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 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 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.

scholarly journals Sub-millisecond conformational transitions of short single-stranded DNA lattices by photon correlation single-molecule FRET

2021 ◽  
Author(s):  
Brett Israels ◽  
Claire S. Albrecht ◽  
Anson Dang ◽  
Megan Barney ◽  
Peter H. von Hippel ◽  
...  
Keyword(s):  
Single Molecule ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Time Correlation ◽  
Distribution Functions ◽  
Ssdna Binding ◽  
Single Molecule Fret ◽  
Thermally Driven ◽  
Ssb Proteins ◽  
Minority Species

Thermally-driven conformational fluctuations (or 'breathing') of DNA plays important roles in the function and regulation of the 'macromolecular machinery of genome expression.' Fluctuations in double-stranded (ds) DNA are involved in the transient exposure of pathways to protein binding sites within the DNA framework, leading to the binding of functional and regulatory proteins to single-stranded (ss) DNA templates. These interactions often require that the ssDNA sequences, as well as the proteins involved, assume transient conformations critical for successful binding. Here we use microsecond-resolved single-molecule F&oumlrster Resonance Energy Transfer (smFRET) experiments to investigate the backbone fluctuations of short (ss) oligo- oligo(dT)n templates within DNA constructs that can also serve as models for ss-dsDNA junctions. Such junctions, as well as the attached ssDNA sequences, are involved in the binding of ssDNA binding (ssb) proteins that control and integrate the mechanisms of DNA replication complexes. We have used these data to determine multi-order time-correlation functions (TCFs) and probability distribution functions (PDFs) that characterize the kinetic and thermodynamic behavior of the system. We find that the oligo(dT)n tails of ss-dsDNA constructs inter-convert, on sub-millisecond time-scales, between three macrostates with distinctly different end-to-end distances. These are: (i) a 'compact' macrostate that represents the dominant species at equilibrium; (ii) a 'partially extended' macrostate that exists as a minority species; and (iii) a 'highly extended' macrostate that is present in trace amounts. We propose a model for ssDNA secondary structure that advances our understanding of how spontaneously formed nucleic acid conformations may facilitate the activities of ssDNA associating proteins.

scholarly journals The HRDC domain oppositely modulates the unwinding activity of E. coli RecQ helicase on duplex DNA and G-quadruplex

2020 ◽  
Vol 295 (51) ◽  
pp. 17646-17658
Author(s):  
Fang-Yuan Teng ◽  
Ting-Ting Wang ◽  
Hai-Lei Guo ◽  
Ben-Ge Xin ◽  
Bo Sun ◽  
...  
Keyword(s):  
Single Molecule ◽  
Genome Stability ◽  
Premature Aging ◽  
Binding Modes ◽  
Ssdna Binding ◽  
Recq Helicases ◽  
E Coli ◽  
Single Molecule Fret ◽  
G4 Dna ◽  
Long Time

RecQ family helicases are highly conserved from bacteria to humans and have essential roles in maintaining genome stability. Mutations in three human RecQ helicases cause severe diseases with the main features of premature aging and cancer predisposition. Most RecQ helicases shared a conserved domain arrangement which comprises a helicase core, an RecQ C-terminal domain, and an auxiliary element helicase and RNaseD C-terminal (HRDC) domain, the functions of which are poorly understood. In this study, we systematically characterized the roles of the HRDC domain in E. coli RecQ in various DNA transactions by single-molecule FRET. We found that RecQ repetitively unwinds the 3′-partial duplex and fork DNA with a moderate processivity and periodically patrols on the ssDNA in the 5′-partial duplex by translocation. The HRDC domain significantly suppresses RecQ activities in the above transactions. In sharp contrast, the HRDC domain is essential for the deep and long-time unfolding of the G4 DNA structure by RecQ. Based on the observations that the HRDC domain dynamically switches between RecA core- and ssDNA-binding modes after RecQ association with DNA, we proposed a model to explain the modulation mechanism of the HRDC domain. Our findings not only provide new insights into the activities of RecQ on different substrates but also highlight the novel functions of the HRDC domain in DNA metabolisms.

scholarly journals MutL traps MutS at a DNA mismatch

2015 ◽  
Vol 112 (35) ◽  
pp. 10914-10919 ◽  
Author(s):  
Ruoyi Qiu ◽  
Miho Sakato ◽  
Elizabeth J. Sacho ◽  
Hunter Wilkins ◽  
Xingdong Zhang ◽  
...  
Keyword(s):  
Single Molecule ◽  
Conformational Changes ◽  
Thermus Aquaticus ◽  
Dna Mismatch ◽  
Sliding Clamp ◽  
Single Molecule Fret ◽  
Mismatch Detection ◽  
Current Thinking ◽  
Processivity Factor ◽  
Subsequent Activation

DNA mismatch repair (MMR) identifies and corrects errors made during replication. In all organisms except those expressing MutH, interactions between a DNA mismatch, MutS, MutL, and the replication processivity factor (β-clamp or PCNA) activate the latent MutL endonuclease to nick the error-containing daughter strand. This nick provides an entry point for downstream repair proteins. Despite the well-established significance of strand-specific nicking in MMR, the mechanism(s) by which MutS and MutL assemble on mismatch DNA to allow the subsequent activation of MutL’s endonuclease activity by β-clamp/PCNA remains elusive. In both prokaryotes and eukaryotes, MutS homologs undergo conformational changes to a mobile clamp state that can move away from the mismatch. However, the function of this MutS mobile clamp is unknown. Furthermore, whether the interaction with MutL leads to a mobile MutS–MutL complex or a mismatch-localized complex is hotly debated. We used single molecule FRET to determine that Thermus aquaticus MutL traps MutS at a DNA mismatch after recognition but before its conversion to a sliding clamp. Rather than a clamp, a conformationally dynamic protein assembly typically containing more MutL than MutS is formed at the mismatch. This complex provides a local marker where interaction with β-clamp/PCNA could distinguish parent/daughter strand identity. Our finding that MutL fundamentally changes MutS actions following mismatch detection reframes current thinking on MMR signaling processes critical for genomic stability.

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 SOS Error-Prone DNA Polymerase V Mutasome and β-Sliding Clamp Acting in Concert on Undamaged DNA and during Translesion Synthesis

Cells ◽  
2021 ◽  
Vol 10 (5) ◽  
pp. 1083
Author(s):  
Adhirath Sikand ◽  
Malgorzata Jaszczur ◽  
Linda B. Bloom ◽  
Roger Woodgate ◽  
Michael M. Cox ◽  
...  
Keyword(s):  
Dna Synthesis ◽  
Dna Polymerase ◽  
Pathogenic Bacteria ◽  
Fold Increase ◽  
Translesion Dna Synthesis ◽  
Sliding Clamp ◽  
Pol V ◽  
Dna Polymerase V ◽  
Acting In Concert

In the mid 1970s, Miroslav Radman and Evelyn Witkin proposed that Escherichia coli must encode a specialized error-prone DNA polymerase (pol) to account for the 100-fold increase in mutations accompanying induction of the SOS regulon. By the late 1980s, genetic studies showed that SOS mutagenesis required the presence of two “UV mutagenesis” genes, umuC and umuD, along with recA. Guided by the genetics, decades of biochemical studies have defined the predicted error-prone DNA polymerase as an activated complex of these three gene products, assembled as a mutasome, pol V Mut = UmuD’2C-RecA-ATP. Here, we explore the role of the β-sliding processivity clamp on the efficiency of pol V Mut-catalyzed DNA synthesis on undamaged DNA and during translesion DNA synthesis (TLS). Primer elongation efficiencies and TLS were strongly enhanced in the presence of β. The results suggest that β may have two stabilizing roles: its canonical role in tethering the pol at a primer-3’-terminus, and a possible second role in inhibiting pol V Mut’s ATPase to reduce the rate of mutasome-DNA dissociation. The identification of umuC, umuD, and recA homologs in numerous strains of pathogenic bacteria and plasmids will ensure the long and productive continuation of the genetic and biochemical journey initiated by Radman and Witkin.

scholarly journals Encoding Multiple Virtual Signals in DNA Barcodes with Single-Molecule FRET

Nano Letters ◽  
2021 ◽  
Vol 21 (4) ◽  
pp. 1694-1701 ◽  
Author(s):  
Sung Hyun Kim ◽  
Hyunwoo Kim ◽  
Hawoong Jeong ◽  
Tae-Young Yoon
Keyword(s):  
Single Molecule ◽  
Dna Barcodes ◽  
Single Molecule Fret
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