scholarly journals Using microsecond single-molecule FRET to determine the assembly pathways of T4 ssDNA binding protein onto model DNA replication forks

2017 ◽  
Vol 114 (18) ◽  
pp. E3612-E3621 ◽  
Author(s):  
Carey Phelps ◽  
Brett Israels ◽  
Davis Jose ◽  
Morgan C. Marsh ◽  
Peter H. von Hippel ◽  
...  
Keyword(s):  
Dna Replication ◽  
Single Molecule ◽  
Bound States ◽  
Function Analysis ◽  
Binding Protein ◽  
Critical Role ◽  
Replication Forks ◽  
Ssdna Binding ◽  
Single Molecule Fret ◽  
Ssdna Binding Protein

DNA replication is a core biological process that occurs in prokaryotic cells at high speeds (∼1 nucleotide residue added per millisecond) and with high fidelity (fewer than one misincorporation event per 107 nucleotide additions). The ssDNA binding protein [gene product 32 (gp32)] of the T4 bacteriophage is a central integrating component of the replication complex that must continuously bind to and unbind from transiently exposed template strands during DNA synthesis. We here report microsecond single-molecule FRET (smFRET) measurements on Cy3/Cy5-labeled primer-template (p/t) DNA constructs in the presence of gp32. These measurements probe the distance between Cy3/Cy5 fluorophores that label the ends of a short (15-nt) segment of ssDNA attached to a model p/t DNA construct and permit us to track the stochastic interconversion between various protein bound and unbound states. The length of the 15-nt ssDNA lattice is sufficient to accommodate up to two cooperatively bound gp32 proteins in either of two positions. We apply a unique multipoint time correlation function analysis to the microsecond-resolved smFRET data obtained to determine and compare the kinetics of various possible reaction pathways for the assembly of cooperatively bound gp32 protein onto ssDNA sequences located at the replication fork. The results of our analysis reveal the presence and translocation mechanisms of short-lived intermediate bound states that are likely to play a critical role in the assembly mechanisms of ssDNA binding proteins at replication forks and other ss duplex junctions.

scholarly journals PCNA monoubiquitination is regulated by diffusion of Rad6/Rad18 complexes along RPA filaments

2020 ◽  
Author(s):  
Mingjie Li ◽  
Bhaswati Sengupta ◽  
Stephen J. Benkovic ◽  
Tae Hee Lee ◽  
Mark Hedglin
Keyword(s):  
Single Molecule ◽  
Posttranslational Modification ◽  
Complex Dynamics ◽  
Translesion Dna Synthesis ◽  
Ssdna Binding ◽  
Sliding Clamp ◽  
Single Molecule Fret ◽  
Dna Damaging Agents ◽  
Ssdna Binding Protein ◽  
Template Dna

ABSTRACTTranslesion DNA synthesis (TLS) enables DNA replication through damaging modifications to template DNA and requires monoubiquitination of the PCNA sliding clamp by the Rad6/Rad18 complex. This posttranslational modification is critical to cell survival following exposure to DNA damaging agents and is tightly regulated to restrict TLS to damaged DNA. RPA, the major single strand DNA (ssDNA) binding protein, forms filaments on ssDNA exposed at TLS sites and plays critical yet undefined roles in regulating PCNA monoubiquitination. Here, we utilize kinetic assays and single molecule FRET microscopy to monitor PCNA monoubiquitination and Rad6/Rad18 complex dynamics on RPA filaments, respectively. Results reveal that a Rad6/Rad18 complex is recruited to an RPA filament via Rad18•RPA interactions and randomly translocates along the filament. These translocations promote productive interactions between the Rad6/Rad18 complex and the resident PCNA, significantly enhancing monoubiquitination. These results illuminate critical roles of RPA in the specificity and efficiency of PCNA monoubiquitination.

scholarly journals Single-molecule mapping of replisome progression

2022 ◽  
Author(s):  
Clemence Claussin ◽  
Jacob Vazquez ◽  
Iestyn Whitehouse
Keyword(s):  
Dna Replication ◽  
Short Duration ◽  
Gene Transcription ◽  
Single Molecule ◽  
Critical Role ◽  
Full Length ◽  
New Method ◽  
Replication Forks ◽  
Origin Of Replication ◽  
Precise Mapping

Fundamental aspects of DNA replication, such as the anatomy of replication stall sites, how replisomes are influenced by gene transcription and whether the progression of sister replisomes is coordinated are poorly understood. Available techniques do not allow the precise mapping of the positions of individual replisomes on chromatin. We have developed a new method called Replicon-seq that entails the excision of full-length replicons by controlled nuclease cleavage at replication forks. Replicons are sequenced using Nanopore, which provides a single molecule readout of long DNA molecules. Using Replicon-seq, we have investigated replisome movement along chromatin. We found that sister replisomes progress with remarkable consistency from the origin of replication but function autonomously. Replication forks that encounter obstacles pause for a short duration but rapidly resume synthesis. The helicase Rrm3 plays a critical role both in mitigating the effect of protein barriers and facilitating efficient termination. Replicon-seq provides an unprecedented means of defining replisome movement across the genome.

scholarly journals Compartmentalization of the replication fork by single-stranded DNA binding protein regulates translesion synthesis

2020 ◽  
Author(s):  
Seungwoo Chang ◽  
Elizabeth S. Thrall ◽  
Luisa Laureti ◽  
Vincent Pagès ◽  
Joseph J. Loparo
Keyword(s):  
Dna Replication ◽  
Binding Protein ◽  
Translesion Synthesis ◽  
Genome Maintenance ◽  
Ssdna Binding ◽  
Epsilon Subunit ◽  
Pol Iv ◽  
Ssdna Binding Protein ◽  
Pol Iii

AbstractDNA replication is mediated by the coordinated actions of multiple enzymes within replisomes. Processivity clamps tether many of these enzymes to DNA, allowing access to the primer/template junction. Many clamp-interacting proteins (CLIPs) are involved in genome maintenance pathways including translesion synthesis (TLS). Despite their abundance, DNA replication in bacteria is not perturbed by these CLIPs. Here we show that while the TLS polymerase Pol IV is largely excluded from moving replisomes, the remodeling of ssDNA binding protein (SSB) upon replisome stalling enriches Pol IV at replication forks. This enrichment is indispensable for Pol IV-mediated TLS on both the leading and lagging strands as it enables Pol IV-processivity clamp binding by overcoming the gatekeeping role of the Pol III epsilon subunit. As we have demonstrated for the Pol IV-SSB interaction, we propose that the binding of CLIPs to the processivity clamp must be preceded by interactions with factors that serve as localization markers for their site of action.

scholarly journals A critical role for Dna2 at unwound telomeres

2018 ◽  
Author(s):  
Marta Markiewicz-Potoczny ◽  
Michael Lisby ◽  
David Lydall
Keyword(s):  
Dna Damage ◽  
Dna Damage Response ◽  
Binding Protein ◽  
Critical Role ◽  
Temperature Sensitive ◽  
Critical Function ◽  
Ssdna Binding ◽  
Damage Response ◽  
Ssdna Binding Protein ◽  
A Subunit

AbstractDna2 is a nuclease and helicase that functions redundantly with other proteins in Okazaki fragment processing, double strand break (DSB) resection and checkpoint kinase activation. Dna2 is an essential enzyme, required for yeast and mammalian cell viability. Here we report that numerous mutations affecting the DNA damage checkpoint suppress dna2Δ lethality in Saccharomyces cerevisiae. dna2Δ cells are also suppressed by deletion of helicases, PIF1 and MPH1, and by deletion of POL32, a subunit of DNA polymerase δ. All dna2Δ cells are temperature sensitive, have telomere length defects, and low levels of telomeric 3’ single stranded DNA (ssDNA). Interestingly, Rfa1, a subunit of the major ssDNA binding protein RPA, and the telomere specific ssDNA binding protein Cdc13, often co-localize in dna2Δ cells. This suggests that telomeric defects often occur in dna2Δ cells. There are several plausible explanations for why the most critical function of Dna2 is at telomeres. Telomeres modulate the DNA damage response (DDR) at chromosome ends, inhibiting resection, ligation and cell cycle arrest. We suggest that Dna2 nuclease activity contributes to modulating the DNA damage response at telomeres by removing telomeric C-rich ssDNA and thus preventing checkpoint activation.

scholarly journals Single-molecule observation of ATP-independent SSB displacement by RecO in Deinococcus radiodurans

eLife ◽  
2020 ◽  
Vol 9 ◽  
Author(s):  
Jihee Hwang ◽  
Jae-Yeol Kim ◽  
Cheolhee Kim ◽  
Soojin Park ◽  
Sungmin Joo ◽  
...  
Keyword(s):  
Single Molecule ◽  
Free Space ◽  
Deinococcus Radiodurans ◽  
Binding Protein ◽  
Fast Diffusion ◽  
Ssdna Binding ◽  
Loading Process ◽  
Double Stranded Dna ◽  
Ssdna Binding Protein

Deinococcus radiodurans (DR) survives in the presence of hundreds of double-stranded DNA (dsDNA) breaks by efficiently repairing such breaks. RecO, a protein that is essential for the extreme radioresistance of DR, is one of the major recombination mediator proteins in the RecA-loading process in the RecFOR pathway. However, how RecO participates in the RecA-loading process is still unclear. In this work, we investigated the function of drRecO using single-molecule techniques. We found that drRecO competes with the ssDNA-binding protein (drSSB) for binding to the freely exposed ssDNA, and efficiently displaces drSSB from ssDNA without consuming ATP. drRecO replaces drSSB and dissociates it completely from ssDNA even though drSSB binds to ssDNA approximately 300 times more strongly than drRecO does. We suggest that drRecO facilitates the loading of RecA onto drSSB-coated ssDNA by utilizing a small drSSB-free space on ssDNA that is generated by the fast diffusion of drSSB on ssDNA.

scholarly journals The Bloom syndrome complex senses RPA-coated single-stranded DNA to restart stalled replication forks

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Ann-Marie K. Shorrocks ◽  
Samuel E. Jones ◽  
Kaima Tsukada ◽  
Carl A. Morrow ◽  
Zoulikha Belblidia ◽  
...  
Keyword(s):  
Replication Stress ◽  
Bloom Syndrome ◽  
Holliday Junctions ◽  
Replication Forks ◽  
Single Stranded Dna ◽  
Ssdna Binding ◽  
Ssdna Binding Protein ◽  
Dna Replication Stress ◽  
Dna End Resection ◽  
Stalled Replication Forks

AbstractThe Bloom syndrome helicase BLM interacts with topoisomerase IIIα (TOP3A), RMI1 and RMI2 to form the BTR complex, which dissolves double Holliday junctions to produce non-crossover homologous recombination (HR) products. BLM also promotes DNA-end resection, restart of stalled replication forks, and processing of ultra-fine DNA bridges in mitosis. How these activities of the BTR complex are regulated in cells is still unclear. Here, we identify multiple conserved motifs within the BTR complex that interact cooperatively with the single-stranded DNA (ssDNA)-binding protein RPA. Furthermore, we demonstrate that RPA-binding is required for stable BLM recruitment to sites of DNA replication stress and for fork restart, but not for its roles in HR or mitosis. Our findings suggest a model in which the BTR complex contains the intrinsic ability to sense levels of RPA-ssDNA at replication forks, which controls BLM recruitment and activation in response to replication stress.

Single-molecule binding characterization of primosomal protein PriA involved in replication restart

2021 ◽  
Author(s):  
Tzu-Yu Lee ◽  
Yi-Ching Li ◽  
Min-Guan Lin ◽  
Chwan-Deng Hsiao ◽  
Hung-Wen Li
Keyword(s):  
Dna Replication ◽  
Dna Synthesis ◽  
Single Molecule ◽  
Replication Forks ◽  
Bacterial Survival ◽  
Dna Damages ◽  
Replication Restart

DNA damages lead to stalled or collapsed replication forks. Replication restart primosomes re-initiate DNA synthesis at these stalled or collapsed DNA replication forks, which is important for bacterial survival. Primosomal...

scholarly journals Imaging and energetics of single SSB-ssDNA molecules reveal intramolecular condensation and insight into RecOR function

eLife ◽  
2015 ◽  
Vol 4 ◽  
Author(s):  
Jason C Bell ◽  
Bian Liu ◽  
Stephen C Kowalczykowski
Keyword(s):  
Long Range ◽  
Single Molecule ◽  
Magnetic Tweezers ◽  
Intramolecular Interactions ◽  
Ssdna Binding ◽  
Ssdna Binding Protein ◽  
Intramolecular Condensation ◽  
Nucleoprotein Complexes ◽  
Ssdna Binding Proteins ◽  
Dna Maintenance

Escherichia coli single-stranded DNA (ssDNA) binding protein (SSB) is the defining bacterial member of ssDNA binding proteins essential for DNA maintenance. SSB binds ssDNA with a variable footprint of ∼30–70 nucleotides, reflecting partial or full wrapping of ssDNA around a tetramer of SSB. We directly imaged single molecules of SSB-coated ssDNA using total internal reflection fluorescence (TIRF) microscopy and observed intramolecular condensation of nucleoprotein complexes exceeding expectations based on simple wrapping transitions. We further examined this unexpected property by single-molecule force spectroscopy using magnetic tweezers. In conditions favoring complete wrapping, SSB engages in long-range reversible intramolecular interactions resulting in condensation of the SSB-ssDNA complex. RecO and RecOR, which interact with SSB, further condensed the complex. Our data support the idea that RecOR--and possibly other SSB-interacting proteins—function(s) in part to alter long-range, macroscopic interactions between or throughout nucleoprotein complexes by microscopically altering wrapping and bridging distant sites.

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