Thrap3 promotes R-loop resolution via interaction with methylated DDX5

Transcription-replication conflicts lead to DNA damage and genomic instability, which are closely related to human diseases. A major source of these conflicts is the formation of R-loops, which consist of an RNA-DNA hybrid and a displaced single-stranded DNA. Although these structures have been studied, many aspects of R-loop biology and R-loop-mediated genome instability remain unclear. Here, we demonstrate that thyroid hormone receptor-associated protein 3 (Thrap3) plays a critical role in regulating R-loop resolution. In cancer cells, Thrap3 interacts with DEAD-box helicase 5 (DDX5) and localizes to R-loops. Arginine-mediated methylation of DDX5 is required for its interaction with Thrap3, and the Thrap3-DDX5 axis induces the recruitment of 5'-3' exoribonuclease 2 (XRN2) into R-loops. Loss of Thrap3 increases R-loop accumulation and DNA damage. These findings suggest that Thrap3 mediates resistance to cell death by preventing R-loop accumulation in cancer cells. Cancer: DNA damage associated with nucleic acid loops A nuclear protein appears to inhibit cancer cell death by preventing the accumulation of nucleic acid structures called R-loops. R-loops are by-products of transcription, comprising two misaligned DNA strands and one RNA strand. They are involved in gene expression, but also threaten genome integrity and have been linked to the onset of neurodegeneration and cancers. A team led by Jang Hyun Choi and Hyug Moo Kwon, Ulsan National Institute of Science and Technology, South Korea, explored the role of Thrap3, a nuclear protein involved in RNA splicing, in R-loop-associated DNA damage. They found that Thrap3 binds to an enzyme essential for resolving R-loops. When the team suppressed Thrap3 expression, they saw an increase in R-loops in both normal and cancer cells. This R-loop accumulation significantly inhibited the growth of breast cancer cells

Ring finger protein 126 (RNF126) suppresses ionizing radiation-induced p53-binding protein 1 (53BP1) focus formation

Cells have evolved sophisticated mechanisms to maintain genomic integrity in response to DNA damage. Ionizing radiation (IR)-induced DNA damage results in the formation of IR-induced foci (iRIF) in the nucleus. The iRIF formation is part of the DNA damage response (DDR), which is an essential signaling cascade that must be strictly regulated because either the loss of or an augmented DDR leads to loss of genome integrity. Accordingly, negative regulation of the DDR is as critical as its activation. In this study, we have identified ring finger protein 126 (RNF126) as a negative regulator of the DDR from a screen of iRIF containing 53BP1. RNF126 overexpression abolishes not only the formation of 53BP1 iRIF but also of RNF168, FK2, RAP80, and BRCA1. However, the iRIF formation of H2AX, MDC1, and RNF8 is maintained, indicating that RNF126 acts between RNF8 and RNF168 during the DDR. In addition, RNF126 overexpression consistently results in the loss of RNF168-mediated H2A monoubiquitination at lysine 13/15 and inhibition of the non-homologous end joining capability. Taken together, our findings reveal that RNF126 is a novel factor involved in the negative regulation of DDR, which is important for sustaining genomic integrity

TonEBP recognizes R-loops and initiates m6A RNA methylation for R-loop resolution

R-loops are three-stranded, RNA???DNA hybrid, nucleic acid structures produced due to inappropriate processing of newly transcribed RNA or transcription-replication collision (TRC). Although R-loops are important for many cellular processes, their accumulation causes genomic instability and malignant diseases, so these structures are tightly regulated. It was recently reported that R-loop accumulation is resolved by methyltransferase-like 3 (METTL3)-mediated m6A RNA methylation under physiological conditions. However, it remains unclear how R-loops in the genome are recognized and induce resolution signals. Here, we demonstrate that tonicity-responsive enhancer binding protein (TonEBP) recognizes R-loops generated by DNA damaging agents such as ultraviolet (UV) or camptothecin (CPT). Single-molecule imaging and biochemical assays reveal that TonEBP preferentially binds a R-loop via both 3D collision and 1D diffusion along DNA in vitro. In addition, we find that TonEBP recruits METTL3 to R-loops through the Rel homology domain (RHD) for m6A RNA methylation. We also show that TonEBP recruits RNaseH1 to R-loops through a METTL3 interaction. Consistent with this, TonEBP or METTL3 depletion increases R-loops and reduces cell survival in the presence of UV or CPT. Collectively, our results reveal an R-loop resolution pathway by TonEBP and m6A RNA methylation by METTL3 and provide new insights into R-loop resolution processes

Direct visualization of replication and R-loop collision using single-molecule imaging

R-loops are three-stranded nucleic acid structures that can cause replication stress by blocking replication fork progression. However, the detailed mechanism underlying the collision of DNA replication forks and R-loops remains elusive. To investigate how R-loops induce replication stress, we use single-molecule fluorescence imaging to directly visualize the collision of replicating Phi29 DNA polymerase (Phi29 DNAp), the simplest replication system, and R-loops. We demonstrate that a single R-loop can block replication, and the blockage is more pronounced when an RNA???DNA hybrid is on the non-template strand. We show that this asymmetry results from secondary structure formation on the non-template strand, which impedes the progression of Phi29 DNAp. We also show that G-quadruplex formation on the displaced single-stranded DNA in an R-loop enhances the replication stalling. Moreover, we observe the collision between Phi29 DNAp and RNA transcripts synthesized by T7 RNA polymerase (T7 RNAp). RNA transcripts cause more stalling because of the presence of T7 RNAp. Our work provides insights into how R-loops impede DNA replication at single-molecule resolution

Merlin inhibits growth hormone-regulated Raf-ERKs pathways by binding to Grb2 protein

Numerous studies have suggested that the NF2 protein merlin is involved in the regulation of abnormal cell growth and proliferation. In this study, to better understand the merlin's mechanisms that contribute to the inhibition of tumorigenesis, we examined the potential action of merlin on the cell proliferative signaling pathways in response to growth hormone (GH). Merlin effectively attenuated the GH-induced serum response element (SRE) and Elk-l-mediated transcriptional activation, as well as the endogenous SRE-regulated gene c-fos expression in NIH3T3 cells. In addition, merlin prevented the Raf-1 complex activation process, which resulted in the suppression of MAP kinase/ERK, extracellular signal-regulated kinase (ERKs), and Elk-1 phosphorylation, which are the downstream signals of Raf-1. Moreover, it was shown that merlin interacted with endogenous growth factor receptor bound 2 (Grb2) protein and inhibited its expression. These results suggest that merlin contributes, via its protein-to-protein interaction with Grb2 and consequent inhibition of the MAPK pathways, to the regulation of the abnormal cell proliferation, and this provides a further mechanism underlying the tumor suppressor function of merlin

Interactome Analysis Reveals that Heterochromatin Protein 1 gamma (HP1 gamma) Is Associated with the DNA Damage Response Pathway

Purpose Heterochromatin protein 1 gamma (HP1 gamma) interacts with chromosomes by binding to lysine 9-methylated histone H3 or DNA/RNA. HP1 gamma is involved in various biological processes. The purpose of this study is to gain an understanding of how HP1 gamma functions in these processes by identifying HP1 gamma-binding proteins using mass spectrometry. Materials and Methods We performed affinity purification of HP1 gamma-binding proteins using G(1)/S phase or prometaphase HEK293T cell lysates that transiently express mock or FLAG-HP1 gamma. Coomassie staining was performed for HP1 gamma-binding complexes, using cell lysates prepared by affinity chromatography FLAG-agarose beads, and the bands were digested and then analyzed using a mass spectrometry. Results We identified 99 HP1 gamma-binding proteins with diverse cellular functions, including spliceosome, regulation of the actin cytoskeleton, tight junction, pathogenic Escherichia coli infection, mammalian target of rapamycin signaling pathway, nucleotide excision repair, DNA replication, homologous recombination, and mismatch repair. Conclusion Our results suggested that HP1 gamma is functionally active in DNA damage response via protein-protein interaction

Correlation between failure and local material property in chopped carbon fiber chip‐reinforced sheet molding compound composites under tensile load

Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/148378/1/pc24767_am.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/148378/2/pc24767.pd

Experimental and numerical evaluations on the effects of adhesive fillet, overlap length and unbonded area in adhesive- bonded joints

To realize robust structural design, the effects of the adhesive fillet, overlap length and unbonded area in adhesive- bonded joints need to be fully understood and incorporated into a fatigue life estimation method. In the present work, both static and fatigue experiments are performed on six types of adhesive- bonded joints to illuminate these effects systematically. A straightforward total fatigue life evaluation method is proposed to address these effects. A statistical crack initiation model is established based on the fatigue data of bulk adhesive specimens. Growth life is calculated using the interfacial crack model and mixed mode crack growth method. Good correlation is observed between the calculated and experimental fatigue lives. Furthermore, the effects of the adhesive fillet, overlap length and unbonded area are analysed based on both calculated and experimental results. Results indicate that adhesive fillet postpones crack initiation by reducing local strain level, both overlap length and unbonded area change the growth life by length. Besides, overlap length promotes the fraction of mode II strain energy release rate in total, reducing crack growth rates and extending growth life.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/162715/2/ffe13294.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/162715/1/ffe13294_am.pd