How Chi Sequence Modifies RecBCD Single‐Stranded DNA Translocase Activity

Cho, Chia-Chuan; Chung, Chia-Hao; Li, Hung‐Wen

doi:10.1002/cphc.201700840

Cited by 3 publications

(2 citation statements)

References 30 publications

(78 reference statements)

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Even without the RecD motor, RecBC is a rapid and processive helicase, although slower than RecBCD . A secondary ssDNA translocase activity, that operates within both RecBC and RecBCD and is controlled by the ATPase activity of the RecB motor, is associated with the RecB arm region that is observed to contact duplex DNA in crystal structures . However, this secondary translocase activity shows no polarity preference on ssDNA and thus may actually reflect a double stranded DNA translocase activity .…”

Section: Recbcd Has Three Translocase Activitiesmentioning

confidence: 99%

How Does a Helicase Unwind DNA? Insights from RecBCD Helicase

Lohman

Fazio

2018

BioEssays

View full text Add to dashboard Cite

DNA helicases are a class of molecular motors that catalyze processive unwinding of double stranded DNA. In spite of much study, we know relatively little about the mechanisms by which these enzymes carry out the function for which they are named. Most current views are based on inferences from crystal structures. A prominent view is that the canonical ATPase motor exerts a force on the ssDNA resulting in "pulling" the duplex across a "pin" or "wedge" in the enzyme leading to a mechanical separation of the two DNA strands. In such models, DNA base pair separation is tightly coupled to ssDNA translocation of the motors. However, recent studies of the Escherichia coli RecBCD helicase suggest an alternative model in which DNA base pair melting and ssDNA translocation occur separately. In this view, the enzyme-DNA binding free energy is used to melt multiple DNA base pairs in an ATP-independent manner, followed by ATP-dependent translocation of the canonical motors along the newly formed ssDNA tracks. Repetition of these two steps results in processive DNA unwinding. We summarize recent evidence suggesting this mechanism for RecBCD helicase action.

show abstract

Section: Recbcd Has Three Translocase Activitiesmentioning

confidence: 99%

How Does a Helicase Unwind DNA? Insights from RecBCD Helicase

Lohman

Fazio

2018

BioEssays

View full text Add to dashboard Cite

show abstract

“…During DNA unwinding, RecB and RecD translocate along the complementary ssDNA strands, such that both motors move in the same net direction [5,6]. In addition to its canonical 3' to 5' translocase activity, the RecB motor also controls a secondary translocase activity involving the RecB arm domain, that may reflect a dsDNA translocase activity that may contribute to DNA base pair melting during DNA unwinding [7] [8][9][10][11][12].…”

Section: Introductionmentioning

confidence: 99%

E. coliRecBCD Nuclease Domain Regulates Helicase Activity but not Single Stranded DNA Translocase Activity

Fazio,

Mersch,

Hao

et al. 2023

Preprint

View full text Add to dashboard Cite

Much is still unknown about the mechanisms by which helicases unwind duplex DNA. Whereas structure-based models describe DNA unwinding as a consequence of mechanically pulling the DNA duplex across a wedge domain in the helicase by the single stranded (ss)DNA translocase activity of the ATPase motors, biochemical data indicate that processive DNA unwinding by the E. coli RecBCD helicase can occur in the absence of ssDNA translocation of the canonical RecB and RecD motors. Here, we present evidence that dsDNA unwinding is not a simple consequence of ssDNA translocation by the RecBCD motors. Using stopped-flow fluorescence approaches, we show that a RecB nuclease domain deletion variant (RecBΔNucCD) unwinds dsDNA at significantly slower rates than RecBCD, while the rate of ssDNA translocation is unaffected. This effect is primarily due to the absence of the nuclease domain and not the absence of the nuclease activity, since a nuclease-dead mutant, which retains the nuclease domain, showed no significant change in rates of ssDNA translocation or dsDNA unwinding relative to RecBCD on short DNA substrates (≤60 base pairs). This indicates that ssDNA translocation is not rate-limiting for DNA unwinding. RecBΔNucCD also initiates unwinding much slower than RecBCD from a blunt-ended DNA, although it binds with higher affinity than RecBCD. RecBΔNucCD also unwinds DNA ~two-fold slower than RecBCD on long DNA (~20 kilo base pair) in single molecule optical tweezer experiments, although the rates for RecBD1080ACD unwinding are intermediate between RecBCD and RecBΔNucCD. Surprisingly, significant pauses occur even in the absence of chi (crossover hotspot instigator) sites. We hypothesize that the nuclease domain influences the rate of DNA base pair melting, rather than DNA translocation, possibly allosterically. Since the rate of DNA unwinding by RecBCD also slows after it recognizes a chi sequence, RecBΔNucCD may mimic a post-chi state of RecBCD.

show abstract