E. coli RecB Nuclease Domain Regulates RecBCD Helicase Activity but not Single Stranded DNA Translocase Activity.

Much is still unknown about the mechanisms by which helicases unwind duplex DNA. Whereas structure-based models describe DNA unwinding as occurring by the ATPase motors mechanically pulling the DNA duplex across a wedge domain in the helicase, biochemical data show that processive DNA unwinding by E. coli RecBCD helicase can occur in the absence of ssDNA translocation by the canonical RecB and RecD motors. Here we show that DNA unwinding is not a simple consequence of ssDNA translocation by the 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 ssDNA translocation rate is unaffected. This effect is primarily due to the absence of the nuclease domain since a nuclease-dead mutant (RecBD1080ACD), which retains the nuclease domain, showed no change in ssDNA translocation or dsDNA unwinding rates relative to RecBCD on short DNA substrates (≤60 base pairs). Hence, ssDNA translocation is not rate-limiting for DNA unwinding. RecBΔNucCD also initiates unwinding much slower than RecBCD from a blunt-ended DNA. 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 in DNA unwinding 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, possibly allosterically and that RecBΔNucCD may mimic a post-chi state of RecBCD.

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

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 (RecB ΔNuc CD) 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 (RecB D1080A CD), 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. RecB ΔNuc CD also initiates unwinding much slower than RecBCD from a blunt-ended DNA, although it binds with higher affinity than RecBCD. RecB ΔNuc CD 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 RecB D1080A CD unwinding are intermediate between RecBCD and RecB ΔNuc CD. 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, RecB ΔNuc CD may mimic a post- chi state of RecBCD.

Heterogeneity in E. coli RecBCD Helicase-DNA Binding and Base Pair Melting.

E. coli RecBCD, a helicase/nuclease involved in double stranded (ds) DNA break repair, binds to a dsDNA end and melts out several DNA base pairs (bp) using only its binding free energy. We examined RecBCD-DNA initiation complexes using thermodynamic and structural approaches. Measurements of enthalpy changes for RecBCD binding to DNA ends possessing pre-melted ssDNA tails of increasing length suggest that RecBCD interacts with ssDNA as long as 17-18 nucleotides and can melt at least 10-11 bp upon binding a blunt DNA end. Cryo-EM structures of RecBCD alone and in complex with a blunt-ended dsDNA show significant conformational heterogeneities associated with the RecB nuclease domain (RecBNuc) and the RecD subunit. In the absence of DNA, 56% of RecBCD molecules show no density for the RecB nuclease domain, RecBNuc, and all RecBCD molecules show only partial density for RecD. DNA binding reduces these conformational heterogeneities, with 63% of the molecules showing density for both RecD and RecBNuc. This suggests that the RecBNuc domain is dynamic and influenced by DNA binding. The major RecBCD-DNA structural class in which RecBNuc is docked onto RecC shows melting of at least 11 bp from a blunt DNA end, much larger than previously observed. A second structural class in which RecBNuc is not docked shows only four bp melted suggesting that RecBCD complexes transition between states with different extents of DNA melting and that the extent of melting regulates initiation of helicase activity.

Processive DNA Unwinding by RecBCD Helicase in the Absence of Canonical Motor Translocation.

Escherichia coli RecBCD is a DNA helicase/nuclease that functions in double-stranded DNA break repair. RecBCD possesses two motors (RecB, a 3' to 5' translocase, and RecD, a 5' to 3' translocase). Current DNA unwinding models propose that motor translocation is tightly coupled to base pair melting. However, some biochemical evidence suggests that DNA melting of multiple base pairs may occur separately from single-stranded DNA translocation. To test this hypothesis, we designed DNA substrates containing reverse backbone polarity linkages that prevent ssDNA translocation of the canonical RecB and RecD motors. Surprisingly, we find that RecBCD can processively unwind DNA for at least 80bp beyond the reverse polarity linkages. This ability requires an ATPase active RecB motor, the RecB "arm" domain, and also the RecB nuclease domain, but not its nuclease activity. These results indicate that RecBCD can unwind duplex DNA processively in the absence of ssDNA translocation by the canonical motors and that the nuclease domain regulates the helicase activity of RecBCD.

A Novel Recombinant DNA System for High Efficiency Affinity Purification of Proteins in Saccharomyces cerevisiae.

Isolation of endogenous proteins from Saccharomyces cerevisiae has been facilitated by inserting encoding polypeptide affinity tags at the C-termini of chromosomal open reading frames (ORFs) using homologous recombination of DNA fragments. Tagged protein isolation is limited by a number of factors, including high cost of affinity resins for bulk isolation and low concentration of ligands on the resin surface, leading to low isolation efficiencies and trapping of contaminants. To address this, we have created a recombinant "CelTag" DNA construct from which PCR fragments can be created to easily tag C-termini of S. cerevisiae ORFs using selection for a nat1 marker. The tag has a C-terminal cellulose binding module to be used in the first affinity step. Microgranular cellulose is very inexpensive and has an effectively continuous ligand on its surface, allowing rapid, highly efficient purification with minimal background. Cellulose-bound proteins are released by specific cleavage of an included site for TEV protease, giving nearly pure product. The tag can be lifted from the recombinant DNA construct either with or without a 13x myc epitope tag between the target ORF and the TEV protease site. Binding of CelTag protein fusions to cellulose is stable to high salt, nonionic detergents, and 1 M urea, allowing stringent washing conditions to remove loosely associated components, as needed, before specific elution. It is anticipated that this reagent could allow isolation of protein complexes from large quantities of yeast extract, including soluble, membrane-bound, or nucleic acid-associated assemblies.