Rrm3 and Pif1 division of labor during replication through leading and lagging strand G-quadruplex.

Members of the conserved Pif1 family of 5'-3' DNA helicases can unwind G4s and mitigate their negative impact on genome stability. In Saccharomyces cerevisiae, two Pif1 family members, Pif1 and Rrm3, contribute to the suppression of genomic instability at diverse regions including telomeres, centromeres and tRNA genes. While Pif1 can resolve lagging strand G4s in vivo, little is known regarding Rrm3 function at G4s and its cooperation with Pif1 for G4 replication. Here, we monitored replication through G4 sequences in real time to show that Rrm3 is essential for efficient replisome progression through G4s located on the leading strand template, but not on the lagging strand. We found that Rrm3 importance for replication through G4s is dependent on its catalytic activity and its N-terminal unstructured region. Overall, we show that Rrm3 and Pif1 exhibit a division of labor that enables robust replication fork progression through leading and lagging strand G4s, respectively.

"Helicase" Activity promoted through dynamic interactions between a ssDNA translocase and a diffusing SSB protein.

Replication protein A (RPA) is a eukaryotic single-stranded (ss) DNA-binding (SSB) protein that is essential for all aspects of genome maintenance. RPA binds ssDNA with high affinity but can also diffuse along ssDNA. By itself, RPA is capable of transiently disrupting short regions of duplex DNA by diffusing from a ssDNA that flanks the duplex DNA. Using single-molecule total internal reflection fluorescence and optical trapping combined with fluorescence approaches, we show that S. cerevisiae Pif1 can use its ATP-dependent 5' to 3' translocase activity to chemomechanically push a single human RPA (hRPA) heterotrimer directionally along ssDNA at rates comparable to those of Pif1 translocation alone. We further show that using its translocation activity, Pif1 can push hRPA from a ssDNA loading site into a duplex DNA causing stable disruption of at least 9 bp of duplex DNA. These results highlight the dynamic nature of hRPA enabling it to be readily reorganized even when bound tightly to ssDNA and demonstrate a mechanism by which directional DNA unwinding can be achieved through the combined action of a ssDNA translocase that pushes an SSB protein. These results highlight the two basic requirements for any processive DNA helicase: transient DNA base pair melting (supplied by hRPA) and ATP-dependent directional ssDNA translocation (supplied by Pif1) and that these functions can be unlinked by using two separate proteins.

Branched unwinding mechanism of the Pif1 family of DNA helicases.

Members of the Pif1 family of helicases function in multiple pathways that involve DNA synthesis: DNA replication across G-quadruplexes; break-induced replication; and processing of long flaps during Okazaki fragment maturation. Furthermore, Pif1 increases strand-displacement DNA synthesis by DNA polymerase δ and allows DNA replication across arrays of proteins tightly bound to DNA. This is a surprising feat since DNA rewinding or annealing activities limit the amount of single-stranded DNA product that Pif1 can generate, leading to an apparently poorly processive helicase. In this work, using single-molecule Förster resonance energy transfer approaches, we show that 2 members of the Pif1 family of helicases, Pif1 from Saccharomyces cerevisiae and Pfh1 from Schizosaccharomyces pombe, unwind double-stranded DNA by a branched mechanism with 2 modes of activity. In the dominant mode, only short stretches of DNA can be processively and repetitively opened, with reclosure of the DNA occurring by mechanisms other than strand-switching. In the other less frequent mode, longer stretches of DNA are unwound via a path that is separate from the one leading to repetitive unwinding. Analysis of the kinetic partitioning between the 2 different modes suggests that the branching point in the mechanism is established by conformational selection, controlled by the interaction of the helicase with the 3' nontranslocating strand. The data suggest that the dominant and repetitive mode of DNA opening of the helicase can be used to allow efficient DNA replication, with DNA synthesis on the nontranslocating strand rectifying the DNA unwinding activity.

Pif1, RPA, and FEN1 modulate the ability of DNA polymerase δ to overcome protein barriers during DNA synthesis.

Successful DNA replication requires carefully regulated mechanisms to overcome numerous obstacles that naturally occur throughout chromosomal DNA. Scattered across the genome are tightly bound proteins, such as transcription factors and nucleosomes, that are necessary for cell function, but that also have the potential to impede timely DNA replication. Using biochemically reconstituted systems, we show that two transcription factors, yeast Reb1 and Tbf1, and a tightly positioned nucleosome, are strong blocks to the strand displacement DNA synthesis activity of DNA polymerase δ. Although the block imparted by Tbf1 can be overcome by the DNA-binding activity of the single-stranded DNA-binding protein RPA, efficient DNA replication through either a Reb1 or a nucleosome block occurs only in the presence of the 5'-3' DNA helicase Pif1. The Pif1-dependent stimulation of DNA synthesis across strong protein barriers may be beneficial during break-induced replication where barriers are expected to pose a problem to efficient DNA bubble migration. However, in the context of lagging strand DNA synthesis, the efficient disruption of a nucleosome barrier by Pif1 could lead to the futile re-replication of newly synthetized DNA. In the presence of FEN1 endonuclease, the major driver of nick translation during lagging strand replication, Pif1-dependent stimulation of DNA synthesis through a nucleosome or Reb1 barrier is prevented. By cleaving the short 5' tails generated during strand displacement, FEN1 eliminates the entry point for Pif1. We propose that this activity would protect the cell from potential DNA re-replication caused by unwarranted Pif1 interference during lagging strand replication.

Complementary roles of Pif1 helicase and single stranded DNA binding proteins in stimulating DNA replication through G-quadruplexes.

G-quadruplexes (G4s) are stable secondary structures that can lead to the stalling of replication forks and cause genomic instability. Pif1 is a 5' to 3' helicase, localized to both the mitochondria and nucleus that can unwind G4s in vitro and prevent fork stalling at G4 forming sequences in vivo. Using in vitro primer extension assays, we show that both G4s and stable hairpins form barriers to nuclear and mitochondrial DNA polymerases δ and γ, respectively. However, while single-stranded DNA binding proteins (SSBs) readily promote replication through hairpins, SSBs are only effective in promoting replication through weak G4s. Using a series of G4s with increasing stabilities, we reveal a threshold above which G4 through-replication is inhibited even with SSBs present, and Pif1 helicase is required. Because Pif1 moves along the template strand with a 5'-3'-directionality, head-on collisions between Pif1 and polymerase δ or γ result in the stimulation of their 3'-exonuclease activity. Both nuclear RPA and mitochondrial SSB play a protective role during DNA replication by preventing excessive DNA degradation caused by the helicase-polymerase conflict.

PCNA accelerates the nucleotide incorporation rate by DNA polymerase δ.

DNA polymerase delta (Pol δ) is responsible for the elongation and maturation of Okazaki fragments in eukaryotic cells. Proliferating cell nuclear antigen (PCNA) recruits Pol δ to the DNA and serves as a processivity factor. Here, we show that PCNA also stimulates the catalytic rate of Saccharomyces cerevisiae Pol δ by >10-fold. We determined template/primer DNA binding affinities and stoichiometries by Pol δ in the absence of PCNA, using electrophoretic mobility shift assays, fluorescence intensity changes and fluorescence anisotropy binding titrations. We provide evidence that Pol δ forms higher ordered complexes upon binding to DNA. The Pol δ catalytic rates in the absence and presence of PCNA were determined at millisecond time resolution using quench flow kinetic measurements. The observed rate for single nucleotide incorporation by a preformed DNA-Pol δ complex in the absence of PCNA was 40 s-1. PCNA enhanced the nucleotide incorporation rate by >10 fold. Compared to wild-type, a growth-defective yeast PCNA mutant (DD41,42AA) showed substantially less stimulation of the Pol δ nucleotide incorporation rate, identifying the face of PCNA that is important for the acceleration of catalysis.

A stable tetramer is not the only oligomeric state that mitochondrial single-stranded DNA binding proteins can adopt.

Mitochondrial single-stranded DNA (ssDNA)-binding proteins (mtSSBs) are required for mitochondrial DNA replication and stability and are generally assumed to form homotetramers, and this species is proposed to be the one active for ssDNA binding. However, we recently reported that the mtSSB from Saccharomyces cerevisiae (ScRim1) forms homotetramers at high protein concentrations, whereas at low protein concentrations, it dissociates into dimers that bind ssDNA with high affinity. In this work, using a combination of analytical ultracentrifugation techniques and DNA binding experiments with fluorescently labeled DNA oligonucleotides, we tested whether the ability of ScRim1 to form dimers is unique among mtSSBs. Although human mtSSBs and those from Schizosaccharomyces pombe, Xenopus laevis, and Xenopus tropicalis formed stable homotetramers, the mtSSBs from Candida albicans and Candida parapsilosis formed stable homodimers. Moreover, the mtSSBs from Candida nivariensis and Candida castellii formed tetramers at high protein concentrations, whereas at low protein concentrations, they formed dimers, as did ScRim1. Mutational studies revealed that the ability to form either stable tetramers or dimers depended on a complex interplay of more than one amino acid at the dimer-dimer interface and the C-terminal unstructured tail. In conclusion, our findings indicate that mtSSBs can adopt different oligomeric states, ranging from stable tetramers to stable dimers, and suggest that a dimer of mtSSB may be a physiologically relevant species that binds to ssDNA in some yeast species.

The telomere-binding protein Rif2 and ATP-bound Rad50 have opposing roles in the activation of yeast Tel1ATM kinase.

Saccharomyces cerevisiae Tel1 is the ortholog of human ATM kinase and initiates a cell cycle checkpoint in response to dsDNA breaks (DSBs). Tel1ATM kinase is activated synergistically by naked dsDNA and the Mre11-Rad50-Xrs2NBS1 complex (MRX). A multisubunit protein complex, which is related to human shelterin, protects telomeres from being recognized as DSBs, thereby preventing a Tel1ATM checkpoint response. However, at very short telomeres, Tel1ATM can be recruited and activated by the MRX complex, resulting in telomere elongation. Conversely, at long telomeres, Rap1-interacting-factor 2 (Rif2) is instrumental in suppressing Tel1 activity. Here, using an in vitro reconstituted Tel1 kinase activation assay, we show that Rif2 inhibits MRX-dependent Tel1 kinase activity. Rif2 discharges the ATP-bound form of Rad50, which is essential for all MRX-dependent activities. This conclusion is further strengthened by experiments with a Rad50 allosteric ATPase mutant that maps outside the conserved ATP binding pocket. We propose a model in which Rif2 attenuates Tel1 activity at telomeres by acting directly on Rad50 and discharging its activated ATP-bound state, thereby rendering the MRX complex incompetent for Tel1 activation. These findings expand our understanding of the mechanism by which Rif2 controls telomere length.

The signature motif of the Saccharomyces cerevisiae Pif1 DNA helicase is essential in vivo for mitochondrial and nuclear functions and in vitro for ATPase activity.

Pif1 family DNA helicases are conserved from bacteria to humans and have critical and diverse functions in vivo that promote genome integrity. Pif1 family helicases share a 23 amino acid region, called the Pif1 signature motif (SM) that is unique to this family. To determine the importance of the SM, we did mutational and functional analysis of the SM from the Saccharomyces cerevisiae Pif1 (ScPif1). The mutations deleted portions of the SM, made one or multiple single amino acid changes in the SM, replaced the SM with its counterpart from a bacterial Pif1 family helicase and substituted an α-helical domain from another helicase for the part of the SM that forms an α helix. Mutants were tested for maintenance of mitochondrial DNA, inhibition of telomerase at telomeres and double strand breaks, and promotion of Okazaki fragment maturation. Although certain single amino acid changes in the SM can be tolerated, the presence and sequence of the ScPif1 SM were essential for all tested in vivo functions. Consistent with the in vivo analyses, in vitro studies showed that the presence and sequence of the ScPif1 SM were critical for ATPase activity but not substrate binding.

The mitochondrial single-stranded DNA binding protein from S. cerevisiae, Rim1, does not form stable homo-tetramers and binds DNA as a dimer of dimers.

Rim1 is the mitochondrial single-stranded DNA binding protein in Saccharomyces cerevisiae and functions to coordinate replication and maintenance of mtDNA. Rim1 can form homo-tetramers in solution and this species has been assumed to be solely responsible for ssDNA binding. We solved structures of tetrameric Rim1 in two crystals forms which differ in the relative orientation of the dimers within the tetramer. In testing whether the different arrangement of the dimers was due to formation of unstable tetramers, we discovered that while Rim1 forms tetramers at high protein concentration, it dissociates into a smaller oligomeric species at low protein concentrations. A single point mutation at the dimer-dimer interface generates stable dimers and provides support for a dimer-tetramer oligomerization model. The presence of Rim1 dimers in solution becomes evident in DNA binding studies using short ssDNA substrates. However, binding of the first Rim1 dimer is followed by binding of a second dimer, whose affinity depends on the length of the ssDNA. We propose a model where binding of DNA to a dimer of Rim1 induces tetramerization, modulated by the ability of the second dimer to interact with ssDNA.

Pif1 is essential for efficient replisome progression through lagging strand G-quadruplex DNA secondary structures.

Pif1 DNA helicase is a potent unwinder of G-quadruplex (G4) structures in vitro and functions to maintain genome stability at G4 sequences in Saccharomyces cerevisiae. Here, we developed and utilized a live-cell imaging approach to quantitatively measure the progression rates of single replication forks through different G4 containing sequences in individual yeast cells. We show that in the absence of Pif1, replication rates through specific lagging strand G4 sequences in vivo is significantly decreased. In contrast, we found that in the absence of Pif1, replication rates through the same G4s on the leading strand are not decreased relative to the respective WT strains, showing that Pif1 is essential only for efficient replication through lagging strand G4s. Additionally, we show that a canonical PIP sequence in Pif1 interacts with PCNA and that replication through G4 structures is significantly slower in the absence of this interaction in vitro and in vivo. Thus, Pif1-PCNA interaction is essential for optimal replisome progression through G4 sequences, highlighting the importance of coupling between Pif1 activity and replisome progression during yeast genome replication.

Chemo-mechanical pushing of proteins along single-stranded DNA.

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.

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.

The wrapping loop and Rap1 C-terminal (RCT) domain of yeast Rap1 modulate access to different DNA binding modes.

Budding yeast Rap1 is a specific double-stranded DNA-binding protein involved in repression and activation of gene transcription and in the establishment of the nucleoprotein complex formed at telomeres. The DNA-binding domain (DBD) of Rap1 forms a high affinity complex with DNA where both Myb-like domains bind to the recognition site. However, we recently showed that the DBD can also access an alternative, lower affinity DNA-binding mode where a single Myb-like domain binds. This results in Rap1-DNA complexes with stoichiometry higher than previously anticipated. In this work, we show that the ability of the DBD to form higher stoichiometry complexes on DNA is maintained also in larger Rap1 constructs. This indicates that transition between at least two DNA-binding modes is a general property of the protein and not a specific feature of the DBD in isolation. The transition between binding modes is modulated by the C-terminal wrapping loop within the DBD, consistent with the proposed model in which the transient opening of this region allows a switch between binding modes. Finally, we provide evidence that the Rap1 C terminus interacts with the DNA-binding domain, suggesting a complex network of interactions that couples changes in conformation of the protein to the binding of its DNA recognition sequence.

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.

The DNA-binding domain of yeast Rap1 interacts with double-stranded DNA in multiple binding modes.

Saccharomyces cerevisiae repressor-activator protein 1 (Rap1) is an essential protein involved in multiple steps of DNA regulation, as an activator in transcription, as a repressor at silencer elements, and as a major component of the shelterin-like complex at telomeres. All the known functions of Rap1 require the known high-affinity and specific interaction of the DNA-binding domain with its recognition sequences. In this work, we focus on the interaction of the DNA-binding domain of Rap1 (Rap1(DBD)) with double-stranded DNA substrates. Unexpectedly, we found that while Rap1(DBD) forms a high-affinity 1:1 complex with its DNA recognition site, it can also form lower-affinity complexes with higher stoichiometries on DNA. These lower-affinity interactions are independent of the presence of the recognition sequence, and we propose they originate from the ability of Rap1(DBD) to bind to DNA in two different binding modes. In one high-affinity binding mode, Rap1(DBD) likely binds in the conformation observed in the available crystal structures. In the other alternative lower-affinity binding mode, we propose that a single Myb-like domain of the Rap1(DBD) makes interactions with DNA, allowing for more than one protein molecule to bind to the DNA substrates. Our findings suggest that the Rap1(DBD) does not simply target the protein to its recognition sequence but rather it might be a possible point of regulation.

Rapid profiling of DNA replication dynamics using mass spectrometry-based analysis of nascent DNA.

The primary method for probing DNA replication dynamics is DNA fiber analysis, which utilizes thymidine analog incorporation into nascent DNA, followed by immunofluorescent microscopy of DNA fibers. Besides being time-consuming and prone to experimenter bias, it is not suitable for studying DNA replication dynamics in mitochondria or bacteria, nor is it adaptable for higher-throughput analysis. Here, we present mass spectrometry-based analysis of nascent DNA (MS-BAND) as a rapid, unbiased, quantitative alternative to DNA fiber analysis. In this method, incorporation of thymidine analogs is quantified from DNA using triple quadrupole tandem mass spectrometry. MS-BAND accurately detects DNA replication alterations in both the nucleus and mitochondria of human cells, as well as bacteria. The high-throughput capability of MS-BAND captured replication alterations in an E. coli DNA damage-inducing gene library. Therefore, MS-BAND may serve as an alternative to the DNA fiber technique, with potential for high-throughput analysis of replication dynamics in diverse model systems.

Direct observation of individual RecA filaments assembling on single DNA molecules.

Escherichia coli RecA is essential for the repair of DNA double-strand breaks by homologous recombination. Repair requires the formation of a RecA nucleoprotein filament. Previous studies have indicated a mechanism of filament assembly whereby slow nucleation of RecA protein on DNA is followed by rapid growth. However, many aspects of this process remain unclear, including the rates of nucleation and growth and the involvement of ATP hydrolysis, largely because visualization at the single-filament level is lacking. Here we report the direct observation of filament assembly on individual double-stranded DNA molecules using fluorescently modified RecA. The nucleoprotein filaments saturate the DNA and extend it approximately 1.6-fold. At early time points, discrete RecA clusters are seen, permitting analysis of single-filament growth from individual nuclei. Formation of nascent RecA filaments is independent of ATP hydrolysis but is dependent on the type of nucleotide cofactor and the RecA concentration, suggesting that nucleation involves binding of approximately 4-5 ATP-RecA monomers to DNA. Individual RecA filaments grow at rates of 3-10 nm s(-1). Growth is bidirectional and, in contrast to nucleation, independent of nucleotide cofactor, suggesting addition of approximately 2-7 monomers s(-1). These results are in accord with extensive genetic and biochemical studies, and indicate that assembly in vivo is controlled at the nucleation step. We anticipate that our approach and conclusions can be extended to the related eukaryotic counterpart, Rad51 (see ref.), and to regulation by assembly mediators.

Shelterin Components Modulate Nucleic Acids Condensation and Phase Separation in the Context of Telomeric DNA.

Telomeres are nucleoprotein complexes that protect the ends of chromosomes and are essential for chromosome stability in Eukaryotes. In cells, individual telomeres form distinct globules of finite size that appear to be smaller than expected for bare DNA. Moreover, telomeres can cluster together, form telomere-induced-foci or co-localize with promyelocytic leukemia (PML) nuclear bodies. The physical basis for collapse of individual telomeres and coalescence of multiple ones remains unclear, as does the relationship between these two phenomena. By combining single-molecule force spectroscopy measurements, optical microscopy, turbidity assays, and simulations, we show that the telomere scaffolding protein TRF2 can condense individual DNA chains and drives coalescence of multiple DNA molecules, leading to phase separation and the formation of liquid-like droplets. Addition of the TRF2 binding protein hRap1 modulates phase boundaries and tunes the specificity of solution demixing while simultaneously altering the degree of DNA compaction. Our results suggest that the condensation of single telomeres and formation of biomolecular condensates containing multiple telomeres are two different outcomes driven by the same set of molecular interactions. Moreover, binding partners, such as other telomere components, can alter those interactions to promote single-chain DNA compaction over multiple-chain phase separation.