ATP-dependent translocation of proteins along single-stranded DNA: Models and methods of analysis of pre-steady state kinetics

Processive DNA helicases are able to translocate along single-stranded DNA (ssDNA) with biased directionality in a nucleoside triphosphate-dependent reaction, although translocation is not generally sufficient for helicase activity. An understanding of the mechanism of protein translocation along ssDNA requires pre-steady state transient kinetic experiments. Although ensemble experimental approaches have been developed recently for the study of translocation of proteins along DNA, quantitative analysis of the complete time-courses from these experiments, which is needed to obtain quantitative estimates of translocation kinetic parameters (rate constants, processivity, step sizes and ATP coupling) has been lacking. We discuss three ensemble transient kinetic experiments that can be used to study protein translocation along ssDNA, along with the advantages and limitations of each approach. We further describe methods to analyze the complete kinetic time-courses obtained from such experiments performed with a series of ssDNA lengths under single-round conditions (i.e. in the absence of re-binding of dissociated protein to DNA). These analysis methods utilize a sequential n-step model for protein translocation along ssDNA and enable quantitative determinations of the rate constant, processivity and step size for translocation through global non-linear least-squares fitting of the full time-courses. (C) 2004 Elsevier Ltd. All rights reserved.