Proliferating Cell Nuclear Antigen Uses Two Distinct Modes to Move along DNA
2009; Elsevier BV; Volume: 284; Issue: 26 Linguagem: Inglês
10.1074/jbc.m109.008706
ISSN1083-351X
AutoresAnna B. Kochaniak, Satoshi Habuchi, Joseph J. Loparo, Debbie J. Chang, Karlene A. Cimprich, Johannes C. Walter, Antoine M. van Oijen,
Tópico(s)DNA and Nucleic Acid Chemistry
ResumoProliferating cell nuclear antigen (PCNA) plays an important role in eukaryotic genomic maintenance by topologically binding DNA and recruiting replication and repair proteins. The ring-shaped protein forms a closed circle around double-stranded DNA and is able to move along the DNA in a random walk. The molecular nature of this diffusion process is poorly understood. We use single-molecule imaging to visualize the movement of individual, fluorescently labeled PCNA molecules along stretched DNA. Measurements of diffusional properties as a function of viscosity and protein size suggest that PCNA moves along DNA using two different sliding modes. Most of the time, the clamp moves while rotationally tracking the helical pitch of the DNA duplex. In a less frequently used second mode of diffusion, the movement of the protein is uncoupled from the helical pitch, and the clamp diffuses at much higher rates. Proliferating cell nuclear antigen (PCNA) plays an important role in eukaryotic genomic maintenance by topologically binding DNA and recruiting replication and repair proteins. The ring-shaped protein forms a closed circle around double-stranded DNA and is able to move along the DNA in a random walk. The molecular nature of this diffusion process is poorly understood. We use single-molecule imaging to visualize the movement of individual, fluorescently labeled PCNA molecules along stretched DNA. Measurements of diffusional properties as a function of viscosity and protein size suggest that PCNA moves along DNA using two different sliding modes. Most of the time, the clamp moves while rotationally tracking the helical pitch of the DNA duplex. In a less frequently used second mode of diffusion, the movement of the protein is uncoupled from the helical pitch, and the clamp diffuses at much higher rates. The proliferating cell nuclear antigen (PCNA) 3The abbreviations used are: PCNAproliferating cell nuclear antigenRFCreplication factor CMSDmean square displacementLMWVlow molecular weight viscogenHMWVhigh molecular weight viscogenPEGpolyethylene glycolCHAPS3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acidQDotquantum dot. 3The abbreviations used are: PCNAproliferating cell nuclear antigenRFCreplication factor CMSDmean square displacementLMWVlow molecular weight viscogenHMWVhigh molecular weight viscogenPEGpolyethylene glycolCHAPS3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acidQDotquantum dot. is a homotrimeric, ring-shaped protein that forms a closed circle around double-stranded DNA. The protein serves as a processivity factor for the eukaryotic replicative polymerases δ and ϵ by tethering them to the DNA (1Garg P. Burgers P.M. Crit. Rev. Biochem. Mol. Biol. 2005; 40: 115-128Crossref PubMed Scopus (213) Google Scholar). Additionally, PCNA interacts with a large number of replication, repair, and signaling factors to coordinate enzymatic processes at sites of replication and repair (2Moldovan G.L. Pfander B. Jentsch S. Cell. 2007; 129: 665-679Abstract Full Text Full Text PDF PubMed Scopus (1326) Google Scholar). This recruitment of nucleic-acid enzymes to a topological clamp around the DNA is a strategy employed in organisms ranging from bacteriophage to humans. The remarkable similarity of the ring-shaped structures of the Escherichia coli and bacteriophage T4 sliding clamps to PCNA underscores the evolutionary success of this molecular approach. proliferating cell nuclear antigen replication factor C mean square displacement low molecular weight viscogen high molecular weight viscogen polyethylene glycol 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid quantum dot. proliferating cell nuclear antigen replication factor C mean square displacement low molecular weight viscogen high molecular weight viscogen polyethylene glycol 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid quantum dot. PCNA is a homotrimer consisting of 37-kDa subunits, each of which comprises two similar globular domains. The PCNA monomers are arrange in head-to-tail fashion, forming a ring with pseudo 6-fold symmetry. The central channel has a diameter of 34 Å, large enough to accommodate double-stranded DNA (3Krishna T.S. Kong X.P. Gary S. Burgers P.M. Kuriyan J. Cell. 1994; 79: 1233-1243Abstract Full Text PDF PubMed Scopus (752) Google Scholar, 4Gulbis J.M. Kelman Z. Hurwitz J. O'Donnell M. Kuriyan J. Cell. 1996; 87: 297-306Abstract Full Text Full Text PDF PubMed Scopus (642) Google Scholar). PCNA forms stable ring-shaped trimers in solution (5Schurtenberger P. Egelhaaf S.U. Hindges R. Maga G. Jónsson Z.O. May R.P. Glatter O. Hübscher U. J. Mol. Biol. 1998; 275: 123-132Crossref PubMed Scopus (58) Google Scholar) that need to be opened to load onto DNA (6Burgers P.M. Yoder B.L. J. Biol. Chem. 1993; 268: 19923-19926Abstract Full Text PDF PubMed Google Scholar). The clamp loader, replication factor C (RFC), mediates the assembly of PCNA onto DNA at primer-template junctions (7Tsurimoto T. Stillman B. J. Biol. Chem. 1991; 266: 1950-1960Abstract Full Text PDF PubMed Google Scholar) or at nicks in the DNA backbone (8Podust L.M. Podust V.N. Sogo J.M. Hübscher U. Mol. Cell. Biol. 1995; 15: 3072-3081Crossref PubMed Scopus (95) Google Scholar) in a process that is dependent on ATP. The PCNA·DNA complex is very stable, exhibiting a half-life of tens of minutes (9Yao N. Turner J. Kelman Z. Stukenberg P.T. Dean F. Shechter D. Pan Z.Q. Hurwitz J. O'Donnell M. Genes Cells. 1996; 1: 101-113Crossref PubMed Scopus (181) Google Scholar, 10Podust V.N. Podust L.M. Müller F. Hübscher U. Biochemistry. 1995; 34: 5003-5010Crossref PubMed Scopus (43) Google Scholar). Although the interactions of various replication and repair proteins with PCNA are well studied (recently reviewed in Ref. 2Moldovan G.L. Pfander B. Jentsch S. Cell. 2007; 129: 665-679Abstract Full Text Full Text PDF PubMed Scopus (1326) Google Scholar), the interactions between PCNA and DNA are less well understood. Structural studies reveal that the central channel of the clamp is lined with highly conserved, positively charged residues (3Krishna T.S. Kong X.P. Gary S. Burgers P.M. Kuriyan J. Cell. 1994; 79: 1233-1243Abstract Full Text PDF PubMed Scopus (752) Google Scholar, 4Gulbis J.M. Kelman Z. Hurwitz J. O'Donnell M. Kuriyan J. Cell. 1996; 87: 297-306Abstract Full Text Full Text PDF PubMed Scopus (642) Google Scholar). Mutational analysis indicates that these residues may be more important for PCNA loading onto DNA than for sliding along DNA (11Fukuda K. Morioka H. Imajou S. Ikeda S. Ohtsuka E. Tsurimoto T. J. Biol. Chem. 1995; 270: 22527-22534Abstract Full Text Full Text PDF PubMed Scopus (151) Google Scholar). Molecular dynamics simulations suggest that the positively charged residues interact with the phosphodiester backbone but that these interactions are highly dynamic and are frequently displaced by ions from solution (12Ivanov I. Chapados B.R. McCammon J.A. Tainer J.A. Nucleic Acids Res. 2006; 34: 6023-6033Crossref PubMed Scopus (65) Google Scholar). The ring structure of PCNA and its relatively weak interaction with DNA allow it to move along the DNA in a diffusive fashion. Early work showed that PCNA can only be stably trapped on linear DNA when bound to polymerase δ on a primer-template (13Ng L. McConnell M. Tan C.K. Downey K.M. Fisher P.A. J. Biol. Chem. 1993; 268: 13571-13576Abstract Full Text PDF PubMed Google Scholar). Further evidence for PCNA moving along DNA stems from a study, which measured UV cross-linking of PCNA to a chemically modified, double-stranded DNA template. On circular DNA, a high degree of cross-linking was observed, but linearization of the template dramatically reduced cross-linking, suggesting a rapid dissociation of PCNA from the DNA ends (14Tinker R.L. Kassavetis G.A. Geiduschek E.P. EMBO J. 1994; 13: 5330-5337Crossref PubMed Scopus (57) Google Scholar). In the absence of a more direct way to assess the motion of PCNA along DNA, it has been difficult to study the molecular nature of the PCNA·DNA interactions and to understand how translocation of the sliding clamp occurs. In recent years, single-molecule techniques have been used to study diffusion of a variety of proteins along DNA. The earliest studies focused on the E. coli RNA polymerase, which is suggested to diffuse along DNA to find promoters (15Kabata H. Kurosawa O. Arai I. Washizu M. Margarson S.A. Glass R.E. Shimamoto N. Science. 1993; 262: 1561-1563Crossref PubMed Scopus (240) Google Scholar, 16Harada Y. Funatsu T. Murakami K. Nonoyama Y. Ishihama A. Yanagida T. Biophys. J. 1999; 76: 709-715Abstract Full Text Full Text PDF PubMed Scopus (237) Google Scholar, 17Kim J.H. Larson R.G. Nucleic Acids Res. 2007; 35: 3848-3858Crossref PubMed Scopus (90) Google Scholar). Other proteins studied include lac repressor (18Wang Y.M. Austin R.H. Cox E.C. Phys. Rev. Lett. 2006; 97 (048302)Google Scholar) and p53 (19Tafvizi A. Huang F. Leith J.S. Fersht A.R. Mirny L.A. van Oijen A.M. Biophys. J. 2008; 95: L01-L03Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar); the DNA damage surveillance and repair proteins oxoguanine DNA glycosylase 1 (hOgg1) (20Blainey P.C. van Oijen A.M. Banerjee A. Verdine G.L. Xie X.S. Proc. Natl. Acad. Sci. U.S.A. 2006; 103: 5752-5757Crossref PubMed Scopus (371) Google Scholar), Rad51 (21Granéli A. Yeykal C.C. Robertson R.B. Greene E.C. Proc. Natl. Acad. Sci. U.S.A. 2006; 103: 1221-1226Crossref PubMed Scopus (140) Google Scholar), and Msh2-Msh6 (22Gorman J. Chowdhury A. Surtees J.A. Shimada J. Reichman D.R. Alani E. Greene E.C. Mol. Cell. 2007; 28: 359-370Abstract Full Text Full Text PDF PubMed Scopus (181) Google Scholar); and the herpes simplex virus processivity factor UL42 (23Komazin-Meredith G. Mirchev R. Golan D.E. van Oijen A.M. Coen D.M. Proc. Natl. Acad. Sci. U.S.A. 2008; 105: 10721-10726Crossref PubMed Scopus (59) Google Scholar). These studies allowed for a detailed biophysical characterization of diffusive protein motion along DNA and revealed a number of different mechanisms of translocation (24Gorman J. Greene E.C. Nat. Struct. Mol. Biol. 2008; 15: 768-774Crossref PubMed Scopus (222) Google Scholar). Here we use a single-molecule approach to show that PCNA diffuses along DNA using two distinct modes. In one diffusion mode, the clamp tracks the helical pitch of the DNA duplex, resulting in a rotational movement of the protein around the DNA. In the second mode, the protein undergoes a faster, predominantly translational motion. We speculate how these two diffusive mechanisms contribute to the activity of the different classes of proteins that are tethered to DNA by PCNA. The human PCNA open reading frame was amplified from Int pET19pps (kind gift from Dr. Tom Ellenberger) and was cloned into pET28b between the NdeI and BamHI restriction sites. The N-terminally hexahistidine-tagged protein was overexpressed in E. coli BL21(DE3) cells, purified over a nickel-nitrilotriacetic acid column (Qiagen), dialyzed into Storage Buffer (50 mm Tris, pH 8, 50 mm NaCl, 1 mm EDTA, 1 mm dithiothreitol, and 10% glycerol) and stored at −80 °C. Purified human RFC complex with an N-terminal RFC1 deletion, which increases PCNA loading efficiency, was a kind gift from Dr. Jerard Hurwitz (25Cai J. Uhlmann F. Gibbs E. Flores-Rozas H. Lee C.G. Phillips B. Finkelstein J. Yao N. O'Donnell M. Hurwitz J. Proc. Natl. Acad. Sci. U.S.A. 1996; 93: 12896-12901Crossref PubMed Scopus (71) Google Scholar, 26Uhlmann F. Cai J. Gibbs E. O'Donnell M. Hurwitz J. J. Biol. Chem. 1997; 272: 10058-10064Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar). The purified PCNA was labeled by coupling AlexaFluor 555-maleimide (Invitrogen) to solvent-exposed cysteine groups on the protein. The human PCNA monomer contains six cysteine residues of which two are surface-exposed (PDB ID: 1AXC) (4Gulbis J.M. Kelman Z. Hurwitz J. O'Donnell M. Kuriyan J. Cell. 1996; 87: 297-306Abstract Full Text Full Text PDF PubMed Scopus (642) Google Scholar), resulting in a total of six solvent-accessible cysteines per trimer. The labeling reaction was performed following the manufacturer's suggested protocol. Briefly, PCNA aliquots were thawed and dialyzed overnight at 4 degrees into labeling buffer (50 mm Hepes, pH 7.4, 50 mm NaCl). After reducing the protein with 10 mm Tris(2-carboxyethyl)phosphine, dye was added at a ratio of 10 dyes per PCNA trimer, and the reaction was allowed to proceed for 2 h at 22 °C. Finally, the reaction was quenched with 2 mm β-mercaptoethanol, and free dye was separated from labeled protein using a size exclusion column (PD10, Amersham Biosciences). Labeling stoichiometry was assessed using UV-visible spectrophotometry and determined to be 0.7 ± 0.3 AlexaFluor 555 per PCNA trimer. Fractions were aliquoted and stored at −80 °C. Full-length Xenopus laevis PCNA (X. laevis PCNA) was cloned and expressed as described previously (27Chang D.J. Lupardus P.J. Cimprich K.A. J. Biol. Chem. 2006; 281: 32081-32088Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar). The His6-X. laevis PCNA was used to generate polyclonal antibodies in rabbits (Josman, LLC). High speed supernatant (egg cytosolic) extract was prepared from X. laevis eggs as described previously (28Walter J. Sun L. Newport J. Mol. Cell. 1998; 1: 519-529Abstract Full Text Full Text PDF PubMed Scopus (220) Google Scholar). Endogenous PCNA was depleted to below 0.25% with a rabbit polyclonal anti-X. laevis PCNA antibody (characterized in supplemental Fig. S1A). The extent of depletion was determined by Western blotting with anti-PCNA mouse monoclonal antibody PC10 (Santa Cruz Biotechnology). Purified and labeled human PCNA was reintroduced to the depleted extract at a concentration of 1 μm monomers, and primer extension was measured. Extracts were supplemented with an ATP-regeneration system (2 mm ATP, 20 mm phosphocreatine, 5 μg/ml creatine kinase (all from Sigma)), 15 μg/ml nocodazole (Sigma), and [α-32P]dATP (PerkinElmer Life Sciences). M13mp18 single-stranded DNA (New England Biolabs) was added at 15 ng/μl extract. Incorporation of radioactivity was measured after running the replication products on a 0.8% agarose-TBE gel. Imaging buffers were based on the work of Ellison and Stillman (29Ellison V. Stillman B. PLoS Biol. 2003; 1: E33Crossref PubMed Scopus (287) Google Scholar). To obtain buffers with varying concentrations of potassium glutamate, stocks of Buffer A (50 mm Hepes, pH 7.5, 7 mm MgCl2, 1 mm CHAPS, 1 mg/ml bovine serum albumin, 1 mm dithiothreitol, 0.001% Nonidet P-40) with 0 mm potassium glutamate and 500 mm potassium glutamate were prepared and mixed to achieve the desired salt concentration. The total ionic strength I of the buffers was calculated by using, l=12∑n=1NCnZ2(eq. 1) where c is the molar concentration of ion n, z is the charge number of that ion, and the sum is taken over all ions n. At pH 7.5, 47% of the 50 mm Hepes (free acid; pKa = 7.55) in our solutions will be charged (z2 = 1), resulting in an ionic strength of 12 mm. The sodium hydroxide used to achieve a pH of 7.5 contributes ∼13 mm to the ionic strength. Similarly, 7 mm MgCl2 adds 21 mm ionic strength (for the divalent magnesium ions, z2 = 4). In the absence of any potassium glutamate, these contributions add up to an ionic strength of 41 mm. Addition of potassium glutamate will change the ionic strength accordingly (z = 1). Viscous sliding buffers were based on Buffer B (50 mm Hepes, pH 7.5, 7 mm MgCl2, 150 mm potassium glutamate) with glycerol, or PEG6000 added by weight to achieve the desired viscosity (5*ηwater or 10*ηwater) (47Sheely M.L. Ind. Eng. Chem. 1932; 24: 1060-1064Crossref Scopus (191) Google Scholar, 49Mei L.H. Lin D.Q. Zhu Z.Q. Han Z.X. J. Chem. Eng. Data. 1995; 40: 1168-1171Crossref Scopus (71) Google Scholar). Streptavidin-coated flow cells were constructed as reported before (20Blainey P.C. van Oijen A.M. Banerjee A. Verdine G.L. Xie X.S. Proc. Natl. Acad. Sci. U.S.A. 2006; 103: 5752-5757Crossref PubMed Scopus (371) Google Scholar, 30Ha T. Rasnik I. Cheng W. Babcock H.P. Gauss G.H. Lohman T.M. Chu S. Nature. 2002; 419: 638-641Crossref PubMed Scopus (394) Google Scholar, 31Lee J.B. Hite R.K. Hamdan S.M. Xie X.S. Richardson C.C. van Oijen A.M. Nature. 2006; 439: 621-624Crossref PubMed Scopus (226) Google Scholar) with a flow-channel width of 1.5 mm and height of 0.12 mm. The inlet tubing had an inner diameter of 0.38 mm (PE20 from BD Biosciences) and 10-cm length to minimize the dead volume. Biotin-lambda-biotin DNA was prepared by ligating custom, 12-mer oligonucleotides with 3′Biotin modification (IDT) (BL1, 5′-AGGTCGCCGCCC-Biotin-3′; and BL2, 5′-GGGCGGCGACT-Biotin-3′) to their complementary, single-stranded ends of lambda DNA (PerkinElmer Life Sciences). To allow single-molecule observations of protein sliding along stretched DNA in the absence of hydrodynamic flow, we assembled stretched DNA onto the surface of the flow cell with both DNA ends coupled to the surface. To achieve this double tethering, 30 pm biotin-lambda-biotin DNA was flow stretched in Buffer A with 35 mm potassium glutamate. After the association of the first biotin with the streptavidin-coated surface, the DNA was stretched by the flow and the second biotin bound to the surface. By changing the flow rate that was applied when introducing the DNA into the flow cell, the length distribution of the DNA could be controlled (results not shown). To visualize DNA, we flow in a 100 nm solution of Sytox Orange (Invitrogen), a DNA intercalating dye, in Buffer A and image with 521 nm laser excitation at variable power. We only stain the DNA at the end of the experiment after PCNA sliding trajectories have been collected. The results described in the main text are obtained on DNA that was immobilized to the surface using a flow rate of 0.05 ml/min, resulting in a mean end-to-end distance of 11.5 ± 0.1 μm (corresponding to 70.3 ± 0.5% of the contour length of lambda-phage B-DNA) for DNA molecules upon which PCNA sliding was measured (see supplemental Fig. S4B). Typically, we observed 10–25 doubly tethered DNA molecules per 80 × 80 μm2 field of view. The typical single-molecule PCNA loading reaction contained 0.4 nm RFC (1/1000 dilution of stock immediately prior to use), 1 nm PCNA (1/2000 dilution immediately prior to use), and 1 mm ATP in 100 μl of Buffer A with 35 mm potassium glutamate. This reaction was drawn into the flow cell containing doubly tethered DNA molecules, was allowed to incubate for 20 min at room temperature, and then was flushed out with 40 flow-cell volumes of Buffer A with 500 mm potassium glutamate. Next, the buffer of interest was exchanged into the flow cell for 40 flow-cell volumes, flow was stopped, and event acquisition would begin. Typically, 0–3 PCNA molecules were observed per doubly tethered DNA molecule. QDots (605 nm) were functionalized with mouse monoclonal anti-histidine tag antibody (MCA1396 from AbD Serotec) using the Invitrogen QDot Antibody Conjugation Kit. For PCNA·QDot experiments, AlexaFluor 555- or mock-labeled PCNA was loaded as usual except that the initial wash included ∼1 nm anti-his QDots for 10 min. Next, the flow cell was washed with 40–100 flow cell volumes of Buffer A + 500 mm potassium glutamate to remove free QDots. Finally, the buffer of interest was exchanged into the flow cell for 40 flow-cell volumes, flow was stopped, and image acquisition was started. Fluorescence imaging of AlexaFluor 555-PCNA and PCNA·QDot complexes moving along DNA or QDot bound to DNA was performed as before (19Tafvizi A. Huang F. Leith J.S. Fersht A.R. Mirny L.A. van Oijen A.M. Biophys. J. 2008; 95: L01-L03Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar). The AlexaFluor 555 dye and QDot (605 nm) emission were excited by the 521-nm line from an Ar/Kr laser (Coherent I-70 Spectrum), and fluorescence was collected by an EM-CCD (Andor iXon) after filtering scattered laser light (Chroma Technology). Typically, 2.5- to 5-fold less power was used for QDot imaging than for AlexaFluor 555 imaging. Typical frame rates were 19 Hz for AlexaFluor 555-PCNA and 4 Hz for PCNA·QDot. Data were analyzed by custom-written particle-tracking MATLAB® code (19Tafvizi A. Huang F. Leith J.S. Fersht A.R. Mirny L.A. van Oijen A.M. Biophys. J. 2008; 95: L01-L03Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar). The positions of labeled particles were determined by fitting each single-molecule fluorescence image to a two-dimensional Gaussian distribution. This procedure allows the determination of a particle's position with a precision that can be better than the diffraction-limited optical resolution of the microscope used. The accuracy of position determination strongly depends on the signal strength corresponding to a single molecule and is given by, σ2=[s2N+a2/12N+8πs4b2a2N2](eq. 2) where N is the number of photons collected (32Thompson R.E. Larson D.R. Webb W.W. Biophys. J. 2002; 82: 2775-2783Abstract Full Text Full Text PDF PubMed Scopus (1831) Google Scholar). Typical signals from individual AlexaFluor 555 and QDot labels corresponded to 1000 ± 500 and 6000 ± 3000 photons per 50-ms integration. Using the standard deviation of the microscope point-spread function s (150 nm), the pixel size a (166 nm), and the background level b (13–19 photons for AlexaFluor 555 imaging and 4–12 photons for QDot imaging), we calculate the standard error of position determination to be σ = 12 nm for AlexaFluor 555-PCNA and σ = 2.2 nm for PCNA·QDot. Using the experimentally obtained trajectories of individual sliding particles, we determined the diffusion coefficient D of each particle by plotting its mean square displacement (MSD) as a function of step interval m times the time interval, Δt, MSD(M,m)=∑i=1M−m(yi+m−yi)2M−m(eq. 3) Here, M represents the total number of steps in the trajectory, m ranges from 1 to M, and yi is the displacement of the particle along the axis of the DNA in step i. Effectively, MSD(M,m) is the average of the squared displacements of the particle in the trajectory with length M, calculated from all pairs of positions that are m steps apart. According to the one-dimensional diffusion equation MSD(M,m) = 2DmΔt, we can obtain D by fitting the resulting data to a straight line using a weighted least-square fitting procedure. We measure the diffusion coefficient of the PCNA as half of the slope of the line fit to the MSD versus mΔt between steps m = 2 through m = 10. The lower end of this range was chosen, because many trajectories exhibit nonlinear behavior in the first step likely due to DNA fluctuations. The top of the range was limited by the shortest trajectories we accepted for fitting. Particles that were apparently immobile on the DNA or that showed bounded diffusion in the time window under consideration were excluded from analysis. Since bounded diffusion is apparent as a nonlinearity of the MSD versus mΔt, we report only particles for which the MSD(M,m) was correlated to mΔt with a Pearson correlation coefficient of 0.9 or higher. Using MATLAB, we simulated a one-dimensional random walk of 100 steps, each consisting of the average of 100 sub-steps with normally distributed step sizes. Sub-steps were used to better approximate the averaging inherent to imaging a single molecule over a finite window of time (the exposure time Δt). Next, a single parameter was used to scale the step size distribution of the particles to a desired diffusion coefficient. As we did with our experimentally observed diffusion trajectories, we fit the mean-square displacement MSD versus time mΔt of these sample trajectories in the range of m = 2 through 10. Finally, we simulated one-dimensional diffusion trajectories of a stepper changing randomly between two different diffusion coefficients on timescales faster than Δt. We used two different step sizes as the only two parameters to scale the trajectories. The two step sizes were chosen such that their resultant diffusion coefficients matched the theoretical limits for helical and non-helical diffusion of PCNA along DNA. Subsequently, we used a probability fhel for the stepper to use the step size associated with helical diffusion (and a probability (1 − fhel) to use the step size corresponding to non-helical diffusion). fhel was varied from 0 (100% maximal non-helical diffusion) to 1 (100% maximal helical diffusion). The mean diffusion coefficient from 100 of these 100-step trajectories was observed to vary linearly with fhel. To visualize the movement of PCNA along DNA, a cysteine-reactive organic dye (AlexaFluor 555-maleimide) was used to fluorescently label His-tagged PCNA at one of its native cysteines. Human PCNA monomers have six cysteine residues of which only two, C27 and C62, are solvent exposed (PD BID: 1AXC) (4Gulbis J.M. Kelman Z. Hurwitz J. O'Donnell M. Kuriyan J. Cell. 1996; 87: 297-306Abstract Full Text Full Text PDF PubMed Scopus (642) Google Scholar). Their positions are in loops distant from the DNA-interaction surface, making it unlikely that labeling would interfere with protein movement along DNA. Spectrophotometry performed after labeling revealed the presence of 0.7 ± 0.3 AlexaFluor 555 per PCNA trimer (see "Experimental Procedures"). To confirm that labeling did not inhibit PCNA function, we performed a primer extension assay using X. laevis egg extracts. A high speed supernatant of Xenopus egg cytoplasm supports PCNA-dependent replication of single-stranded DNA (33Méchali M. Harland R.M. Cell. 1982; 30: 93-101Abstract Full Text PDF PubMed Scopus (86) Google Scholar). Upon depletion of the endogenous X. laevis PCNA to <0.25% (supplemental Fig. S1A), primer extension on M13 single-stranded DNA was reduced ∼30-fold. Supplementing the depleted extracts with mock-labeled or AlexaFluor 555-labeled PCNA restored synthesis to 40% of the level seen in mock depleted extract (supplemental Fig. S1, B and C). This result shows that AlexaFluor 555-PCNA is able to support processive DNA synthesis just as actively as unmodified PCNA. To track fluorescently labeled PCNA on DNA, we stretched and immobilized λ phage DNA on the glass surface of a microfluidic flow cell. To this end, we functionalized both ends of linearized λ DNA with biotin to allow binding to a streptavidin-coated surface. Introducing the biotinylated λ DNA into the flow cell at high flow rates caused the DNA to bind to the surface via streptavidin in a stretched state, with a length corresponding to ∼70% of its contour length. To minimize nonspecific interactions between the surface and protein or DNA, the glass was chemically functionalized with high molecular weight, biotinylated poly(ethylene glycol) (PEG) on top of which the streptavidin was deposited (30Ha T. Rasnik I. Cheng W. Babcock H.P. Gauss G.H. Lohman T.M. Chu S. Nature. 2002; 419: 638-641Crossref PubMed Scopus (394) Google Scholar). PCNA was loaded onto the stretched λ DNA in the flow cell. PCNA requires RFC and ATP for loading around DNA in the absence of DNA ends (6Burgers P.M. Yoder B.L. J. Biol. Chem. 1993; 268: 19923-19926Abstract Full Text PDF PubMed Google Scholar, 7Tsurimoto T. Stillman B. J. Biol. Chem. 1991; 266: 1950-1960Abstract Full Text PDF PubMed Google Scholar, 8Podust L.M. Podust V.N. Sogo J.M. Hübscher U. Mol. Cell. Biol. 1995; 15: 3072-3081Crossref PubMed Scopus (95) Google Scholar, 34Fien K. Stillman B. Mol. Cell. Biol. 1992; 12: 155-163Crossref PubMed Scopus (190) Google Scholar). We reduced the concentrations of both RFC and PCNA in the loading reaction to limit the number of PCNA trimers loaded per λ DNA to less than one. After loading is complete, the RFC clamp loader releases from the DNA-bound clamp (35Podust V.N. Tiwari N. Stephan S. Fanning E. J. Biol. Chem. 1998; 273: 31992-31999Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar, 36Gomes X.V. Burgers P.M.J. J. Biol. Chem. 2001; 276: 34768-34775Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar, 37Gomes X.V. Schmidt S.L. Burgers P.M. J. Biol. Chem. 2001; 276: 34776-34783Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar). However, to ensure that the molecules visualized were PCNA alone and not PCNA bound by the clamp loader, we washed the flow cell with 0.5 m potassium glutamate buffer to remove any residual RFC complexes or incompletely loaded PCNA (8Podust L.M. Podust V.N. Sogo J.M. Hübscher U. Mol. Cell. Biol. 1995; 15: 3072-3081Crossref PubMed Scopus (95) Google Scholar). Furthermore, omission of ATP in the washing and imaging steps prevents residual RFC from binding to DNA or PCNA (36Gomes X.V. Burgers P.M.J. J. Biol. Chem. 2001; 276: 34768-34775Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar). Following these stringent washing steps, individual fluorescent complexes could be observed moving along the DNA for several hours. When ATP or RFC was omitted from the loading reaction, no sliding events were observed (data not shown). Taken together, these observations suggest successful loading and observation of individual fluorescently labeled PCNA complexes on DNA. Once PCNA was loaded onto the stretched λ DNA, it was visualized using wide-field fluorescence microscopy. The fluorescence of individual, labeled PCNA molecules was imaged as a function of time with a charge-coupled device camera. Fig. 1A shows a representative time series of images of a single molecule of PCNA moving along λ DNA. The position of the clamp on the DNA through time was determined using particle-tracking algorithms (see "Experimental Procedures"). Fitting every diffraction-limited, fluorescence image with a two-dimensional Gaussian function determined the position of the protein. The precision of position determination is determined by the total amount of signal collected from a single molecule and can be much better than the resolution of th
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