Direct observation of the wrapping/unwrapping kinetics of ssDNA around SSB.  The Escherichia coli single stranded DNA binding (SSB) protein binds selectively to ssDNA intermediates during DNA replication, recombination and repair.  Each subunit of the homo-tetrameric protein contains a potential ssDNA binding site. It has been proposed that SSB, while bound to the ssDNA, could take advantage of its four potential binding sites and translocate along the ssDNA via a rolling mechanism. Since the protein cannot translocate any faster than the rate at which ssDNA wraps and unwraps from around SSB, a mechanistic picture of the rolling mechanism requires knowledge of the wrapping/unwrapping rates.

            In vitro, SSB binds to ssDNA in two or more distinct binding modes. In the fully wrapped (SSB)₆₅ mode, the two ends of a ~70-nt strand are expected to be in close contact (1). Thus, FRET measurements between donor and acceptor molecules attached at either end of the ~70-nt long ssDNA strand provide a sensitive probe of the wrapping and unwrapping kinetics (2). Previous stopped-flow studies of SSB binding to (dT)₇₀ were unable to resolve the bimolecular association step from the unimolecular wrapping/unwrapping step, due to limited time-resolution (2, 3). In my laboratory, we made the first direct observation of the wrapping/unwrapping kinetics, in response to a laser T-jump perturbation of a pre-formed SSB-ssDNA complex (4). Our measurements revealed that the wrapping/unwrapping process occurs on time-scales of tens of microseconds, which is fast enough not to be the rate-limiting step for processes such as DNA replication in E. coli, which proceed at much slower rates of ~1000 nucleotides/s. Thus, this study demonstrated that a rolling mechanism may provide SSB protein with the ability to rapidly move along ssDNA during DNA metabolic processes.

These kinetics measurements represent a novel application of T-jump techniques to extend the study of the dynamics of protein-DNA interactions to the microsecond-to-millisecond time-scales, and hold promise for similar studies on other systems. Our success in applying this technique to probe the dynamics of another protein-DNA complex is described below.

Direct observation of DNA bending/unbending kinetics in complex with DNA-bending protein IHF: Regulation of gene transcription frequently requires specific proteins that can kink, bend or curve DNA. In many protein-DNA complexes, the protein also undergoes conformational rearrangements that facilitate favorable interactions with DNA. These concerted changes in proteins and DNA are believed to be a key feature underlying the induced-fit mechanism proposed for the recognition of specific binding sites on DNA by proteins. The details of the recognition mechanism remain elusive. In particular, very little is known about the dynamics of the conformational rearrangements that lead to the precise recognition.

E. coli Integration Host Factor (IHF) is a member of a family of DNA-bending proteins that are ubiquitous in prokaryotes and aid in chromosomal compaction as well as in the assembly of higher-order nucleoprotein complexes necessary for replication initiation and site-specific recombination. Members of this family of proteins find their preferred sites by "indirect readout" of the DNA sequence.  Rather than direct interactions between the protein and specific DNA base-pairs, this mechanism relies on the sequence-dependence of the DNA's conformation and deformability. Thus, these proteins have become paradigms for investigating indirect readout mechanism for recognition of DNA binding sites as well as the mechanics of DNA bending.

Our laser T-jump measurements, in combination with stopped-flow measurements in the laboratory of Prof. Donald Crothers at Yale, provided the first direct observation of the bending/unbending kinetics of a cognate DNA sequence bound to IHF (5, 6). These measurements resolved a long-standing question in this field as to whether binding and bending of DNA by DNA-bending proteins occurs in a concerted manner, or sequentially. More interestingly, these measurements indicated that the rate-limiting step in the bending of DNA in the complex may well be spontaneous bending/kinking of the DNA, presumably occurring at the sites of kinks in the DNA that are observed in the crystal structure of the complex (6). Spontaneous bending/kinking of DNA, in the absence of a bound protein, has not been directly observed, although it has been implicated in recent studies, raising the question as to whether the DNA is capable of undergoing spontaneous fluctuations in which distorted conformations are energetically accessible.

1.         Raghunathan, S., Kozlov, A. G., Lohman, T. M. & Waksman, G. (2000) Nat. Struct. Biol. 7, 648-652.

2.         Kozlov, A. G. & Lohman, T. M. (2002) Biochemistry 41, 6032-6044.

3.         Kozlov, A. G. & Lohman, T. M. (2002) Biochemistry 41, 11611-11627.

4.         Kuznetsov, S. V., Kozlov, A. G., Lohman, T. M. & Ansari, A. (2006) J. Mol. Biol. 359, 55-65.

5.         Sugimura, S. & Crothers, D. M. (2006) Proc. Natl. Acad. Sci. U S A 103, 18510-4.

6.         Kuznetsov, S. V., Sugimura, S., Vivas, P., Crothers, D. M. & Ansari, A. (2006) Proc. Natl. Acad. Sci. U S A 103, 18515-20.