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Hopping of a Processivity Factor on DNA Revealed by Single-Molecule Assays of Diffusion

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Hopping of a Processivity Factor on DNA Revealed by Single-Molecule Assays of Diffusion Hopping of a processivity factor on DNA revealed by single-molecule assays of diffusion Gloria Komazin-Meredith*, Rossen Mirchev*, David E. Golan, Antoine M. van Oijen, and Donald M. Coen† Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, 250 Longwood Avenue, Boston, MA 02115 Edited by Charles C. Richardson, Harvard Medical School, Boston, MA, and approved May 16, 2008 (received for review March 17, 2008) Many DNA-interacting proteins diffuse on DNA to perform their sliding clamp (8)]. Indeed, biochemical studies have shown that biochemical functions. Processivity factors diffuse on DNA to per- sliding clamps can diffuse on DNA (9, 10); however, to our mit unimpeded elongation by their associated DNA polymerases, knowledge, their rates and mechanisms of diffusion have not but little is known regarding their rates and mechanisms of been identified. Similarly, HSV UL42, despite its tight binding to diffusion. The processivity factor of herpes simplex virus DNA DNA, does not slow the elongation of the viral polymerase (5) polymerase, UL42, unlike ‘‘sliding clamp’’ processivity factors that and does diffuse on DNA (11). Its diffusion coefficient (D) on normally form rings around DNA, binds DNA directly and tightly as DNA has been estimated to be Ϸ10 bp2/s from ensemble studies a monomer, but can still diffuse on DNA. To investigate the measuring the dependence of the off-rate of UL42 from DNA mechanism of UL42 diffusion on DNA, we examined the effects of of different lengths (7, 11). This value is consistent with the rate salt concentration on diffusion coefficient. Ensemble studies, em- of HSV polymerase translocation (5, 7). However, as noted in ploying electrophoretic mobility shift assays on relatively short ref. 11, this estimate was based on an indirect analysis that relied DNAs, showed that off-rates of UL42 from DNA depended on DNA on several assumptions. length at higher but not lower salt concentrations, consistent with Berg et al. (12) have defined two major mechanisms of the diffusion coefficient being salt-dependent. Direct assays of the diffusion on DNA, three-dimensional and one-dimensional. In motion of single fluorescently labeled UL42 molecules along DNA three-dimensional diffusion, proteins dissociate from DNA and revealed increased diffusion at higher salt concentrations. Remark- rebind a distance away on the same DNA or on another DNA ably, the diffusion coefficients observed in these assays were in a manner that is not positionally correlated; i.e., not adjacent Ϸ104-fold higher than those calculated from ensemble experi- to or near the starting point of diffusion. The folding of DNA in ments. Discrepancies between the single-molecule and ensemble solution increases the likelihood of three-dimensional diffusion results were resolved by the observation, in single-molecule ex- (12). By contrast, in one-dimensional or linear diffusion, which periments, that UL42 releases relatively slowly from the ends of is the mechanism that is relevant to processivity factor function, DNA in a salt-dependent manner. The results indicate that UL42 the protein samples sites on the DNA in a positionally correlated ‘‘hops’’ rather than ‘‘slides,’’ i.e., it microscopically dissociates from manner. Berg et al. describe two mechanisms of one-dimensional and reassociates with DNA as it diffuses rather than remaining so diffusion, ‘‘sliding’’ and ‘‘hopping,’’ and provide an experimental intimately associated with DNA that cation condensation on the test to distinguish these mechanisms (12). Sliding is defined as phosphate backbone does not affect its motion. These findings motion along the contour length of the DNA via transfer of may be relevant to mechanisms of other processivity factors and bound protein between linearly contiguous sites, implying a DNA-binding proteins. helical path of movement. Hopping is defined as microscopic dissociation of the protein from DNA to a point where the herpes simplex virus ͉ linear diffusion protein becomes free to move but can quickly and with high probability reassociate with the same or a nearby site. In effect, NA polymerases are central to DNA replication. Most the protein remains macroscopically bound but can test nearby Dreplicative DNA polymerases include accessory subunits binding sites through repeated microscopic dissociations. Mi- that promote replication of long stretches of DNA without croscopic dissociations can be defined as ones in which the dissociating from the template. The best known of these pro- protein is removed just far enough from DNA to permit recon- densation of cations onto the phosphate backbone of DNA. cessivity factors are the ‘‘sliding clamps’’ (reviewed in ref. 1), Thus, operationally, diffusion mediated by hopping is acceler- which include polymerase subunits of bacteria, eukaryotes, and ated by higher salt concentrations, whereas sliding is unaffected. archaea that form multimeric rings around DNA with the aid of Compared with our knowledge of processivity factors, we ATP-dependent clamp-loaders. These rings then tether their know much more about the mechanisms and rates of diffusion cognate catalytic subunits to DNA, permitting processive DNA on DNA of other DNA binding proteins. By the criteria de- synthesis. scribed above, certain of these proteins have been shown to slide A variety of cellular and viral polymerases include processivity on DNA by either indirect measurements or by direct single- subunits that do not use ATP or other proteins for loading onto molecule observations (e.g., refs. 13–15). Other proteins have DNA. Of these, herpes simplex virus (HSV) UL42 is one of the been shown to exhibit salt-dependent diffusion (e.g., ref. 16), but best characterized. This protein’s structure resembles that of a monomer of the sliding clamp proliferating cell nuclear antigen (2), yet UL42 binds directly to DNA as a monomer with Author contributions: G.K.-M., R.M., D.E.G., A.M.v.O., and D.M.C. designed research; relatively high affinity (apparent dissociation constant (Kd)in G.K.-M. and R.M. performed research; A.M.v.O. contributed new reagents/analytic tools; the nanomolar range) (3–5). This direct binding of DNA by G.K.-M., R.M., D.E.G., A.M.v.O., and D.M.C. analyzed data; and G.K.-M., R.M., D.E.G., UL42 tethers the catalytic subunit of HSV DNA polymerase A.M.v.O., and D.M.C. wrote the paper. (Pol) to DNA, thereby enabling processivity (3, 5–7). The authors declare no conflict of interest. An important attribute of processivity factors is their ability to This article is a PNAS Direct Submission. diffuse on DNA. Such diffusion permits tethering of the catalytic *G.K.-M. and R.M. contributed equally to this work. subunit without impeding translocation of the enzyme. The loose †To whom correspondence should be addressed. E-mail: don࿝[email protected]. topological association of sliding clamps with DNA, as opposed This article contains supporting information online at www.pnas.org/cgi/content/full/ to direct binding, is widely thought to permit diffusion [although 0802676105/DCSupplemental. a recent study has shown direct binding to DNA by the E. coli © 2008 by The National Academy of Sciences of the USA 10720–10725 ͉ PNAS ͉ August 5, 2008 ͉ vol. 105 ͉ no. 31 www.pnas.org͞cgi͞doi͞10.1073͞pnas.0802676105 Downloaded by guest on September 24, 2021 Fig. 1. Effect of salt concentration on DNA length dependence of UL42 dissociation from DNA. Half-lives of UL42-DNA complexes were measured by EMSAs ϭ 2 ϩ at 10 mM (A), 25 mM (B), and 50 mM (C) NaCl. Data were fitted using the equation t1͞2 ln 2/((12D/b ) koff(internal)), relating the length of DNA, b, and the half-life of the protein on DNA, t1͞2, to permit calculation of the diffusion coefficient (D) and koff(internal) (7) (continuous line). In A and C, data for krelease(ends) (Table 1) ϭ ⅐ ϩ Ϫ1 from single-molecule experiments at 10 mM (A) and 50 mM (C) NaCl were fitted using the equation t1͞2 ln2 (2krelease(ends)/b koff(internal)) , where the koff(internal) values were derived from either single-molecule studies (dotted line) or EMSA studies (dashed line). it has been difficult to distinguish between hopping and three- value calculated from data obtained at 75 mM NaCl and 3 mM dimensional diffusion events, especially on longer DNAs in MgCl2 (7). However, at 10 mM and 25 mM NaCl, no dependence solution (17, 18). In at least some cases, three-dimensional of D on DNA length could be ascertained (Fig. 1 A and B). This diffusion has been shown to dominate at lengths Ͼ50 bp, even result differs from previous observations at 10 mM NaCl and 3 at low ionic strengths (19). However, to our knowledge, no mM MgCl2 [ref. 11 and G.K.-M. and D.H.C., unpublished examples of direct observation of hopping at the single molecule observations), which can be attributed to the lack of MgCl2 in the level have been reported. Moreover, despite the terminology present assay. Using the equation above, we calculated from sliding clamps, to our knowledge, no processivity factor has been these data that D is Ͻ1bp2/s at 10 mM and 25 mM NaCl, and examined for its mechanism of diffusion thus at least 10 times lower than D at 50 mM NaCl, implying a To address the mechanism of one-dimensional diffusion by very strong dependence of diffusion on salt concentration. In UL42, we examined its diffusion in the presence of different salt contrast, only a Ͻ2-fold difference in koff(internal) between 25 and concentrations, first in ensemble studies employing relatively 50 mM NaCl was calculated from these assays (Table S1). In short DNAs, and then, to permit direct measurements in the these ensemble experiments, the strong dependence of D on absence of three-dimensional diffusion, using single-molecule NaCl concentration was consistent with UL42 diffusion on DNA methods on long-stretched DNAs.
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