Dynamic DNA binding licenses a repair factor to bypass roadblocks in search of DNA lesions

Brown, Maxwell W.; Kim, Yoori; Williams, Gregory M.; Huck, John D.; Surtees, Jennifer A.; Finkelstein, Ilya J.

doi:10.1038/ncomms10607

Cited by 49 publications

(56 citation statements)

References 70 publications

(136 reference statements)

Supporting

Mentioning

Contrasting

Order By: Relevance

“…All single-molecule data was collected at 37°C using a Nikon Ti-E microscope in a prism-TIRF configuration (Brown et al, 2016; Myler et al, 2016). Data were collected with a 200 ms exposure through a 60X water-immersion objective (1.2NA, Nikon), a 500 nm long-pass (Chroma) and a 638 nm dichroic beam splitter (Chroma) for two-channel detection through two EMCCD cameras (Andor iXon DU897, cooled to −80°C).…”

Section: Star Methodsmentioning

confidence: 99%

“…Nucleosomes were reconstituted via stepwise salt dialysis using 1.5 M, 1.0 M, 0.8 M, 0.6 M, 0.4 M, and 0.2 M NaCl concentrations each for 2 hours (Brown et al, 2016). …”

Section: Star Methodsmentioning

confidence: 99%

“…Human RPA and RPA-GFP were purified from E. coli using a pET expression vector (Myler et al, 2016). Human histones were individually purified from inclusion bodies, assembled into octamers, and reconstituted on DNA curtains via step-wise salt dialysis (Brown et al, 2016). …”

Section: Star Methodsmentioning

confidence: 99%

See 2 more Smart Citations

Single-Molecule Imaging Reveals How Mre11-Rad50-Nbs1 Initiates DNA Break Repair

et al. 2017

Self Cite

View full text Add to dashboard Cite

Summary DNA double-strand break (DSB) repair is essential for maintaining our genomes. Mre11-Rad50-Nbs1 (MRN) and Ku70-Ku80 (Ku) direct distinct DSB repair pathways, but the interplay between these complexes at a DSB remains unclear. Here, we use high-throughput single-molecule microscopy to show that MRN searches for free DNA ends by one-dimensional facilitated diffusion, even on nucleosome-coated DNA. Rad50 binds homoduplex DNA and promotes facilitated diffusion, whereas Mre11 is required for DNA end recognition and nuclease activities. MRN gains access to occluded DNA ends by removing Ku or other DNA adducts via an Mre11-dependent nucleolytic reaction. Next, MRN loads Exonuclease 1 (Exo1) onto the free DNA ends to initiate DNA resection. In the presence of Replication Protein A (RPA), MRN acts as a processivity factor for Exo1, retaining the exonuclease on DNA for long-range resection. Our results provide a mechanism for how MRN promotes homologous recombination on nucleosome-coated DNA.

show abstract

Section: Star Methodsmentioning

confidence: 99%

“…Nucleosomes were reconstituted via stepwise salt dialysis using 1.5 M, 1.0 M, 0.8 M, 0.6 M, 0.4 M, and 0.2 M NaCl concentrations each for 2 hours (Brown et al, 2016). …”

Section: Star Methodsmentioning

confidence: 99%

See 1 more Smart Citation

Single-Molecule Imaging Reveals How Mre11-Rad50-Nbs1 Initiates DNA Break Repair

et al. 2017

Self Cite

View full text Add to dashboard Cite

show abstract

“…After slide fabrication, the flowcells and SLBs are assembled as described previously (Brown et al, 2016; Finkelstein, Visnapuu, & Greene, 2010; Gallardo et al, 2015; Myler et al, 2016). In brief, the flowcell is constructed using a clean microfabricated quartz slide and a glass coverslip using a double-sided tape, and then connectors are sealed to the quartz slide using a glue gun.…”

Section: Methodsmentioning

confidence: 99%

“…Wash and assemble flowcells as previously described (Brown et al, 2016; Finkelstein et al, 2010; Gallardo et al, 2015; Myler et al, 2016). See Note 1 .…”

Section: Methodsmentioning

confidence: 99%

Next-Generation DNA Curtains for Single-Molecule Studies of Homologous Recombination

Soniat

Myler

Schaub

et al. 2017

Methods in Enzymology

Self Cite

View full text Add to dashboard Cite

Homologous recombination (HR) is a universally conserved DNA double-strand break repair pathway. Single-molecule fluorescence imaging approaches have revealed new mechanistic insights into nearly all aspects of HR. These methods are especially suited for studying protein complexes because multicolor fluorescent imaging can parse out subassemblies and transient intermediates that associate with the DNA substrates on the millisecond to hour timescales. However, acquiring single-molecule datasets remains challenging because most of these approaches are designed to measure one molecular reaction at a time. The DNA curtains platform facilitates high-throughput single-molecule imaging by organizing arrays of DNA molecules on the surface of a microfluidic flowcell. Here, we describe a second-generation UV lithography-based protocol for fabricating flowcells for DNA curtains. This protocol greatly reduces the challenges associated with assembling DNA curtains and paves the way for the rapid acquisition of large datasets from individual single-molecule experiments. Drawing on our recent studies of human HR, we also provide an overview of how DNA curtains can be used for observing facilitated protein diffusion, processive enzyme translocation, and nucleoprotein filament dynamics on single-stranded DNA. Together, these protocols and case studies form a comprehensive introduction for other researchers that may want to adapt DNA curtains for high-throughput single-molecule studies of DNA replication, transcription, and repair.

show abstract

Insights into DNA cleavage by MutL homologs from analysis of conserved motifs in eukaryotic Mlh1

Putnam

Kolodner

2023

BioEssays

View full text Add to dashboard Cite

MutL family proteins contain an N‐terminal ATPase domain (NTD), an unstructured interdomain linker, and a C‐terminal domain (CTD), which mediates constitutive dimerization between subunits and often contains an endonuclease active site. Most MutL homologs direct strand‐specific DNA mismatch repair by cleaving the error‐containing daughter DNA strand. The strand cleavage reaction is poorly understood; however, the structure of the endonuclease active site is consistent with a two‐ or three‐metal ion cleavage mechanism. A motif required for this endonuclease activity is present in the unstructured linker of Mlh1 and is conserved in all eukaryotic Mlh1 proteins, except those from metamonads, which also lack the almost absolutely conserved Mlh1 C‐terminal phenylalanine‐glutamate‐arginine‐cysteine (FERC) sequence. We hypothesize that the cysteine in the FERC sequence is autoinhibitory, as it sequesters the active site. We further hypothesize that the evolutionary co‐occurrence of the conserved linker motif with the FERC sequence indicates a functional interaction, possibly by linker motif‐mediated displacement of the inhibitory cysteine. This role is consistent with available data for interactions between the linker motif with DNA and the CTDs in the vicinity of the active site.

show abstract

Dynamic DNA binding licenses a repair factor to bypass roadblocks in search of DNA lesions

Cited by 49 publications

References 70 publications

Single-Molecule Imaging Reveals How Mre11-Rad50-Nbs1 Initiates DNA Break Repair

Single-Molecule Imaging Reveals How Mre11-Rad50-Nbs1 Initiates DNA Break Repair

Next-Generation DNA Curtains for Single-Molecule Studies of Homologous Recombination

Insights into DNA cleavage by MutL homologs from analysis of conserved motifs in eukaryotic Mlh1

Contact Info

Product

Resources

About