Single-molecule imaging of DNA curtains reveals mechanisms of KOPS sequence targeting by the DNA translocase FtsK

Lee, Ja Yil; Finkelstein, Ilya J.; Crozat, Estelle; Sherratt, David J.; Greene, Eric C.

doi:10.1073/pnas.1201613109

Cited by 58 publications

(86 citation statements)

References 31 publications

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“…For experiments at a higher ionic strength, the NaCl concentration was increased to 130 mM. Human or yeast Exo1-biotin was prelabeled with streptavidin QDs (Qdot 705; Life Tech) before use, as previously described (72). To make the DNA curtain, λ-DNA was hybridized with a biotinylated oligonucleotide on one end and a 3′ overhang-generating oligonucleotide on the other end (Table S2).…”

Section: Methodsmentioning

confidence: 99%

Single-molecule imaging reveals the mechanism of Exo1 regulation by single-stranded DNA binding proteins

Myler

Gallardo

Zhou

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

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104

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Exonuclease 1 (Exo1) is a 5′→3′ exonuclease and 5′-flap endonuclease that plays a critical role in multiple eukaryotic DNA repair pathways. Exo1 processing at DNA nicks and double-strand breaks creates long stretches of single-stranded DNA, which are rapidly bound by replication protein A (RPA) and other single-stranded DNA binding proteins (SSBs). Here, we use single-molecule fluorescence imaging and quantitative cell biology approaches to reveal the interplay between Exo1 and SSBs. Both human and yeast Exo1 are processive nucleases on their own. RPA rapidly strips Exo1 from DNA, and this activity is dependent on at least three RPA-encoded single-stranded DNA binding domains. Furthermore, we show that ablation of RPA in human cells increases Exo1 recruitment to damage sites. In contrast, the sensor of single-stranded DNA complex 1-a recently identified human SSB that promotes DNA resection during homologous recombination-supports processive resection by Exo1. Although RPA rapidly turns over Exo1, multiple cycles of nuclease rebinding at the same DNA site can still support limited DNA processing. These results reveal the role of single-stranded DNA binding proteins in controlling Exo1-catalyzed resection with implications for how Exo1 is regulated during DNA repair in eukaryotic cells.A ll DNA maintenance processes require nucleases, which enzymatically cleave the phosphodiester bonds in nucleic acids. Exo1, a member of the Rad2 family of nucleases, participates in DNA mismatch repair (MMR), double-strand break (DSB) repair, nucleotide excision repair (NER), and telomere maintenance (1-3). Exo1 is the only nuclease implicated in MMR, where its 5ʹ to 3ʹ exonuclease activity is used to remove long tracts of mismatch-containing single-stranded DNA (ssDNA) (2, 4-7). In addition, functionally deficient Exo1 variants have been identified in familial colorectal cancers, and Exo1-null mice exhibit a significant increase in tumor development, decreased lifespan, and sterility (8, 9). Exo1 also promotes DSB repair via homologous recombination (HR) by processing the free DNA ends to generate kilobase-length ssDNA resection products (1, 10-12). The resulting ssDNA is paired with a homologous DNA sequence located on a sister chromatid, and the missing genetic information is then restored via DNA synthesis. The central role of Exo1 in DNA repair is highlighted by the large set of genetic interactions between Exo1 and nearly all other DNA maintenance and metabolism pathways (13).Exo1 generates long tracts of ssDNA in both MMR and DSB repair (3). This ssDNA is rapidly bound by replication protein A (RPA), a ubiquitous heterotrimeric protein that participates in all DNA transactions that generate ssDNA intermediates (14). RPA protects the ssDNA from degradation, participates in DNA damage response signaling, and acts as a loading platform for downstream DSB repair proteins (15-17). RPA also coordinates DNA resection by removing secondary ssDNA structures and by modulating the Bloom syndrome, RecQ helicase-like (BLM)/ DNA2-and Ex...

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Section: Methodsmentioning

confidence: 99%

Single-molecule imaging reveals the mechanism of Exo1 regulation by single-stranded DNA binding proteins

Myler

Gallardo

Zhou

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

104

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“…It is important to consider that when the ATPase subunits were fused into a concatemer, there might have been unknown and unintended alterations either to the ATPase activity of individual monomers or to their ability to form higher-order multimers, which could mask the effect of mutant subunits. Conversely, a number of single-molecule studies have now shown that the linked trimeric FtsK proteins appear to translocate and respond to nucleotides similarly to unlinked hexamers (77,179). Nevertheless, in order to explain these results, a new model in which more than one subunit within a hexameric ring would contact DNA concurrently was proposed.…”

Section: Fig 18mentioning

confidence: 99%

“…This ensures that the motor is loaded correctly onto DNA in a specific orientation and that the subsequent translocation is toward the XerCD-dif site. Directional translocation of DNA is, therefore, sequence dependent at the motor-loading step; further, a translocating FtsK appears to ignore additional KOPS and reads through them (77,84).…”

Section: Revolution Motorsmentioning

confidence: 99%

Biological Nanomotors with a Revolution, Linear, or Rotation Motion Mechanism

Guo

Noji

Yengo

et al. 2016

Microbiol Mol Biol Rev

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SUMMARYThe ubiquitous biological nanomotors were classified into two categories in the past: linear and rotation motors. In 2013, a third type of biomotor, revolution without rotation (http://rnanano.osu.edu/movie.html), was discovered and found to be widespread among bacteria, eukaryotic viruses, and double-stranded DNA (dsDNA) bacteriophages. This review focuses on recent findings about various aspects of motors, including chirality, stoichiometry, channel size, entropy, conformational change, and energy usage rate, in a variety of well-studied motors, including FoF1ATPase, helicases, viral dsDNA-packaging motors, bacterial chromosome translocases, myosin, kinesin, and dynein. In particular, dsDNA translocases are used to illustrate how these features relate to the motion mechanism and how nature elegantly evolved a revolution mechanism to avoid coiling and tangling during lengthy dsDNA genome transportation in cell division. Motor chirality and channel size are two factors that distinguish rotation motors from revolution motors. Rotation motors use right-handed channels to drive the right-handed dsDNA, similar to the way a nut drives the bolt with threads in same orientation; revolution motors use left-handed motor channels to revolve the right-handed dsDNA. Rotation motors use small channels (<2 nm in diameter) for the close contact of the channel wall with single-stranded DNA (ssDNA) or the 2-nm dsDNA bolt; revolution motors use larger channels (>3 nm) with room for the bolt to revolve. Binding and hydrolysis of ATP are linked to different conformational entropy changes in the motor that lead to altered affinity for the substrate and allow work to be done, for example, helicase unwinding of DNA or translocase directional movement of DNA.

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“…However, FtsK C stops at least transiently and/or dissociates at XerCD bound to dif (27,32). Reversals in translocation direction have been observed to occur spontaneously (18,28,30,31) and in response to XerCD bound to dif (32). The use of a fluorophore label, along with singly tethered DNA, has allowed us to observe FtsK C without requiring any loop extrusion, and to observe its interaction with synaptic complexes of XerCD, where previous single-molecule work has only dealt with unsynapsed XerCD-dif (32).…”

mentioning

confidence: 99%

“…Previous studies of FtsK C assembly and translocation have used biochemical methods (26,27) and single-molecule techniques, including magnetic and optical tweezers (18,(28)(29)(30); tethered particle motion (TPM) (18); and, more recently, DNA curtains (31,32). Optical/magnetic tweezers and TPM experiments have relied on loop extrusion by FtsK C to observe its action (looping by FtsK shortens the length between two DNA ends, hence displacing the bead used in TPM or optical/magnetic tweezers).…”

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confidence: 99%

Assembly, translocation, and activation of XerCD- dif recombination by FtsK translocase analyzed in real-time by FRET and two-color tethered fluorophore motion

May¹,

Zawadzki²,

Sherratt³

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

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The FtsK dsDNA translocase functions in bacterial chromosome unlinking by activating XerCD-dif recombination in the replication terminus region. To analyze FtsK assembly and translocation, and the subsequent activation of XerCD-dif recombination, we extended the tethered fluorophore motion technique, using two spectrally distinct fluorophores to monitor two effective lengths along the same tethered DNA molecule. We observed that FtsK assembled stepwise on DNA into a single hexamer, and began translocation rapidly (∼0.25 s). Without extruding DNA loops, single FtsK hexamers approached XerCD-dif and resided there for ∼0.5 s irrespective of whether XerCD-dif was synapsed or unsynapsed. FtsK then dissociated, rather than reversing. Infrequently, FtsK activated XerCD-dif recombination when it encountered a preformed synaptic complex, and dissociated before the completion of recombination, consistent with each FtsK-XerCD-dif encounter activating only one round of recombination.

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Single-molecule imaging of DNA curtains reveals mechanisms of KOPS sequence targeting by the DNA translocase FtsK

Cited by 58 publications

References 31 publications

Single-molecule imaging reveals the mechanism of Exo1 regulation by single-stranded DNA binding proteins

Single-molecule imaging reveals the mechanism of Exo1 regulation by single-stranded DNA binding proteins

Biological Nanomotors with a Revolution, Linear, or Rotation Motion Mechanism

Assembly, translocation, and activation of XerCD- dif recombination by FtsK translocase analyzed in real-time by FRET and two-color tethered fluorophore motion

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