Bre1/RNF20 promotes Rad51-mediated strand exchange and antagonizes the Srs2/FBH1 helicases.

Central to homologous recombination (HR) is the assembly of Rad51 recombinase on single-strand DNA (ssDNA), forming the Rad51-ssDNA filament. How the Rad51 filament is efficiently established and sustained remains partially understood. Here, we find that the yeast ubiquitin ligase Bre1 and its human homolog RNF20, a tumor suppressor, function as recombination mediators, promoting Rad51 filament formation and subsequent reactions via multiple mechanisms independent of their ligase activities. We show that Bre1/RNF20 interacts with Rad51, directs Rad51 to ssDNA, and facilitates Rad51-ssDNA filament assembly and strand exchange in vitro. In parallel, Bre1/RNF20 interacts with the Srs2 or FBH1 helicase to counteract their disrupting effect on the Rad51 filament. We demonstrate that the above functions of Bre1/RNF20 contribute to HR repair in cells in a manner additive to the mediator protein Rad52 in yeast or BRCA2 in human. Thus, Bre1/RNF20 provides an additional layer of mechanism to directly control Rad51 filament dynamics.

5-Methyl-cytosine stabilizes DNA but hinders DNA hybridization revealed by magnetic tweezers and simulations.

5-Methyl-cytosine (5mC) is one of the most important DNA modifications and plays versatile biological roles. It is well known that 5mC stabilizes DNA duplexes. However, it remains unclear how 5mC affects the kinetics of DNA melting and hybridization. Here, we studied the kinetics of unzipping and rezipping using a 502-bp DNA hairpin by single-molecule magnetic tweezers. Under constant loading rates, 5mC increases the unzipping force but counterintuitively decreases the rezipping force at various salt and temperature conditions. Under constant forces, the non-methylated DNA hops between metastable states during unzipping and rezipping, which implies low energy barriers. Surprisingly, the 5mC DNA can't rezip after fully unzipping unless much lower forces are applied, where it rezips stochastically in a one-step manner, which implies 5mC kinetically hinders DNA hybridization and high energy barriers in DNA hybridization. All-atom molecular dynamics simulations reveal that the 5mC kinetically hinders DNA hybridization due to steric effects rather than electrostatic effects caused by the additional methyl groups of cytosines. Considering the possible high speed of DNA unzipping and zipping during replication and transcription, our findings provide new insights into the biological roles of 5mC.

Rtt105 promotes high-fidelity DNA replication and repair by regulating the single-stranded DNA-binding factor RPA.

Single-stranded DNA (ssDNA) covered with the heterotrimeric Replication Protein A (RPA) complex is a central intermediate of DNA replication and repair. How RPA is regulated to ensure the fidelity of DNA replication and repair remains poorly understood. Yeast Rtt105 is an RPA-interacting protein required for RPA nuclear import and efficient ssDNA binding. Here, we describe an important role of Rtt105 in high-fidelity DNA replication and recombination and demonstrate that these functions of Rtt105 primarily depend on its regulation of RPA. The deletion of RTT105 causes elevated spontaneous DNA mutations with large duplications or deletions mediated by microhomologies. Rtt105 is recruited to DNA double-stranded break (DSB) ends where it promotes RPA assembly and homologous recombination repair by gene conversion or break-induced replication. In contrast, Rtt105 attenuates DSB repair by the mutagenic single-strand annealing or alternative end joining pathway. Thus, Rtt105-mediated regulation of RPA promotes high-fidelity replication and recombination while suppressing repair by deleterious pathways. Finally, we show that the human RPA-interacting protein hRIP-α, a putative functional homolog of Rtt105, also stimulates RPA assembly on ssDNA, suggesting the conservation of an Rtt105-mediated mechanism.

Yeast Bromodomain Factor 1 and Its Human Homolog TAF1 Play Conserved Roles in Promoting Homologous Recombination.

Histone acetylation is a key histone post-translational modification that shapes chromatin structure, dynamics, and function. Bromodomain (BRD) proteins, the readers of acetyl-lysines, are located in the center of the histone acetylation-signaling network. How they regulate DNA repair and genome stability remains poorly understood. Here, a conserved function of the yeast Bromodomain Factor 1 (Bdf1) and its human counterpart TAF1 is reported in promoting DNA double-stranded break repair by homologous recombination (HR). Depletion of either yeast BDF1 or human TAF1, or disruption of their BRDs impairs DNA end resection, Replication Protein A (RPA) and Rad51 loading, and HR repair, causing genome instability and hypersensitivity to DNA damage. Mechanistically, it is shown that Bdf1 preferentially binds the DNA damage-induced histone H4 acetylation (H4Ac) via the BRD motifs, leading to its chromatin recruitment. Meanwhile, Bdf1 physically interacts with RPA, and this interaction facilitates RPA loading in the chromatin context and the subsequent HR repair. Similarly, TAF1 also interacts with H4Ac or RPA. Thus, Bdf1 and TAF1 appear to share a conserved mechanism in linking the HR repair to chromatin acetylation in preserving genome integrity.

How do drivers respond to driving risk during car-following? Risk-response driver model and its application in human-like longitudinal control.

The blooming of intelligent connected vehicle (ICV) has been continuously shaping a hybrid traffic environment in which the road is shared among ICVs and vehicles driven by human drivers. However, due to the insufficient understanding of the human driving strategy and style, the conflicts between ICVs and human drivers have arisen public attention, threatening the road safety and bottlenecking the development of ICV. In order to embed the human driving strategy in the intelligent driving system, researchers have been rolling out efforts on driver modeling. Most driver models, however, still suffer from the limited application scope or poor transparency. Within our finite horizons, a unified and readable driver model for various driving scenarios is generally unobtainable. In this work, we tried to model the human driving strategy from an aspect of human nature, that is, the way human drivers respond to the driving risk. We employed the risk field theory (also known as the safety field theory) to model the environmental risk in a comprehensive manner. By studying the risk-response strategy from the driving data of 24 human drivers, we proposed a unified structure, which we call the risk-response driver model (RRDM), to model the human driving strategy. This model provides access to learning not only the average driving strategy of a group of human drivers but also the specific driving style of a single driver. The explicit and readable driving strategy produced by RRDM can be directly employed to reproduce human-like longitudinal driving control. We verified the performance of our model in car-following tasks and found that its human-like driving performance is recoverable among the human drivers who participated in the tests.

Single-molecule probing the duplex and G4 unwinding patterns of a RecD family helicase.

RecD family helicases play an important role in prokaryotic genome stability and serve as the structural models for studying superfamily 1B (SF1B) helicases. However, RecD-catalyzed duplex DNA unwinding behavior and the underlying mechanism are still elusive. RecD family helicases share a common proto-helicase with eukaryotic Pif1 family helicases, which are well known for their outstanding G-quadruplex (G4) unwinding ability. However, there are still controversial points as to whether and how RecD helicases unfold G4 structures. Here, single-molecule fluorescence resonance energy transfer (smFRET) and magnetic tweezers (MT) were used to study Deinococcus radiodurans RecD2 (DrRecD2)-mediated duplex DNA unwinding and resolution of G4 structures. A symmetric, repetitive unwinding phenomenon was observed on duplex DNA, revealed from the strand switch and translocation of one monomer. Furthermore, we found that DrRecD2 was able to unwind both parallel and antiparallel G4 structures without obvious topological preferences. Surprisingly, the unwinding properties of RecD on duplex and G4 DNA are different from those of Pif1. The findings provide an example, in which the patterns of two molecules derived from a common ancestor deviate during evolution, and they are of significance for understanding the unwinding mechanism and function of SF1B helicases.

The Effect of Nano-Particles and Water Glass on the Water Stability of Magnesium Phosphate Cement Based Mortar.

This paper experimentally presented the water stability of magnesium phosphate cement (MPC) modified by nano-Al2O3 (NA), nano-Fe2O3 (NF) and water glass (WG). The optimal addition of 6% NA, 2% NF and 1% WG significantly improved the water stability of MPC mortar by 86%, 101% and 96% after 28 days of water immersion, respectively. X-Ray Diffraction (XRD) and Scanning Electron Microscope (SEM) were used to analyze the water stability of MPC modified by NA, NF and WG. The results of the micrograph and composition analysis revealed that the proper amount of NA, NF or WG could fill the micro pores and improve the hydration of interior structures of MPC mortar. Thus, the microstructural compactness was satisfied to keep a good water stability of MPC mortar.

A Universal Assay for Making DNA, RNA, and RNA-DNA Hybrid Configurations for Single-Molecule Manipulation in Two or Three Steps without Ligation.

Despite having a great variety of topologies, most DNA, RNA, and RNA-DNA hybrid (RDH) configurations for single-molecule manipulation are composed of several single-stranded (ss) DNA and ssRNA strands, with functional labels at the two ends for surface tethering. On this basis, we developed a simple, robust, and universal amplification-annealing (AA) assay for making all these configurations in two or three steps without inefficient digestion and ligation reactions. As examples, we made ssDNA, short ssDNA with double-stranded (ds) DNA handles, dsDNA with ssDNA handles, replication-fork shaped DNA/RDH/RNA, DNA holiday junction, three-site multiple-labeled and nicked DNA, torsion-constrained RNA/RDH, and short ssRNA with RDH handles. In addition to single-molecule manipulation techniques including optical tweezers, magnetic tweezers, and atomic force microscopy, these configurations can be applied in other surface-tethering techniques as well.

S-DNA and RecA/RAD51-Mediated Strand Exchange in Vitro.

S-DNA (stretched DNA) is an elongated base-paired DNA conformation under high tension. Because the RecA/Rad51 family DNA recombinases form helical filaments on DNA and mediate the formation of the DNA triplex (D-loop), in which the DNA is stretched, and because the extension of these nucleoprotein filaments is similar to the extension of S-DNA, S-DNA has long been hypothesized as a possible state of DNA that participants in RecA/Rad51-mediated DNA strand exchange in homologous recombination. Such a hypothesis, however, is still lacking direct experimental studies. In this work, we have studied the polymerization and strand exchange on S-DNA mediated by Escherichia coli RecA, human Rad51, and Saccharomyces cerevisiae Rad51 by single-molecule magnetic tweezers. We report that RecA/Rad51 polymerizes faster on S-DNA than on B-DNA with the same buffer conditions. Furthermore, the RecA/Rad51-mediated DNA triplex forms faster from S-DNA than from B-DNA together with the homologous single-stranded DNA. These results provide evidence that S-DNA can interact with RecA and Rad51 and shed light on the possible functions of S-DNA.

Age-Dependent Neuroimmune Modulation of IGF-1R in the Traumatic Mice.

BACKGROUND: Age-dependent neuroimmune modulation following traumatic stress is accompanied by discordant upregulation of Fyn signaling in the frontal cortex, but the mechanistic details of the potential cellular behavior regarding IGF-1R/Fyn have not been established. METHODS: Trans-synaptic IGF-1R signaling during the traumatic stress was comparably examined in wild type, Fyn (-/-) and MOR (-/-) mice. Techniques included primary neuron culture, in vitro kinase activity, immunoprecipitation, Western Blot, sucrose discontinuous centrifugation. Besides that, [3 H] incorporation was used to assay lymphocyte proliferation and NK cell activity. RESULTS: We demonstrate robust upregulation of synaptic Fyn activity following traumatic stress, with higher amplitude in 2-month mice than that in 1-year counterpart. We also established that the increased Fyn signaling is accompanied by its molecular connection with IGF-1R within the synaptic zone. Detained analysis using Fyn (-/-) and MOR (-/-) mice reveal that IGF-1R/Fyn signaling is governed to a large extent by mu opioid receptor (MOR), and with age-dependent manner; these signaling cascades played a central role in the modulation of lymphocyte proliferation and NK cell activity. CONCLUSIONS: Our data argued for a pivotal role of synaptic IGF-1R/Fyn signaling controlled by MOR downstream signaling cascades were crucial for the age-dependent neuroimmune modulation following traumatic stress. The result here might present a new quality of synaptic cellular communication governing the stress like events and have significant potential for the development of therapeutic approaches designed to minimize the heightened vulnerability during aging.