Aaron L. Lucius

General Methods for Analysis of Sequential “n-step” Kinetic Mechanisms: Application to Single Turnover Kinetics of Helicase-Catalyzed DNA Unwinding

Helicase-catalyzed DNA unwinding is often studied using all or none assays that detect only the final product of fully unwound DNA. Even using these assays, quantitative analysis of DNA unwinding time courses for DNA duplexes of different lengths, L, using n-step sequential mechanisms, can reveal information about the number of intermediates in the unwinding reaction and the kinetic step size, m, defined as the average number of basepairs unwound between two successive rate limiting steps in the unwinding cycle. Simultaneous nonlinear least-squares analysis using n-step sequential mechanisms has previously been limited by an inability to float the number of unwinding steps, n, and m, in the fitting algorithm. Here we discuss the behavior of single turnover DNA unwinding time courses and describe novel methods for nonlinear least-squares analysis that overcome these problems. Analytic expressions for the time courses, f(ss)(t), when obtainable, can be written using gamma and incomplete gamma functions. When analytic expressions are not obtainable, the numerical solution of the inverse Laplace transform can be used to obtain f(ss)(t). Both methods allow n and m to be continuous fitting parameters. These approaches are generally applicable to enzymes that translocate along a lattice or require repetition of a series of steps before product formation.

DNA Unwinding Step-size of E. coli RecBCD Helicase Determined from Single Turnover Chemical Quenched-flow Kinetic Studies

The mechanism by which Escherichia coli RecBCD DNA helicase unwinds duplex DNA was examined in vitro using pre-steady-state chemical quenched-flow kinetic methods. Single turnover DNA unwinding experiments were performed by addition of ATP to RecBCD that was pre-bound to a series of DNA substrates containing duplex DNA regions ranging from 24 bp to 60 bp. In each case, the time-course for formation of completely unwound DNA displayed a distinct lag phase that increased with duplex length, reflecting the transient formation of partially unwound DNA intermediates during unwinding catalyzed by RecBCD. Quantitative analysis of five independent sets of DNA unwinding time courses indicates that RecBCD unwinds duplex DNA in discrete steps, with an average unwinding step-size, m=3.9(+/-1.3)bp step(-1), with an average unwinding rate of k(U)=196(+/-77)steps s(-1) (mk(U)=790(+/-23)bps(-1)) at 25.0 degrees C (10mM MgCl(2), 30 mM NaCl (pH 7.0), 5% (v/v) glycerol). However, additional steps, not linked directly to DNA unwinding are also detected. This kinetic DNA unwinding step-size is similar to that determined for the E.coli UvrD helicase, suggesting that these two SF1 superfamily helicases may share similar mechanisms of DNA unwinding.

DNA helicases, motors that move along nucleic acids: Lessons from the SF1 helicase superfamily

Publisher Summary This chapter focuses on the mechanistic aspects of superfamily 1 (SFl) DNA helicases. Helicases are allosteric enzymes, many of which are known to function as oligomeric assemblies. Such oligomerization is exemplified by the class of hexameric DNA helicases, including the Escherichia coli DnaB helicase, the phage T7 gene 4 helicase, and the SV40 large T antigen. The initial characterizations of a DNA helicase generally include studies of the features of DNA substrates that are required for efficient unwinding by the helicase. With few exceptions, DNA helicases show a preference for unwinding duplex DNA possessing an single-stranded (ss)-DNA flanking region or tail in vitro . In fact, the unwinding reaction generally displays a defined polarity of unwinding with respect to the backbone polarity of the ss-DNA tail that flanks the duplex DNA. Two operational classes of helicases are helicases that initiate unwinding more efficiently on DNA substrates with a 3-ss-DNA tail and are referred to as “3 to 5 helicases,” whereas those that prefer DNA substrates possessing a 5-ss-DNA tail are referred to as “5 to 3 helicases.”

An Examination of the Kinetic Mechanism of the ATP-Dependent Protease ClpAP

Motor proteins perform functions within the cell by converting the energy of ATP into mechanical work. One such example of this can be found in the ATP-dependent protease from Escherichia coli, ClpAP, which is assembled from two distinct enzymes, a protein unfoldase, ClpA, and a protease, ClpP. The biologically active complex functions through a coordinated action in which ClpA is responsible for enzyme catalyzed protein unfolding and ATP-dependent polypeptide translocation, while ClpP will proteolytically degrade polypeptide substrates that have been translocated into its central cavity. Without such systems, deleterious effects are often observed within the cell as a result of either the accumulation of misfolded proteins or from unregulated activity from partially synthesized proteins.Of interest is the kinetic mechanism of ClpAP, and more specifically, the possibility of allosteric effects of ClpP upon ClpA. Kinetic parameters such as the step-size of polypeptide translocation, processvity, macroscopic rate, and microscopic rate constants have been measured for ClpA in the absence of ClpP under single-turnover conditions, but quantitative estimates of these parameters have not been determined in the presence of the proteolytic component, ClpP. In an effort to address this issue, single-turnover chemical quenched-flow methods in conjunction with stopped-flow fluorescence techniques have been used to examine the molecular mechanism of ClpAP catalyzed polypeptide translocation and degradation. The single-turnover methods are performed with enzyme in large excess over substrates and allow for the observation of the conversion of substrates into intermediates or products in a single cycle of catalysis. We have shown that the kinetic parameters are dramatically different in the presence of the proteolytic component and ClpP has a clear allosteric effect on the mechanism.

Structural and Functional Studies of the E. Coli ClpA Molecular Motor

ATP dependent proteases, such as the E. coli ClpAP and eukaryotic 26 S proteasome are critical components of protein quality control pathways. These proteases have the responsibility of removing misfolded proteins that can occur during heat shock or stress. ClpAP is composed of a tetradecameric serine protease, ClpP (21.6 kDa monomer), and either the hexameric ClpA (84.2 kDa monomer) or ClpX (46.2 kDa monomer) ATPase/protein unfoldase. In addition to its proteolytic activity, ClpA has protein remodeling activity and therefore, in the absence of ClpP, is considered a molecular chaperone. From sequence analysis, ClpA has been found to be a member of the ATPases Associated with various Activities (AAA+) family of proteins. This family of proteins includes a number of oligomeric chaperones and some DNA helicases. Here we report a rigorous investigation of the self association properties of the E. coli ClpA chaperone, including the ligand linked assembly of the structure active in polypeptide translocation. This has been done by employing sedimentation velocity, sedimentation equilibrium and dynamic light scattering experiments. We also employ rapid mixing kinetic approaches to examine the polypeptide translocation activities of ClpA and ClpAP. Thus far we have shown that ClpA translocates from the carboxy- to amino-terminus without dissociating under single-turnover conditions and thus we consider ClpA to be a processive and directional polypeptide translocase. In addition, we will discuss the first determination of a kinetic step-size for a polypeptide translocase, where the kinetic step-size is defined as the number of amino acids translocated per repeating step.