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Using Microsecond Single-Molecule FRET to Determine the Assembly

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Using Microsecond Single-Molecule FRET to Determine the Assembly Using microsecond single-molecule FRET to determine PNAS PLUS the assembly pathways of T4 ssDNA binding protein onto model DNA replication forks Carey Phelpsa,b,1, Brett Israelsa,b, Davis Josea, Morgan C. Marsha,b, Peter H. von Hippela,2, and Andrew H. Marcusa,b,2 aDepartment of Chemistry and Biochemistry, Institute of Molecular Biology, University of Oregon, Eugene, OR 97403; and bDepartment of Chemistry and Biochemistry, Oregon Center for Optical, Molecular and Quantum Science, University of Oregon, Eugene, OR 97403 Edited by Stephen C. Kowalczykowski, University of California, Davis, CA, and approved March 20, 2017 (received for review December 2, 2016) DNA replication is a core biological process that occurs in pro- complete coverage of the exposed ssDNA templates at the precisely karyotic cells at high speeds (∼1 nucleotide residue added per regulated concentration of protein that needs to be maintained in millisecond) and with high fidelity (fewer than one misincorpora- the infected Escherichia coli cell (8, 9). The gp32 protein has an tion event per 107 nucleotide additions). The ssDNA binding pro- N-terminal domain, a C-terminal domain, and a core domain. tein [gene product 32 (gp32)] of the T4 bacteriophage is a central The N-terminal domain is necessary for the cooperative binding integrating component of the replication complex that must con- of the gp32 protein through its interactions with the core domain of tinuously bind to and unbind from transiently exposed template an adjacent gp32 protein. To bind to ssDNA, the C-terminal domain strands during DNA synthesis. We here report microsecond single- of the gp32 protein must undergo a conformational change that molecule FRET (smFRET) measurements on Cy3/Cy5-labeled primer- exposes the positively charged region of its core domain, which in template (p/t) DNA constructs in the presence of gp32. These mea- turn, interacts with the negatively charged ssDNA backbone. The surements probe the distance between Cy3/Cy5 fluorophores that binding site size of the gp32 protein is seven nucleotide residues label the ends of a short (15-nt) segment of ssDNA attached to a (10). Although many of the ssbs of higher organisms have larger model p/t DNA construct and permit us to track the stochastic in- binding footprints, the functional role of binding cooperativity in terconversion between various protein bound and unbound states. these systems is less straightforward. Thus, oligomers of E. coli ssb BIOPHYSICS AND The length of the 15-nt ssDNA lattice is sufficient to accommodate up to two cooperatively bound gp32 proteins in either of two can bind to ssDNA in more than one binding mode (11), whereas COMPUTATIONAL BIOLOGY positions. We apply a unique multipoint time correlation function mammalian replication protein As (RPAs) may bind and function as E. coli analysis to the microsecond-resolved smFRET data obtained to de- monomers (12). Both ssb and human RPA have been shown termine and compare the kinetics of various possible reaction path- to diffuse on ssDNA, and this sliding motion may be important in ways for the assembly of cooperatively bound gp32 protein onto discharging their functions (13, 14). Although sliding of the ssDNA sequences located at the replication fork. The results of our gp32 protein along the template strand is expected to be important analysis reveal the presence and translocation mechanisms of to its biological function, there is currently little direct information short-lived intermediate bound states that are likely to play a crit- availableabouttheroleofslidingintheprocessofgp32cluster ical role in the assembly mechanisms of ssDNA binding proteins at assembly on ssDNA template sequences during replication. replication forks and other ss duplex junctions. Significance microsecond single-molecule FRET | multidimensional time correlation functions | ssDNA binding protein A microsecond-resolved single-molecule FRET method was used to monitor the binding and unbinding of the ssDNA he DNA replication complex of the T4 bacteriophage is an binding protein (gene product 32) of the T4 bacteriophage Texcellent model to understand the mechanistic details of replication complex to biologically relevant primer-template DNA synthesis, because it uses the same three protein subas- DNA constructs. A unique multitime correlation function anal- semblies as found in higher organisms, albeit without the many ysis was applied to the resulting sparse data, which permitted additional layers of regulatory complexity (1–5). These subas- the investigation of the kinetics and mechanisms of non- semblies are (i) the helicase/primase (primosome) complex that cooperative and cooperative protein binding, unbinding, and unwinds the dsDNA genome and synthesizes pentameric RNA “sliding.” Our results indicate that noncooperatively bound primer strands while exposing the leading and lagging ssDNA monomer proteins dissociate on the timescale of tens of milli- templates, (ii) the DNA polymerases that use the exposed tem- seconds, which is consistent with the known rate of nucleotide plates to synthesize cDNA daughter strands, and (iii) the replica- addition during DNA replication. The rapid dissociation of the tion clamp–clamp loader complexes that load and unload the monomer suggests that sliding is a much more likely mecha- sliding clamps from the functioning polymerases and thereby, nism for translocation of cooperatively bound clusters of control the processivity of DNA synthesis. indeterminate size. An integral component of DNA replication is the ssDNA binding protein (ssb) (6, 7). The ssbs bind to the exposed ssDNA Author contributions: C.P., P.H.v.H., and A.H.M. designed research; C.P., B.I., D.J., and M.C.M. templates during the critical period after the helicase has un- performed research; D.J. contributed new reagents/analytic tools; C.P., B.I., M.C.M., and A.H.M. analyzed data; and C.P., B.I., D.J., P.H.v.H., and A.H.M. wrote the paper. wound these sequences and before the DNA polymerases have incorporated complementary paired nucleotides into the newly The authors declare no conflict of interest. formed daughter strands. The ssbs are thought to protect ssDNA This article is a PNAS Direct Submission. from nuclease activity and remove unfavorable secondary struc- 1Present address: Department of Biomedical Engineering, Knight Cancer Institute and Oregon Health and Science University Center for Spatial Systems Biomedicine, Oregon tures that would otherwise hinder the efficiency of the replication Health and Science University, Portland, OR 97201. process. The T4 ssb is referred to as the gene product 32 (gp32), 2To whom correspondence may be addressed. Email: [email protected] or ahmarcus@ and it is known to form association complexes with ssDNA uoregon.edu. through a cooperative binding mechanism. The cooperative This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. binding mode of gp32 is thought to be important to achieve 1073/pnas.1619819114/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1619819114 PNAS Early Edition | 1of10 Downloaded by guest on September 26, 2021 In this work, we use microsecond-resolved single-molecule that appear “sparse” and are dominated by stochastic noise when FRET (smFRET) experiments to study the kinetics and mech- viewed with high temporal resolution. Such trajectories are not anism of gp32 dimer assembly on a short 15-nt ssDNA template. amenable to analysis by “direct visualization methods,” which at- Such ssDNA-(gp32)2 complexes can serve as simple model sys- tempt to assign discrete smFRET efficiency values to corresponding tems to examine the basic biochemical steps involved in ssb fil- conformational states and thus, observe and resolve sequences of ament assembly and sliding. Our experiments used fluorescently state to state transitions that make up a biochemical reaction labeled primer-template (p/t) DNA constructs, in which a Cy3 pathway. For example, hidden Markov model (HMM) analysis is a donor chromophore was attached to the 3′ end of the template particularly useful method to evaluate smFRET trajectories that use strand and a Cy5 acceptor chromophore was attached to the 5′ tens of milliseconds resolution (19), but this approach becomes less end of the primer strand near the p/t junction (Fig. 1A). The accurate for microsecond-resolved experiments. template strand contains a 15-nt poly(deoxythymidine) [p(dT)15] In this work, we apply generalized concepts of time correlation sequence that can form an association complex with up to two functions (TCFs) to study the ssDNA-(gp32)2 assembly pathways cooperatively bound gp32 protein monomers at one time. Re- (20–30). We assume that our single-molecule experiments probe cently, Lee et al. (15) showed that smFRET signals observed the instantaneous conformational state of the system at equi- from this same p/t DNA construct in the presence of gp32 un- librium and that the stochastically fluctuating smFRET effi- dergo intermittent protein-induced fluctuations, which reflect ciency, EFRET, can be directly mapped onto this state. A TCF is a changes in the end to end distance of the ssDNA template time-dependent moment of the variable EFRETðtÞ that, when caused by binding and dissociation of gp32 proteins. used to its full potential, can provide a statistically meaningful The experiments performed by Lee et al. (15) were sensitive to way to characterize the dynamics of the interconverting species changes in ssDNA template conformation, which could be ob- lying along the reaction pathway. In general, the nth-order TCF, ðnÞ served using the tens of milliseconds time resolution of standard C ðτ1, τ2, ..., τn−1Þ, can be written as the average product of n smFRET experiments. Although these studies could distinguish successive observations hEFRET ðt1Þ, EFRETðt2Þ, ..., EFRET ðtnÞi,which between unbound template conformations and those with two depends on the n – 1 time intervals τ1 = t2 − t1, τ2 = t3 − t2, ..., cooperatively bound gp32 proteins, they could not clearly resolve τn−1 = tn − tn−1. The complexity of information available from a short-lived singly bound intermediate states.
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