Author's School

Graduate School of Arts & Sciences

Author's Department/Program

Biology and Biomedical Sciences: Biochemistry

Language

English (en)

Date of Award

January 2010

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Chair and Committee

Timothy Lohman

Abstract

This thesis presents mechanistic studies of the E. coli RecBC and RecBCD helicases using transient-state kinetic approaches to understand the relationship between DNA unwinding and ssDNA translocation. RecBC initiates unwinding from duplex DNA ends with pre-existing 5'-(dT)6 and 3'-(dT)6 ssDNA tails using a series of repeated rate-limiting steps with a rate of 345 ┬▒ 8 bp/sec and an average kinetic step-size of ~4 bp: 20 mM Mops-KOH, pH 7.0 at 25°C, 30 mM NaCl, 10 mM MgCl2, 5% glycerol, 1 mM 2-mercaptoethanol) while RecBCD unwinds these same DNA substrates with a rate of 745 ┬▒ 18 bp/sec using a more complicated mechanism which involves the loading of the 5' ssDNA end onto the RecD subunit. After DNA unwinding, RecBC can continue to translocate along a ssDNA extension in the 3' to 5' direction with a rate of 909 ┬▒ 51 nt/sec, consistent with the directionality of the RecB motor subunit. Surprisingly, RecBC also possesses a secondary translocase activity that enables it to move along a ssDNA extension of the opposite DNA strand with a similar rate: 990 ┬▒ 49 nt/sec). Both translocase activities are coupled to ATP hydrolysis from the RecB motor, and the primary translocase is sensitive to the polarity of the ssDNA backbone while the secondary translocase is not. RecBC can also translocate along two non-complementary ssDNA extensions simultaneously using both translocase activities. During DNA unwinding, RecBC consumes an average of 0.95 ┬▒ 0.08 ATP/bp unwound. The primary translocase activity utilizes 0.81 ┬▒ 0.05 ATP/nt translocated while its secondary translocase activity is less tightly coupled and requires 1.12 ┬▒ 0.06 ATP/nt. Translocation along two non-complementary ssDNA extensions has an ATP coupling stoichiometry of 1.07 ┬▒ 0.09 ATP/nt. These data indicate that the large majority, possibly all, of the ATP hydrolyzed by RecBC during DNA unwinding is used to fuel RecBC translocation along the nucleic acid rather than to facilitate base pair melting. These results suggest that RecBC uses a two step active mechanism to unwind DNA by first destabilizing the duplex using its binding free energy in an ATP-independent process, followed by ATP-dependent translocation along the resulting ssDNA.

Comments

Permanent URL: http://dx.doi.org/10.7936/K7BV7DMN

Download

Share

COinS