Single-Molecule Imaging Reveals the Mechanism Underlying Histone Loading of Schizosaccharomyces pombe AAA+ ATPase Abo1

Kang, Young-Seok; Cho, Carol; Lee, Kyung Suk; Song, Ji-Joon; Lee, Ja Yil

doi:10.14348/molcells.2021.2242

Cited by 6 publications

(6 citation statements)

References 26 publications

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“…In this study, we directly visualized the DNA replication catalyzed by Phi29 DNAp using DNA curtain, a high-throughput single-molecule imaging technique that combines lipid fluidity, nano-fabrication, microfluidics and total internal reflection fluorescence microscopy (TIRFM) ( 29 , 30 ). We first characterized the biophysical properties of Phi29 DNAp at the single-molecule level.…”

Section: Introductionmentioning

confidence: 99%

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

Kim,

Shin,

Kang

et al. 2023

Nucleic Acids Research

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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.

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Section: Introductionmentioning

confidence: 99%

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

Kim,

Shin,

Kang

et al. 2023

Nucleic Acids Research

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“…The liposome without biotin was injected into the flowcell with 10 min incubation. The lipid bilayer was promoted on the slide surface for 20 min after washing out excessive liposomes by Lipid buffer (32). The lipid bilayer passivated the surface to prevent nonspecific binding of proteins.…”

Section: Methodsmentioning

confidence: 99%

Abo1 ATPase facilitates the dissociation of FACT from chromatin

Jang,

Kang,

Zofall

et al. 2024

Preprint

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The histone chaperone FACT is a heterodimeric complex consisting of Spt16 and Pob3, crucial for preserving nucleosome integrity during transcription and DNA replication. Loss of FACT leads to cryptic transcription and heterochromatin defects. FACT was shown to interact with Abo1, an AAA+ family histone chaperone involved in nucleosome dynamics. Depletion of Abo1 causes FACT to stall at transcription start sites (TSS) and mimics FACT mutants, indicating a functional association between Abo1 and FACT. However, the precise role of Abo1 in FACT function remains poorly understood. Here, we reveal that Abo1 directly interacts with FACT and facilitates the dissociation of FACT from chromatin. Specifically, the N-terminal region of Abo1 utilizes its FACT interacting (FIN) helix to bind to the N-terminal domain (NTD) of Spt16. In addition, using single-molecule fluorescence imaging, we discovered that Abo1 facilitates the ATP-dependent dissociation of FACT from nucleosomes. Furthermore, we demonstrate that the interaction between Abo1 and FACT is essential for maintaining heterochromatin in fission yeast. In summary, our findings suggest that Abo1 regulates FACT turnover in an ATP-dependent manner, proposing a model of histone chaperone recycling driven by inter-chaperone interactions.

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“…Single-molecule photobleaching assay . The single-molecule photobleaching assay was conducted as described previously ( 36 ). All reactions were performed at 23°C in reaction buffer.…”

Section: Methodsmentioning

confidence: 99%

Alteration of replication protein A binding mode on single-stranded DNA by NSMF potentiates RPA phosphorylation by ATR kinase

Kang

Han

Khim

et al. 2023

Nucleic Acids Research

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Replication protein A (RPA), a eukaryotic single-stranded DNA (ssDNA) binding protein, dynamically interacts with ssDNA in different binding modes and plays essential roles in DNA metabolism such as replication, repair, and recombination. RPA accumulation on ssDNA due to replication stress triggers the DNA damage response (DDR) by activating the ataxia telangiectasia and RAD3-related (ATR) kinase, which phosphorylates itself and downstream DDR factors, including RPA. We recently reported that the N-methyl-D-aspartate receptor synaptonuclear signaling and neuronal migration factor (NSMF), a neuronal protein associated with Kallmann syndrome, promotes RPA32 phosphorylation via ATR upon replication stress. However, how NSMF enhances ATR-mediated RPA32 phosphorylation remains elusive. Here, we demonstrate that NSMF colocalizes and physically interacts with RPA at DNA damage sites in vivo and in vitro. Using purified RPA and NSMF in biochemical and single-molecule assays, we find that NSMF selectively displaces RPA in the more weakly bound 8- and 20-nucleotide binding modes from ssDNA, allowing the retention of more stable RPA molecules in the 30-nt binding mode. The 30-nt binding mode of RPA enhances RPA32 phosphorylation by ATR, and phosphorylated RPA becomes stabilized on ssDNA. Our findings provide new mechanistic insight into how NSMF facilitates the role of RPA in the ATR pathway.

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Single-Molecule Imaging Reveals the Mechanism Underlying Histone Loading of Schizosaccharomyces pombe AAA+ ATPase Abo1

Cited by 6 publications

References 26 publications

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

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

Abo1 ATPase facilitates the dissociation of FACT from chromatin

Alteration of replication protein A binding mode on single-stranded DNA by NSMF potentiates RPA phosphorylation by ATR kinase

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