Roberto Galletto

Diffusion of human Replication Protein A along single stranded DNA

Replication protein A (RPA) is a eukaryotic single-stranded DNA (ssDNA) binding protein that plays critical roles in most aspects of genome maintenance, including replication, recombination and repair. RPA binds ssDNA with high affinity, destabilizes DNA secondary structure and facilitates binding of other proteins to ssDNA. However, RPA must be removed from or redistributed along ssDNA during these processes. To probe the dynamics of RPA-DNA interactions, we combined ensemble and single-molecule fluorescence approaches to examine human RPA (hRPA) diffusion along ssDNA and find that an hRPA heterotrimer can diffuse rapidly along ssDNA. Diffusion of hRPA is functional in that it provides the mechanism by which hRPA can transiently disrupt DNA hairpins by diffusing in from ssDNA regions adjacent to the DNA hairpin. hRPA diffusion was also monitored by the fluctuations in fluorescence intensity of a Cy3 fluorophore attached to the end of ssDNA. Using a novel method to calibrate the Cy3 fluorescence intensity as a function of hRPA position on the ssDNA, we estimate a one-dimensional diffusion coefficient of hRPA on ssDNA of D1~5000nt(2) s(-1) at 37°C. Diffusion of hRPA while bound to ssDNA enables it to be readily repositioned to allow other proteins access to ssDNA.

E. coli DNA associated with isolated Hfq interacts with Hfq's distal surface and C-terminal domain

The RNA-binding protein Hfq has been studied extensively for its function as a modulator of gene expression at the post-transcriptional level. While most Hfq studies have focused on the proteins interaction with sRNAs and mRNAs, Hfq binding to DNA has been observed but is less explored. During the isolation of Hfq from Escherichiacoli, we found genomic DNA fragments associated with the protein after multiple steps of purification. Sequences of 41 amplified segments from the DNA fragments associated with Hfq were determined. A large fraction of the DNA segments were predicted to have significant helical axis curvature and were from genes associated with membrane proteins, characteristics unexpected for non-specific binding. Analysis by analytical ultracentrifugation indicated that rA(18) binding to Hfq disrupts Hfq-DNA interactions. The latter observation suggests Hfq binding to DNA involves its distal surface. This was supported by a gel mobility shift assay that showed single amino acid mutations on the distal surface of Hfq inhibited Hfq binding to duplex DNA, while six of seven mutations on the proximal surface and outer circumference of the hexamer did not prevent Hfq binding. Two mutated Hfq which have portions of their C-terminal domain removed also failed to bind to DNA. The apparent K(d) for binding wild type Hfq to several duplex DNA was estimated from a gel mobility shift assay to be ~400nM.

DNA Binding Induces Dimerization of Saccharomyces cerevisiae Pif1

In Saccharomyces cerevisiae, Pif1 is involved in a wide range of DNA transactions. It operates both in mitochondria and in the nucleus, where it has telomeric and non-telomeric functions. All of the activities of Pif1 rely on its ability to bind to DNA. We have determined the mode of Pif1 binding to different DNA substrates. While Pif1 is a monomer in solution, we show that binding of ssDNA to Pif1 induces protein dimerization. DNA-induced dimerization of Pif1 is also observed on tailed- and forked-dsDNA substrates, suggesting that on the latter formation of a Pif1 dimer prevents binding of additional Pif1 molecules. A dimer of Pif1 also forms on ssDNA of random composition and in the presence of saturating concentrations of nonhydrolyzable ATP analogues. The observation that a Pif1 dimer is formed on unwinding substrates in the presence of ATP analogues suggests that a dimeric form of the enzyme might constitute the pre-initiation complex leading to its unwinding activity.

Translocation of Saccharomyces cerevisiae Pif1 helicase monomers on single-stranded DNA

In Saccharomyces cerevisiae Pif1 participates in a wide variety of DNA metabolic pathways both in the nucleus and in mitochondria. The ability of Pif1 to hydrolyse ATP and catalyse unwinding of duplex nucleic acid is proposed to be at the core of its functions. We recently showed that upon binding to DNA Pif1 dimerizes and we proposed that a dimer of Pif1 might be the species poised to catalysed DNA unwinding. In this work we show that monomers of Pif1 are able to translocate on single-stranded DNA with 5′ to 3′ directionality. We provide evidence that the translocation activity of Pif1 could be used in activities other than unwinding, possibly to displace proteins from ssDNA. Moreover, we show that monomers of Pif1 retain some unwinding activity although a dimer is clearly a better helicase, suggesting that regulation of the oligomeric state of Pif1 could play a role in its functioning as a helicase or a translocase. Finally, although we show that Pif1 can translocate on ssDNA, the translocation profiles suggest the presence on ssDNA of two populations of Pif1, both able to translocate with 5′ to 3′ directionality.

Dynamics of gapped DNA recognition by human polymerase β

Kinetics of human polymerase β binding to gapped DNA substrates having single stranded (ss) DNA gaps with five or two nucleotide residues in the ssDNA gap has been examined, using the fluorescence stopped-flow technique. The mechanism of the recognition does not depend on the length of the ssDNA gap. Formation of the enzyme complex with both DNA substrates occurs by a minimum three-step reaction, with the bimolecular step followed by two isomerization steps. The results indicate that the polymerase initiates the association with gapped DNA substrates through the DNA-binding subsite located on the 8-kDa domain of the enzyme. This first association step is independent of the length of the ssDNA gap and is characterized by similar rate constants for both examined DNA substrates. The subsequent, first-order transition occurs at the rate of ∼600–1200 s−1. This is the major docking step accompanied by favorable free energy changes in which the 31-kDa domain engages in interactions with the DNA. The 5′-terminal PO 4 − group downstream from the primer is not a specific recognition element of the gap. However, the phosphate group affects the enzyme orientation in the complex with the DNA, particularly, for the substrate with a longer gap.

SSB-DNA binding monitored by fluorescence intensity and anisotropy.

Fluorescence methods have proven to be extremely useful tools for quantitative studies of the equilibria and kinetics of protein-DNA interactions. If the protein contains tryptophan (Trp), as is often the case, and there is a change in intrinsic Trp fluorescence of the protein, one can use this change in signal (quenching/enhancement) to monitor binding. One can also attach an extrinsic fluorophore to either the protein or the DNA and monitor binding due to a change in fluorescence intensity or a change in fluorescence anisotropy. Such equilibrium studies can provide important quantitative information on stoichiometries (occluded site size, number of binding sites) and energetics (affinities and cooperativities) of the interactions. This information is needed to understand the mechanisms of protein-DNA interactions. A critical aspect of such approaches for systems that have non-unity stoichiometries (e.g., a protein that binds multiple ligands) is knowledge of the relationship between the change in fluorescence signal (intensity or anisotropy) and the average extent of binding. Here we describe procedures for using fluorescence approaches to examine the stoichiometries and equilibrium binding affinities of Escherichia coli single-stranded DNA-binding protein (SSB) and Deinococcus radiodurans SSB with long polymeric ssDNA to determine an occluded site size. We also provide examples of studies of SSB binding to shorter oligonucleotides to demonstrate analysis and fitting of the data to an appropriate model (monitoring fluorescence intensity or anisotropy) to obtain quantitative estimates of equilibrium binding parameters. We emphasize that the solution conditions (especially salt concentration and type) can influence not only the binding affinity, but also the mode by which an SSB oligomer binds ssDNA.

Chemo-mechanical pushing of proteins along single-stranded DNA

Significance Cellular processes take place in dynamic, crowded environments. Single-stranded DNA binding (SSB) proteins are ubiquitous in cells, serving to protect the transient single-stranded (ss)DNA formed during DNA replication, recombination, and repair and recruit numerous other proteins to the ssDNA. SSBs must be displaced or otherwise moved in order for DNA to be replicated or repaired. The motor protein activity of ssDNA translocases could serve in this capacity to facilitate directional movement of SSBs along ssDNA. In this work, we show that high-affinity SSBs can be moved directionally along ssDNA and eventually displaced via the ATP-driven action of ssDNA translocases. This process occurs via nonspecific chemo-mechanical pushing of the SSB along the ssDNA in the direction of translocation. Single-stranded (ss)DNA binding (SSB) proteins bind with high affinity to ssDNA generated during DNA replication, recombination, and repair; however, these SSBs must eventually be displaced from or reorganized along the ssDNA. One potential mechanism for reorganization is for an ssDNA translocase (ATP-dependent motor) to push the SSB along ssDNA. Here we use single molecule total internal reflection fluorescence microscopy to detect such pushing events. When Cy5-labeled Escherichia coli (Ec) SSB is bound to surface-immobilized 3′-Cy3–labeled ssDNA, a fluctuating FRET signal is observed, consistent with random diffusion of SSB along the ssDNA. Addition of Saccharomyces cerevisiae Pif1, a 5′ to 3′ ssDNA translocase, results in the appearance of isolated, irregularly spaced saw-tooth FRET spikes only in the presence of ATP. These FRET spikes result from translocase-induced directional (5′ to 3′) pushing of the SSB toward the 3′ ssDNA end, followed by displacement of the SSB from the DNA end. Similar ATP-dependent pushing events, but in the opposite (3′ to 5′) direction, are observed with EcRep and EcUvrD (both 3′ to 5′ ssDNA translocases). Simulations indicate that these events reflect active pushing by the translocase. The ability of translocases to chemo-mechanically push heterologous SSB proteins along ssDNA provides a potential mechanism for reorganization and clearance of tightly bound SSBs from ssDNA.

Rat polymerase β gapped DNA interactions

The role of the 5′ terminal phosphate group downstream from the primer and magnesium cations in the energetics and dynamics of the gapped DNA recognition by rat polymerase β have been examined, using the fluorescence titration and stopped-flow techniques. The analyses have been performed with the entire series of gapped DNA substrates differing in the size of the ssDNA gap. The 5′ terminal phosphate group and magnesium cations exert antagonistic effect on enzyme binding to gapped DNA that depends on the length of the ssDNA gap. The PO4− group amplifies the differences between the substrates with different ssDNA gaps, while in the presence of magnesium, affinities and structural changes induced in the DNA are very similar among examined DNA substrates. Both, the phosphate group and Mg+2 differ dramatically in affecting the thermodynamic response of the gapped DNA-rat pol β system to the salt concentration. The data indicate that these distinct effects result from affecting the structure of the DNA, in the case of the phosphate group, and from direct magnesium binding to the protein. The mechanism of rat enzyme binding depends on the length of the ssDNA gap and the presence of the 5′ terminal phosphate group. Complex formation with DNAs having three, four, and five residues in the gap occurs by a minimum three-step sequential mechanism. Depending on the presence of the 5′ terminal phosphate group and/or magnesium, binding of the enzyme to a DNA containing two residues in the ssDNA gap is described by the same three-step or by a simpler two-step mechanism. With the DNA containing only one residue in the gap, binding is always described by only a two-step mechanism. The PO4− group and magnesium cations have opposite effects on internal stability of the complexes with different length of the ssDNA gap. While the PO4− group increases the stability of internal intermediates with the increasing length of the gap, Mg+2 decreases the stability of the intermediates with longer ssDNA gap. As a result, the combined favorable orientation effect of the phosphate group and the unfavorable Mg+2 effect lead to the optimal docking of the ssDNA gaps with three and four residues by the enzyme.

Pif1 removes a Rap1-dependent barrier to the strand displacement activity of DNA polymerase δ

Using an in vitro reconstituted system in this work we provide direct evidence that the yeast repressor/activator protein 1 (Rap1), tightly bound to its consensus site, forms a strong non-polar barrier for the strand displacement activity of DNA polymerase δ. We propose that relief of inhibition may be mediated by the activity of an accessory helicase. To this end, we show that Pif1, a 5′–3′ helicase, not only stimulates the strand displacement activity of Pol δ but it also allows efficient replication through the block, by removing bound Rap1 in front of the polymerase. This stimulatory activity of Pif1 is not limited to the displacement of a single Rap1 molecule; Pif1 also allows Pol δ to carry out DNA synthesis across an array of bound Rap1 molecules that mimics a telomeric DNA-protein assembly. This activity of Pif1 represents a novel function of this helicase during DNA replication.

Regulation of yeast DNA polymerase δ-mediated strand displacement synthesis by 5′-flaps

The strand displacement activity of DNA polymerase δ is strongly stimulated by its interaction with proliferating cell nuclear antigen (PCNA). However, inactivation of the 3′–5′ exonuclease activity is sufficient to allow the polymerase to carry out strand displacement even in the absence of PCNA. We have examined in vitro the basic biochemical properties that allow Pol δ-exo− to carry out strand displacement synthesis and discovered that it is regulated by the 5′-flaps in the DNA strand to be displaced. Under conditions where Pol δ carries out strand displacement synthesis, the presence of long 5′-flaps or addition in trans of ssDNA suppress this activity. This suggests the presence of a secondary DNA binding site on the enzyme that is responsible for modulation of strand displacement activity. The inhibitory effect of a long 5′-flap can be suppressed by its interaction with single-stranded DNA binding proteins. However, this relief of flap-inhibition does not simply originate from binding of Replication Protein A to the flap and sequestering it. Interaction of Pol δ with PCNA eliminates flap-mediated inhibition of strand displacement synthesis by masking the secondary DNA site on the polymerase. These data suggest that in addition to enhancing the processivity of the polymerase PCNA is an allosteric modulator of other Pol δ activities.