Direct Observation of RecBCD Helicase as Single-Stranded DNA Translocases

Chung, Chia-Hao; Li, Hung‐Wen

doi:10.1021/ja401626t

Cited by 20 publications

(29 citation statements)

References 30 publications

(45 reference statements)

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“…Increasing evidence suggests that many helicases function as ssDNA translocases (35,36). By translocating along single-stranded DNA, helicase exerts force on the DNA substrates.…”

Section: Discussionmentioning

confidence: 99%

Pif1 regulates telomere length by preferentially removing telomerase from long telomere ends

Yu²,

Chien³

et al. 2014

Nucleic Acids Res

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Telomerase, a ribonucleoprotein complex, is responsible for maintaining the telomere length at chromosome ends. Using its RNA component as a template, telomerase uses its reverse transcriptase activity to extend the 3′-end single-stranded, repetitive telomeric DNA sequence. Pif1, a 5′-to-3′ helicase, has been suggested to regulate telomerase activity. We used single-molecule experiments to directly show that Pif1 helicase regulates telomerase activity by removing telomerase from telomere ends, allowing the cycling of the telomerase for additional extension processes. This telomerase removal efficiency increases at longer ssDNA gaps and at higher Pif1 concentrations. The enhanced telomerase removal efficiency by Pif1 at the longer single-stranded telomeric DNA suggests a way of how Pif1 regulates telomerase activity and maintains telomere length.

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“…Increasing evidence suggests that many helicases function as ssDNA translocases (35,36). By translocating along single-stranded DNA, helicase exerts force on the DNA substrates.…”

Section: Discussionmentioning

confidence: 99%

Pif1 regulates telomere length by preferentially removing telomerase from long telomere ends

Yu²,

Chien³

et al. 2014

Nucleic Acids Res

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“…To monitor the ssDNA translocation activities of RecBCD, we designed DNA substrates containing chi 3 or chi 0 sequence along with a long secondary‐structure‐free ssDNA of different polarities. The chi‐free and chi‐containing plasmids used for polymerase chain reaction (PCR) reaction were the same as previous described . We first prepared the DNA handle with chi 3 or chi 0 using PCR, followed by BfuAI (NEB) digestion to produce a sticky end for ligation (see Figure S1 A).…”

Section: Methodsmentioning

confidence: 99%

“…Single‐molecule tethered particle motion (TPM) experiments were carried out as previously described . We anchored digoxigenin‐labeled DNA substrates on the anti‐digoxigenin decorated coverslips.…”

Section: Methodsmentioning

confidence: 99%

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How Chi Sequence Modifies RecBCD Single‐Stranded DNA Translocase Activity

Cho

Chung

2017

ChemPhysChem

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E. coli RecBCD initiates homologous repair as well as degrades foreign DNA. Recognition of chi sequence (5'-GCTGGTGG-3') switches RecBCD from a destructive, nucleolytic mode into a repair-active one that promotes RecA-mediated recombination. RecBCD includes a 3'-to-5' single-stranded DNA (ssDNA) translocase in RecB subunit, a 5'-to-3' translocase in RecD, and a secondary translocase activity associated with RecBC. To understand how chi specifically affects each translocase activity, we directly visualized individual RecBCD translocating along DNA substrates containing a ssDNA gap of different polarities, with or without chi. Disappearance of RecBCD from the ssDNA signals the loss of the ssDNA translocase activity. For substrates containing a ssDNA gap that RecBCD encounters in the 3'-to-5' polarity (3'-to-5' ssDNA), wild-type RecBCD disappears from the DNA substrates with similarly high percentage, either with chi or without. This suggests that (1) the 3'-to-5' translocase in RecB is unaffected by chi and (2) it is low in processivity. With substrates containing a ssDNA gap that RecBCD encounters in the 5'-to-3' polarity (5'-to-3' ssDNA), we found that the leaving percentage increases significantly with chi, implying inactivation of the 5'-to-3' translocase of RecD upon chi recognition. Surprisingly, the RecD defective mutant RecBCD showed only ≈50 % leaving on 5'-to-3' ssDNA, directly revealing the presence of RecBC secondary translocase and its activity is unaffected by chi. Multiple ssDNA translocases within the RecBCD complex both before and after chi ensures processive unwinding of DNA substrates required for efficient recombination events.

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“…Streptavidin‐coated 220 nm beads and the reaction chambers were prepared as previously described . For the tether Brownian motion (BM) measurement, we utilized an inverted optical microscope (IX‐71, Olympus) using a differential interference contrast (DIC) imaging method.…”

Section: Dna Topology Strongly Affects the Strand‐exchange Efficiencymentioning

confidence: 99%

DNA with Different Local Torsional States Affects RecA‐Mediated Recombination Progression

2017

ChemPhysChem

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DNA topology is thought to affect DNA enzyme activity. The helical structure of duplex DNA dictates the change of topological states during strand separation when DNA is constrained. During the repair of DNA double-stranded breaks, the RecA nucleoprotein filament invades DNA and carries out consecutive strand exchange reactions coupled with duplex DNA strand separation. It has been suggested that torsional strain could be generated and its accumulation could inhibit strand exchange. We used hairpin and nicked DNA substrates to test how torsional strain alters the RecA-mediated strand exchange efficiency. Single-molecule tethered particle motion (TPM) experiments showed that torsionally constrained hairpin DNA substrates returned nearly no successful strand exchange events catalyzed by RecA. Surprisingly, the strand exchange efficiencies increase in the presence of DNA nicks or loop disruption. The dwell time of transient RecA events in hairpin is shorter compared to those found in nicked or fork DNA substrates, which suggests a limited strand exchange progression in hairpin substrates. Our observation shows that RecA generates local torsional strain during strand exchange, and the inability to dissipate this torsional strain inhibits homologous recombination progression. DNA topological states are thus important regulation measures of DNA recombination.

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Direct Observation of RecBCD Helicase as Single-Stranded DNA Translocases

Cited by 20 publications

References 30 publications

Pif1 regulates telomere length by preferentially removing telomerase from long telomere ends

Pif1 regulates telomere length by preferentially removing telomerase from long telomere ends

How Chi Sequence Modifies RecBCD Single‐Stranded DNA Translocase Activity

DNA with Different Local Torsional States Affects RecA‐Mediated Recombination Progression

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