A conformational switch in response to Chi converts RecBCD from phage destruction to DNA repair

Cheng, Kaiying; Wilkinson, M. K.; Chaban, Yuriy; Wigley, Dale B.

doi:10.1038/s41594-019-0355-2

Cited by 62 publications

(74 citation statements)

References 46 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…This interpretation accounts for the Chi-dependent band being much sharper than the NSAC1003-or RecB helicase mutant-dependent bands (Figure 3) (14). An alternative interpretation is that Chi may form a kinked configuration in the RecC tunnel (20) and slow or stop DNA translocation until the DNA is cut near Chi; if so, a longer nuclease swing-time would still result in cuts only a few nucleotides from the Chi sequence. In experiments without Chi, the DNA in the RecC tunnel would not typically be kinked when RecD reaches the DNA end, and NSAC1003-or helicase mutant-induced cuts would be spread over a larger region from the same nuclease swing-time.…”

Section: Our Results Presented Here Show a Remarkable Similarity Betwmentioning

confidence: 99%

See 1 more Smart Citation

Small-molecule sensitization of RecBCD helicase-nuclease to a Chi hotspot-activated state

Karabulut

Cirz²,

Smith

2020

Preprint

View full text Add to dashboard Cite

Coordination of multiple activities of complex enzymes is critical for life, including transcribing, replicating, and repairing DNA. Bacterial RecBCD helicase-nuclease must coordinate DNA unwinding and cutting to repair broken DNA. Starting at a DNA end, RecBCD unwinds DNA with its fast RecD helicase on the 5'-ended strand and its slower RecB helicase on the 3'-ended strand. At Chi hotspots (5'GCTGGTGG3'), RecB's nuclease cuts the 3'-ended strand and loads RecA strand-exchange protein onto it. We report here that a small molecule NSAC1003, a sulfanyltriazolobenzimidazole, mimics Chi sites by sensitizing RecBCD to cut DNA at a Chi-independent position a certain percent of the DNA substrate's length. This percent decreases with increasing NSAC1003 concentration. Our data indicate that NSAC1003 slows RecB and sensitizes it to cut DNA when the leading helicase RecD stops at the DNA distal end. Two previously described RecBCD mutants altered in the RecB ATP-binding site also have this property, but uninhibited wild-type RecBCD lacks it. Computation docks NSAC1003 into the ATPbinding site, suggesting that NSAC1003 acts directly on RecB. NSAC1003 will help elucidate the molecular mechanisms of RecBCD-Chi regulation and DNA repair. Similar studies could help elucidate other DNA enzymes whose activities are coordinated at chromosomal sites.

show abstract

Section: Our Results Presented Here Show a Remarkable Similarity Betwmentioning

confidence: 99%

“…In the current studies we noted that it induces RecBCD to cut DNA at a novel position that depends on the NSAC1003 concentration and on the length of the substrate. The two RecB helicase mutants are altered in single amino acids (Y803H and V804E) near RecB's ATP-binding site in a cryoEM structure (20) (see Discussion and Figure 5).…”

Section: Introductionmentioning

confidence: 99%

Small-molecule sensitization of RecBCD helicase-nuclease to a Chi hotspot-activated state

Karabulut

Cirz²,

Smith

2020

Preprint

View full text Add to dashboard Cite

show abstract

“…In the published crystal and cryoEM structures (Figs. 2A and 2B) [26,37,42], four contiguous amino acids (here named C2) in RecC make intimate contact with three contiguous amino acids (named D2) in RecD ( Fig. 2C; Table 1).…”

Section: Recc-recd Interaction For Model Step 2 -Recc Signals Recd Tomentioning

confidence: 99%

“…In the cryoEM structures of RecBCD bound to ds DNA [37,42], four contiguous amino acids with overall positive charge (QPSR in the center of D3) in RecD contact an α-helix of 13 amino acids (B3), six with negative charge (D or E), in RecB ( Fig. 2D; Tables 1 and 3).…”

Section: Recd-recb Interaction For Model Step 3 -Recd Signals Recb Tomentioning

confidence: 99%

See 1 more Smart Citation

Chi hotspot control of RecBCD helicase-nuclease by long-range intramolecular signaling

Amundsen

Taylor

Smith

2020

Preprint

View full text Add to dashboard Cite

Life-saving repair of broken DNA by homologous recombination requires coordinated enzymatic reactions to prepare it for interaction with intact DNA. The multiple activities of enterobacterial RecBCD helicase-nuclease are coordinated by Chi recombination hotspots (5' GCTGGTGG 3') recognized during DNA unwinding. Our previous studies led to a signal transduction model in which the Chi-bound RecC subunit signals RecD, a fast helicase, to stop; RecD then signals RecB, a slower helicase, to swing its nuclease domain into position to cut DNA at Chi and load RecA protein onto the newly generated 3'-ended single-stranded DNA. Here, we report a set of mutants blocked at each step in this pathway. The mutants have helicase and degradative nuclease activities, but most have lost or reduced coordination by Chi and have reduced recombination and DNA repair proficiency. The mutant alterations are widely scattered throughout the three subunits of RecBCD; each alters the intimate contact between two subunits in the crystal or cryoEM structures, supporting the signal transduction model. Similar large conformational changes in other vital enzymes, such as DNA and RNA polymerases, may make RecBCD a paradigm for elucidating their control by the nucleic acid sites they recognize. Author summaryRepair of broken DNA is essential for life. Faithful repair occurs by homologous genetic recombination, which also generates new gene allele combinations important for evolution. In bacteria the RecBCD helicase-nuclease enzyme is essential for most DNA break repair and is regulated by special DNA sites (Chi recombination hotspots). As it unwinds DNA, RecBCD's activities are dramatically altered at Chi to prepare the broken DNA for interaction with intact DNA to finish repair. Here, we describe RecBCD mutants that respond weakly, or not at all, to Chi; the alterations are scattered widely throughout the enzyme, supporting our previous model of large conformational changes of RecBCD at Chi. Similar methods may help understand how other large enzyme complexes are regulated.

show abstract

RecBCD Enzyme and Pathway

Smith

2024

Reference Module in Life Sciences

View full text Add to dashboard Cite

A conformational switch in response to Chi converts RecBCD from phage destruction to DNA repair

Cited by 62 publications

References 46 publications

Small-molecule sensitization of RecBCD helicase-nuclease to a Chi hotspot-activated state

Small-molecule sensitization of RecBCD helicase-nuclease to a Chi hotspot-activated state

Chi hotspot control of RecBCD helicase-nuclease by long-range intramolecular signaling

RecBCD Enzyme and Pathway

Contact Info

Product

Resources

About