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Small-molecule sensitization of RecBCD helicase-nuclease to a Chi hotspot-activated state.
Nucleic Acids Res. 2020 Jun 29 [Online ahead of print]NA

Abstract

Coordinating 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' GCTGGTGG 3'), RecB's nuclease cuts the 3'-ended strand and loads RecA strand-exchange protein onto it. We report 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 relative to RecD and sensitizes it to cut DNA when the leading helicase RecD stops at the DNA end. Two previously described RecBCD mutants altered in the RecB ATP-binding site also have this property, but uninhibited wild-type RecBCD lacks it. ATP and NSAC1003 are competitive; computation docks NSAC1003 into RecB's ATP-binding site, suggesting NSAC1003 acts directly on RecB. NSAC1003 will help elucidate molecular mechanisms of RecBCD-Chi regulation and DNA repair. Similar studies could help elucidate other DNA enzymes with activities coordinated at chromosomal sites.

Authors+Show Affiliations

Division of Basic Sciences, Fred Hutchinson Cancer Research Center, Seattle, WA, USA.Achaogen, Inc., San Francisco, CA, USA.Division of Basic Sciences, Fred Hutchinson Cancer Research Center, Seattle, WA, USA.Division of Basic Sciences, Fred Hutchinson Cancer Research Center, Seattle, WA, USA.

Pub Type(s)

Journal Article

Language

eng

PubMed ID

32597964

Citation

Karabulut, Ahmet C., et al. "Small-molecule Sensitization of RecBCD Helicase-nuclease to a Chi Hotspot-activated State." Nucleic Acids Research, 2020.
Karabulut AC, Cirz RT, Taylor AF, et al. Small-molecule sensitization of RecBCD helicase-nuclease to a Chi hotspot-activated state. Nucleic Acids Res. 2020.
Karabulut, A. C., Cirz, R. T., Taylor, A. F., & Smith, G. R. (2020). Small-molecule sensitization of RecBCD helicase-nuclease to a Chi hotspot-activated state. Nucleic Acids Research. https://doi.org/10.1093/nar/gkaa534
Karabulut AC, et al. Small-molecule Sensitization of RecBCD Helicase-nuclease to a Chi Hotspot-activated State. Nucleic Acids Res. 2020 Jun 29; PubMed PMID: 32597964.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Small-molecule sensitization of RecBCD helicase-nuclease to a Chi hotspot-activated state. AU - Karabulut,Ahmet C, AU - Cirz,Ryan T, AU - Taylor,Andrew F, AU - Smith,Gerald R, Y1 - 2020/06/29/ PY - 2020/06/10/accepted PY - 2020/05/22/revised PY - 2020/04/06/received PY - 2020/6/30/entrez JF - Nucleic acids research JO - Nucleic Acids Res. N2 - Coordinating 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' GCTGGTGG 3'), RecB's nuclease cuts the 3'-ended strand and loads RecA strand-exchange protein onto it. We report 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 relative to RecD and sensitizes it to cut DNA when the leading helicase RecD stops at the DNA end. Two previously described RecBCD mutants altered in the RecB ATP-binding site also have this property, but uninhibited wild-type RecBCD lacks it. ATP and NSAC1003 are competitive; computation docks NSAC1003 into RecB's ATP-binding site, suggesting NSAC1003 acts directly on RecB. NSAC1003 will help elucidate molecular mechanisms of RecBCD-Chi regulation and DNA repair. Similar studies could help elucidate other DNA enzymes with activities coordinated at chromosomal sites. SN - 1362-4962 UR - https://www.unboundmedicine.com/medline/citation/32597964/Small-molecule_sensitization_of_RecBCD_helicase-nuclease_to_a_Chi_hotspot-activated_state L2 - https://academic.oup.com/nar/article-lookup/doi/10.1093/nar/gkaa534 DB - PRIME DP - Unbound Medicine ER -
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