G4 DNA unwinding by BLM and Sgs1p: substrate specificity and substrate-specific inhibition.
To understand the specific genetic instabilities associated with deficiencies in RecQ family helicases, we have studied the substrate preferences of two closely related members of this family, human BLM and Saccharomyces cerevisiae Sgs1p. Here we show that both BLM and Sgs1p preferentially unwind G4 DNA relative to Holliday junction substrates, and that substrate preference reflects binding affinity and maps to the conserved central helicase domain. We identify the porphyrin N-methyl mesoporphyrin IX (NMM) as a specific inhibitor of G4 DNA unwinding, and show that in the presence of NMM the helicase becomes trapped on the NMM-G4 DNA complex, consuming ATP but unable to unwind or dissociate. These results suggest that BLM and Sgs1p function proactively in replication to remove G4 DNA structures which would otherwise present obstacles to fork progression, rather than by promoting recombination to restart a fork that has stalled.
Huber
Michael D
MD
Department of Biochemistry, University of Washington Medical School, 1959 NE Pacific Street, Seattle, WA 98195-7650, USA.
Lee
Damian C
DC
Maizels
Nancy
N
D000251:Adenosine Triphosphatases / Q000378:metabolism*; D001667:Binding, Competitive / Q000187:drug effects; D004247:DNA / Q000737:chemistry / Q000378:metabolism*; D004265:DNA Helicases / Q000378:metabolism*; D006801:Humans; D008652:Mesoporphyrins / Q000378:metabolism / Q000494:pharmacology; D009690:Nucleic Acid Conformation; D053484:RecQ Helicases; D012441:Saccharomyces cerevisiae / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins; D013379:Substrate Specificity
D008652:Mesoporphyrins; D029701:Saccharomyces cerevisiae Proteins; C031468:mesoporphyrin IX; D004247:DNA; D000251:Adenosine Triphosphatases; C097151:Bloom syndrome protein; C090472:SGS1 protein, S cerevisiae; D004265:DNA Helicases; D053484:RecQ Helicases