also mostly located in correct regions. We successfully built a deep learning model to predict the DNA binding sites on target proteins. It demonstrates that 3D protein structures plus atom-type information on protein surfaces can be used to predict the potential binding sites on a protein. This approach should be further extended to develop the binding sites of other important biological molecules.

Three ParA dimers cooperatively assemble on type Ia partition promoters

Accurate DNA segregation is essential for faithful inheritance of genetic material. In bacteria, this process is mainly ensured by a partition system (Par) composed of two proteins, ParA and ParB, and a centromere site. The auto-regulation of Par operon expression is important for efficient partitioning, and is primarily mediated by ParA for type Ia plasmid partition systems. For the plasmid F, four ParAF monomers were proposed to bind to four repeated sequences in the promoter region. By contrast, using quantitative surface plasmon resonance, we showed that three ParAF dimers bind to this region. We uncovered that one perfect inverted repeat (IR) motif, consisting of two hexamer sequences spaced by 28-bp, constitutes the primary ParAF DNA binding site. A similar but degenerated motif overlaps the former. ParAF binding to these motifs is well supported by biochemical and modeling analyses. In addition, molecular dynamics simulations predict that the winged-HTH domain displays high flexibility, which may favor the cooperative ParA binding to the promoter region. We propose that three ParAF dimers bind cooperatively to overlapping motifs thus covering the promoter region. A similar organization is found on both closely related and distant plasmid partition systems, suggesting that such promoter organization for auto-regulated Par operons is widespread and may have evolved from a common ancestor.

Exosomal circEhmt1 Released from Hypoxia-Pretreated Pericytes Regulates High Glucose-Induced Microvascular Dysfunction via the NFIA/NLRP3 Pathway

Diabetic retinopathy (DR) is a frequently occurring microvascular complication induced by long-term hyperglycemia. Pericyte-endothelial cell crosstalk is critical for maintaining vascular homeostasis and remodeling; however, the molecular mechanism underlying that crosstalk remains unknown. In this study, we explored the crosstalk that occurs between endothelial cells and pericytes in response to diabetic retinopathy. Pericytes were stimulated with cobalt chloride (CoCl2) to activate the HIF pathway. Hypoxia-stimulated pericytes were cocultured with high glucose- (HG-) induced endotheliocytes. Cell viability was determined using the CCK-8 assay. Western blot studies were performed to detect the expression of proteins associated with apoptosis, hypoxia, and inflammation. ELISA assays were conducted to analyze the release of IL-1β and IL-18. We performed a circRNA microarray analysis of exosomal RNAs expressed under normoxic or hypoxic conditions. A FISH assay was performed to identify the location of circEhmt1 in pericytes. Chromatin immunoprecipitation (CHIP) was used to identify the specific DNA-binding site on the NFIA-NLRP3 complex. We found that pericyte survival was negatively correlated with the angiogenesis activity of endotheliocytes. We also found that hypoxia upregulated circEhmt1 expression in pericytes, and circEhmt1 could be transferred from pericytes to endotheliocytes via exosomes. Moreover, circEhmt1 overexpression protected endotheliocytes against HG-induced injury in vitro. Mechanistically, circEhmt1 was highly expressed in the nucleus of pericytes and could upregulate the levels of NFIA (a transcription factor) to suppress NLRP3-mediated inflammasome formation. Our study revealed a critical role for circEhmt1-mediated NFIA/NLRP3 signaling in retinal microvascular dysfunction and suggests that signaling pathway as a target for treating DR.

VirB, a key transcriptional regulator of virulence plasmid genes in Shigella flexneri, forms DNA-binding site dependent foci in the bacterial cytoplasm

VirB is a key regulator of genes located on the large virulence plasmid (pINV) in the bacterial pathogen Shigella flexneri. VirB is unusual; it is not related to other transcriptional regulators, instead, it belongs to a family of proteins that primarily function in plasmid and chromosome partitioning; exemplified by ParB. Despite this, VirB does not function to segregate DNA, but rather counters transcriptional silencing mediated by the nucleoid structuring protein, H-NS. Since ParB localizes subcellularly as discrete foci in the bacterial cytoplasm, we chose to investigate the subcellular localization of VirB to gain novel insight into how VirB functions as a transcriptional anti-silencer. To do this, a GFP-VirB fusion that retains the regulatory activity of VirB and yet, does not undergo significant protein degradation in S. flexneri, was used. Surprisingly, discrete fluorescent foci were observed in live wild-type S. flexneri cells and an isogenic virB mutant using fluorescence microscopy. In contrast, foci were rarely observed (<10%) in pINV-cured cells or in cells expressing a GFP-VirB fusion carrying amino acid substitutions in the VirB DNA binding domain. Finally, the 25 bp VirB-binding site was demonstrated to be sufficient and necessary for GFP-VirB focus formation using a set of small surrogate plasmids. Combined, these data demonstrate that the VirB:DNA interactions required for the transcriptional anti-silencing activity of VirB on pINV are a prerequisite for the subcellular localization of VirB in the bacterial cytoplasm. The significance of these findings, in light of the anti-silencing activity of VirB, is discussed. Importance This study reveals the subcellular localization of VirB, a key transcriptional regulator of virulence genes found on the large virulence plasmid (pINV) in Shigella. Fluorescent signals generated by an active GFP-VirB fusion form 2, 3, or 4 discrete foci in the bacterial cytoplasm, predominantly at the quarter cell position. These signals are completely dependent upon VirB interacting with its DNA binding site found either on the virulence plasmid or an engineered surrogate. Our findings: 1) provide novel insight into VirB:pINV interactions, 2) suggest that VirB may have utility as a DNA marker, and 3) raise questions about how and why this anti-silencing protein that controls virulence gene expression on pINV of Shigella spp. forms discrete foci/hubs within the bacterial cytoplasm.