Molecular mechanisms underlying the deoxyribonucleic acid helicase activity of Escherichia coli RecQ

RecQ helicases catalyze critical genome maintenance reactions and play key roles in DNA metabolism. Although their precise cellular roles remain elusive, the importance of RecQ proteins is clear; mutations in any of three human RecQ genes lead to genomic instability and cancer. In Chapter 2 of this thesis, the role of the RecQ-conserved-domain in E. coli RecQ stability and function is examined. From this analysis, I concluded that RecQ binds a Zn2+ ion using a conserved cysteine motif located within the RecQ-conserved-domain and that this Zn2+-binding-domain is an important folding determinant for RecQ.;As helicases, RecQ proteins use the energy of ATP hydrolysis to drive DNA unwinding; however, the mechanisms by which RecQ links ATPase activity to DNA binding/unwinding are unknown. In Chapter 3, the role of a conserved aromatic-rich-loop (ARL) in the ability of RecQ to couple DNA binding to ATP hydrolysis is examined. Single amino acid substitutions made within this loop lead to a decrease in coupling stringency, indicating that the RecQ ARL is critical for coupling the helicase's activities.;Although they are superfamily-2 (SF-2) helicases, RecQ proteins also exhibit similarities to SF-1 helicases. Both SF-1 and -2 helicases use the energy of ATP hydrolysis to drive DNA unwinding; however, the mechanisms by which members of each superfamily accomplish this coupling differ. In Chapter 4 of this thesis, I test the hypothesis that RecQ uses its ARL as a DNA sensor to communicate DNA binding to two other conserved helicase motifs (motifs II and VI), which directly facilitate ATP hydrolysis. My results support a model in which these three components work together in RecQ to couple DNA binding, ATP hydrolysis, and DNA translocation in a hybrid of SF-1 and -2 helicase mechanisms.;RecQ family helicases play important roles at the intersection of DNA replication, recombination, and repair, and this thesis contributes to the growing body of research surrounding the founding member of the family, E. coli RecQ. Understanding the role of the Zn2+-binding-domain in RecQ stability, as well as defining details of its helicase mechanism, provides a clearer view of RecQ's role in genome maintenance in E. coli..