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A Second, Extended PIP Motif in Yeast CAF-1 Mediates Multivalent Recognition by PCNA

Submitted:

19 July 2026

Posted:

21 July 2026

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Abstract
Proliferating cell nuclear antigen (PCNA) is an essential sliding clamp that coordinates nearly all DNA-templated processes. It does so by recruiting a diverse array of factors to DNA through PCNA-interacting protein (PIP) motifs on PCNA-binding proteins. Chromatin assembly factor 1 (CAF-1) is a histone chaperone that deposits histones onto silent regions of the genome immediately following DNA replication. PCNA recruitment of CAF-1 to the replication fork is essential for nucleosome assembly and epigenetic inheritance. In yeast, CAF-1 recruitment to PCNA is assumed to be facilitated using one PIP motif. Here, we identify a second PIP motif in CAF-1, designated PIP1, located within the N-terminal intrinsically disordered region of the protein. Binding kinetics demon-strate the PIP1 motif sequence binds PCNA with substantially lower affinity than the previously characterized PIP motif, designated PIP2. Structural and binding data reveal PIP1 as an extended PIP motif, where downstream residues make extensive, atypical contacts with PCNA that significantly increase its affinity. Neither PIP1 nor PIP2 alone is required for CAF-1–mediated gene silencing in vivo, but simultaneous disruption of both abolishes CAF-1 function. Together, these findings suggest CAF-1 engages PCNA multivalently and provide new insight into how PCNA selectively recognizes binding partners during nucleosome assembly.
Keywords: 
Chromatin assembly factor 1 (CAF-1)
;  
proliferating cell nuclear antigen (PCNA)
;  
nucleosome assembly
;  
PCNA-interacting peptide (PIP)
Subject: 
Biology and Life Sciences  -   Biochemistry and Molecular Biology

1. Introduction

Proliferating cell nuclear antigen (PCNA) is a key modulator of DNA metabolic processes [1]. It acts as a sliding clamp that encircles DNA and tethers numerous proteins to replication forks. The structure of PCNA is homotrimeric, where each subunit of PCNA contains two similar domains that are linked to each other by an interdomain connecting loop (IDCL) [2,3]. In addition to its canonical role in enhancing DNA polymerase processivity, PCNA serves as a molecular scaffold that recruits and regulates the function of a diverse array of factors involved in DNA replication, repair, and chromatin assembly. This recruitment is largely mediated through short interaction motifs on PCNA-binding proteins called PCNA-interacting protein (PIP) motifs. A PIP motif is a conserved sequence of eight amino acids, typically represented by the consensus sequence Qxx[L/I/M]xx[F/Y][F/Y], where “x” represents any amino acid. A majority of PCNA-binding motifs in PCNA-binding proteins are located within intrinsically disordered regions (IDRs) or are flanked by IDRs [4].
Canonical PIP motifs bind a hydrophobic pocket on the front face of PCNA near the IDCL through the conserved glutamine, aliphatic, and aromatic residues in positions one, four, seven, and eight. Upon binding PCNA, these motifs form a characteristic 310 helix (Figure 1A). Despite the short length and the high degree of sequence conservation among PIP motifs, PCNA is able to reliably discriminate between these motifs and coordinate an extraordinary number of factors that compete for access to DNA. This raises fundamental questions about how PCNA can achieve selectivity of binding partners at the replication fork during DNA-templated processes. Accumulating evidence indicates variability between PIP-PCNA interactions may be due to differences in amino acids immediately flanking the PIP motifs [4,5,6,7].
Chromatin assembly factor 1 (CAF-1) is a key PCNA-binding protein that directly links DNA synthesis to chromatin assembly. CAF-1 is a heterotrimeric histone chaperone complex responsible for depositing newly synthesized, modified histones H3 and H4 onto nascent DNA immediately following replication, thereby maintaining nucleosome organization and preserving epigenetic information. The interaction between CAF-1 and PCNA is essential for replication-coupled nucleosome assembly and heterochromatin maintenance [8,9,10]. Disruption of this interaction impairs chromatin formation and gene silencing [9,10]. In yeast, CAF-1 is composed of subunits Cac1, Cac2, and Cac3. CAF-1 recruitment to PCNA at replication forks is believed to be mediated through a PIP motif located near the middle of its largest subunit, Cac1 [1,10,11,12]. We solved the structure of this motif bound to PCNA previously (Figure 1A) [13].
Previous studies of PIP-PCNA interactions have demonstrated residues immediately flanking PIP motifs are important in modulating PCNA interactions [4,5,6,7]. These sequences may influence both affinity and specificity by binding secondary surfaces on PCNA, such as the C-terminus or IDCL [3,14,15]. In the case of CAF-1, residues on the N-terminal and C-terminal side of the PIP motif in Cac1 make contacts with both the C-terminus and the IDCL of PCNA, respectively (Figure 1B) [13]. Furthermore, positively charged residues adjacent to the PIP motif appear to enhance PCNA binding, whereas negatively charged residues can reduce a more robust interaction [13].
In humans, CAF-1 consists of the three subunits p150, p60, and p48. The largest subunit, p150, contains two PIP motifs, denoted PIP1 and PIP2 [12,16]. Previous studies have shown these two PIP motifs are distinct, where the canonical PIP2 motif exhibits low affinity binding to PCNA and is critical for replication-coupled chromatin assembly, while the non-canonical PIP1 motif exhibits higher affinity to PCNA and may be dispensable for CAF-1 recruitment and gene silencing [16]. The significance of possessing two PIP motifs with differing affinities and functions is not clear.
Here, we identified a second PIP motif in yeast Cac1. This motif is located within the N-terminal, intrinsically disordered region of the protein. Therefore, we denote the motif as PIP1. Biochemical analyses show the PIP1 motif binds PCNA with substantially lower affinity than the previously characterized PIP2 motif. However, data suggest the PIP1 motif adopts an extended conformation, possibly forming a β-sheet interaction with the IDCL of PCNA that increases the affinity of the interaction and distinguishing it from most other known PIP motif-PCNA interactions. Binding studies show the presence of the extended residues increases the affinity of the PIP1 motif for PCNA by approximately four-fold. Functional assays indicate neither the extended PIP1 motif nor the PIP2 motif alone is required for CAF-1–mediated gene silencing in vivo; however, CAF-1 is not functional when both motifs are disrupted. Together, these studies suggest a multivalent role for the PIP motifs in CAF-1 and enhance our understanding of how PCNA recognizes its binding partners at the replication fork.

2. Materials and Methods

2.1. Protein Expression and Purification

Wild type yeast PCNA was N-terminally His12-tagged, overexpressed in Bl21(DE3) bacteria harboring plasmid pKW336, and purified as described previously [34]. To produce the PCNA-PIP1 fusion proteins, a five amino acid linker (GGSGG) and the sequence of the extended PIP1 motif of yeast CAF-1 were cloned, respectively, in frame, at the C-terminus of the gene encoding PCNA in the plasmid pKW336. The fusion protein was overexpressed in BL21(DE3) cells. Cells were lysed via sonication and the cell lysate was subjected to centrifugation for clarification. The PCNA-PIP fusion protein was purified using Ni-NTA agarose affinity chromatography (Thermo Scientific), DEAE anion exchange chromatography (GE Healthcare), and a Superdex 200 size exclusion chromatography column (GE Healthcare).

2.2. Peptide Synthesis

The CAF-1 PIP1 peptides used in the initial SPR experiments (residues 12-26, Figure 3) and the PIP1β peptides used for Figure 7C-7E were commercially synthesized by GenScript. All other CAF-1 PIP1 peptides were manually synthesized by Fmoc solid-phase peptide synthesis on Rink Amide Protide resin from Fisher Scientific. Before synthesis, the resin was swelled in N, N-dimethylformamide (DMF) for 30 min. Each coupling reaction utilized 4 equivalents of Fmoc-protected amino acid from Aapptec, Novabiochem, Chem-Impex or Anaspec, 4 equivalents of HCTU (O-(1H-6-chlorobenzotriazole-1-yl)-1,1,3,3- tetramethyluronium hexafluorophosphate), and 8 equivalents of N, N-diisopropylethylamine in 4 mL of DMF. Coupling reactions were conducted at room temperature for 40 min to 24 hours with constant stirring, followed by rinsing the resin with 4 mL of DMF three times. Fmoc deprotection reactions were carried out using 20% piperidine in DMF for 20 min at room temperature with constant stirring, followed by rinsing the resin with 4 mL of DMF three times. After coupling the last amino acid, peptides were cleaved from the resin using 95% trifluoroacetic acid (TFA), 2.5% triisopropylsilane, and 2.5% water (H2O) for a duration of 4-6 hours with constant stirring. Cleaved peptides were precipitated with ice-cold diethyl ether followed by centrifugation at 3,000 × g for 10 min at 4 ºC. The diethyl ether was then removed by decantation, followed by air-drying to obtain a pellet. The peptide pellet was then dissolved in 50% acetonitrile (ACN)/H2O mixture and filtered using a 0.22 µm syringe filter from Fisher Scientific. After filtration, the peptides were purified by reverse-phase high-performance liquid chromatography (HPLC) using a 1260 Infinity II Preparative LC System from Agilent Technologies fitted with a Pursuit 5 µm, 200 Å, 250 × 21.2 mm C18 column from Phenomenex. The final purity of peptides was determined by analytical-scale HPLC using a 1260 Infinity II LC System from Agilent Technologies fitted with a Pursuit 3 200 Å, 150 × 4.6 mm C18 column from Phenomenex. The mobile phase for both types of HPLC runs consisted of Solvent A (H2O + 0.1% TFA) and Solvent B (acetonitrile + 0.1% TFA). HPLC runs were accomplished by gradient elution with increasing concentration of Solvent B. For analytical HPLC runs, the concentration of Solvent B was increased in a linear gradient from 5 to 55% over the course of 30 min or 60 min at a constant flow rate of 1 mL/min. The identity of the synthesized peptides were confirmed via matrix-assisted laser desorption ionization mass spectrometry using a Bruker solariX XR Fourier transform ion cyclotron resonance mass spectrometer (see Supplemental Figure 1). Peptides were quantified through UV-Visible spectroscopy using a VWR UV-3100PC spectrophotometer. For quantification, a tryptophan was added to WT PIP1 (10 – 27), WT PIP1 (10 – 32), and WT PIP1 (10 – 40). These were dissolved in water and quantified using ε280 nm = 5690 M−1cm−1 [35]. The peptide used in Figure 7B (*β; residues 10 – 40) lacked a tryptophan residue. This was dissolved in water and quantified using an estimated ε214 nm based on the contributions from all peptide bond linkages [36,37].

2.3. Surface Plasmon Resonance

Surface plasmon resonance (SPR) assays were performed using a Biacore 8K (Cytiva). Experiments were carried out at a flow rate of 30 μL/min at 25oC in 10 mM HEPES, pH 7.4, 300 mM NaCl, 50 μM EDTA, 0.05% surfactant P20, and 1 mM DTT as the running buffer. His12-tagged PCNA was immobilized on an NiHC200M sensor chip (XanTec bioanalytics). The chip surface was first activated using an injection of 350 mM EDTA (25 µL/min) followed by an injection of 0.5 mM NiCl2 (15 µL/min). Increasing concentrations of the PIP1 peptide in running buffer were injected over the immobilized PCNA. Contact time for the PIP1 peptide was set to 60 s and dissociation time was 180 s. The signal from buffer alone (containing no peptide) over a reference flow cell was subtracted from all responses. Kinetic binding constants were determined using non-linear least-squares fitting to a 1:1 binding model with the Biacore Insight Evaluation 5.0 software. Steady state binding constants were obtained by fitting equilibrium data to a non-linear regression with GraphPad Prism 11.0.2. Experiments were performed using a minimum of seven replicates.

2.4. Small-Angle X-ray Scattering

Wildtype and mutant fusion proteins used in SAXS contained residues 10 - 37 of CAF-1 fused to the C-terminus of a PCNA monomer via a five amino acid linker, GGSGG. SAXS was performed at BioCAT (beamline 18ID at the Advanced Photon Source, Chicago) with in-line size exclusion chromatography (SEC) to separate sample from aggregates and other contaminants thus ensuring optimal sample quality and multiangle light scattering (MALS), dynamic light scattering (DLS) and refractive index measurement (RI)) for additional biophysical characterization (SEC-MALS-SAXS). The samples were loaded on a Superdex 200 Increase 10/300 GL column (Cytiva) run by a 1260 Infinity II HPLC (Agilent Technologies) at 0.6 ml/min. The flow passed through (in order) a MALS detector and a DLS detector (DAWN Helios II, Wyatt Technologies), an RI detector (Optilab T-rEX, Wyatt), and a fiber coupled UV cell. The flow then went through the SAXS flow cell. The flow cell consists of a 1.0 mmID quartz capillary with ~20 μm walls. A coflowing buffer sheath is used to separate sample from the capillary walls, helping prevent radiation damage (Kirby et al., 2016). Scattering intensity was recorded using an PILATUS3 X 1M (Dectris) detector which was placed 3.6 m from the sample giving us access to a q-range of 0.003 Å-1 to 0.33 Å-1 at an energy of 12 keV. 0.5 s exposures were acquired every 1 s during elution and data was reduced using BioXTAS RAW 2.3.0 (Hopkins, 2024). Buffer blanks were created by averaging regions flanking the elution peak and subtracted from exposures selected from the elution peak to create the I(q) vs. q curves used for subsequent analyses. REGALS was used to properly subtract buffer regions from samples with sloping baselines. Molecular weights and hydrodynamic radii were calculated from the MALS and DLS data respectively using the ASTRA 8 software (Wyatt).

2.5. Nano Differential Scanning Fluorimetry

Nano differential scanning fluorimetry (nanoDSF) was performed using a Prometheus Panta instrument (NanoTemper Technologies, Munich, Germany). PCNA was buffer-exchanged into 50 mM TrisCl, pH 8.0, and 150 mM NaCl and diluted to varying concentrations ranging from 15 to 30 µM. The PIP1 motif peptide was dissolved in the same buffer and added in molar excess of at least 3.33-fold relative to PCNA. PCNA and peptide were mixed and incubated at 25°C for 10 minutes prior to loading into standard nanoDSF grade capillaries (NanoTemper Technologies). Samples were subjected to a thermal ramp from 25°C to 95°C at a rate of 1°C/min. Intrinsic tryptophan fluorescence was monitored at 330 nm and 350 nm, and the ratio of fluorescence intensities (F350/F330) was recorded as a function of temperature. Melting temperatures (Tm) were determined from the first derivative of the F350/F330 ratio using the Prometheus Panta analysis software. Shifts in Tm (ΔTm) relative to PCNA alone were used as an indicator of binding, with a positive ΔTm indicating stabilization of PCNA upon peptide binding. Experiments were performed with n = 4 independent experiments and mean ΔTm values are reported with standard deviation.

2.6. Gene Silencing Assays using Flow Cytometry

A yeast strain containing a deletion of the endogenous cac1 gene (W303, cac1D::leu2, rtt106::kan, hmr::GFP/URA3) and a pRS313-derived yeast expression plasmid for the wildtype Cac1 protein were kindly provided by Bruce Stillman. A Q5 Site-Directed Mutagenesis Kit (NEB) was used with the yeast expression plasmid as a template to generate a plasmid with the ΔPIP2 motif mutation in the cac1 gene. All other plasmids encoding for mutated Cac1 proteins were purchased from GenScript. The cac1Δ yeast cells were then transformed with either the wildtype plasmid or one of the mutant Cac1 plasmids using the Frozen-EZ Yeast Transformation kit (Zymo Research). For controls, yeast cells were left untransformed or transformed with the pRS313 empty vector. Cells were grown in 3 mL of liquid yeast peptone dextrose (YPD) media overnight with incubation at 30 °C and shaking at 200 rpm. Overnight cultures were used to inoculate fresh 10 mL cultures to OD600 ≈ 0.2 and were grown with the same incubation conditions to OD600 ≈ 0.7 (early log phase). Cells were pelleted by centrifugation at 4500 rpm for 5 minutes at 4 °C. The cell pellets were washed 3x with 2 mL of 1x PBS (137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 1.8 mM KH2PO4 pH = 7.4) by resuspension and pelleting using the centrifugation conditions described above. Intercellular aggregates were diminished by water bath sonication (Bransonic CPX2800H) for 5 minutes at 25 °C on the low power setting. 20 – 50 μL of sonicated yeast cells were added to 1 mL 1x PBS and 1 μL of propidium iodide (PI) (Invitrogen) and incubated at 25 °C in the dark for 15 minutes. The cells were pelleted by centrifugation at 1200 rpm for 5 minutes and excess PI in the supernatant was decanted. An additional 1 mL of fresh 1x PBS was used to resuspend the PI treated yeast. For each culture, 200 ul of cell suspension was acquired using a ZE5 flow cytometer (Biorad) enabled with a Forward Scatter Small Particle Detector (FSc spd). Data analysis of generated Flow Cytometry Standard (FCS) files was performed using Flowjo Version 10. For this, sequential gating was carried out to first exclude doublets. Next, dead cells were removed through exclusion of PI positive staining cells. Finally, estimations of the percent frequency of GFP expressing yeast were carried out. Experiments were run a minimum of three times using independent yeast preparations.

3. Results

3.1. Identification of a Second PIP Motif in Cac1

A small number of PCNA-binding proteins are known to contain more than one PIP motif, including human and yeast DNA polymerases and human CAF-1 [16,17,18,19,20]. We sought to determine whether yeast CAF-1 also contained more than one PIP motif. Using sequence analysis, we identified a second, putative PIP motif near the N-terminus of the Cac1 subunit with the sequence KKGILSFF (Figure 2A). Because this motif is upstream of the known PIP motif in Cac1, we named the N-terminal motif the “PIP1 motif” and the previously determined motif the “PIP2 motif.” Similarly, the PIP1 and PIP2 motifs of human CAF-1 exist near the N-terminus and near the center of the largest subunit, p150. As observed for most PIP motifs, both motifs in the yeast and human CAF-1 protein exist within predicted intrinsically unstructured regions (Figure 2B and 2C).

3.2. The PIP1 Motif of Cac1 Binds PCNA with Low Affinity

Binding between the PIP1 motif of yeast CAF-1 and PCNA was measured quantitatively using surface plasmon resonance (SPR). PCNA was captured on the surface of a sensor chip and increasing concentrations of a peptide containing the PIP1 motif (residues 12-26) were added to the chip (Figure 3A). The binding affinity of the PCNA-CAF-1 interaction, obtained by plotting the steady state response units vs. ligand concentration, was measured as Kd = 120 ± 10 μM (Figure 3B). This value was surprising, as this is significantly lower than all other known PIP-PCNA interactions. Binding affinities between other PIP motifs and PCNA have been determined to be as high as 80 nM or as low as 60 μM, with most interactions in the mid-micromolar range (1-60 μM) [4,15,21]. For comparison, the PIP2 motif of yeast CAF-1 binds PCNA approximately 30-fold tighter than the PIP1 motif, with an affinity of 4 μM [13].
Evaluation of the kinetics of the sensorgrams fit best to a 1:1 binding model. The association and dissociation rate constants were outside the lower and upper detection limits of the instrument, respectively, for a majority of the experiments. We did obtain reliable kinetic constants from one experiment (Table 1). The quality of the fit was confirmed by a low χ2 value of 0.01 (which was 0.02% of the Rmax), supporting the validity of the model, suggesting these values may be close to the true parameters. Importantly, the Kd obtained from kinetic rate constants (120 µM) was identical to the steady-state Kd (120 µM) determined across multiple replicates, providing confidence in the overall affinity measurement independent of the kinetic analysis. Overall, these results are consistent with a weak, transient interaction typical of PIP-PCNA binding.
These kinetic analyses differ drastically from the PIP2 motif of CAF-1 binding to PCNA, which exhibited a two-step binding mechanism (Table 1). This suggests association of the PIP2 motif to PCNA begins with a step that is relatively fast, followed by a much slower step involving a conformational change [13]. In contrast, the PIP1 motif binds PCNA using a one-step mechanism, without an additional, conformational step.

3.3. Identification of a Putative, Extended PIP1 Motif

We employed small-angle X-ray scattering (SAXS) to characterize the solution conformation of the PIP1-PCNA complex at low resolution. One PCNA trimer contains three identical binding sites for PIP motifs. In addition, the C-terminus of PCNA is in very close proximity to the PIP-binding region in each subunit (see Figure 1B). Therefore, to ensure a stoichiometric ratio of three PIP motifs to one PCNA trimer in these complexes, we employed a method we previously developed for PIP-PCNA structural determination [13]. Here, a peptide sequence containing the PIP1 motif of CAF-1 was fused to the C-terminus of a PCNA monomer via a flexible linker (Figure 4A). To compare conformational changes between the PIP-bound and unbound states of PCNA, we also generated a mutant form of the PCNA-PIP1 fusion protein where the PIP1 motif was unable to bind PCNA. For this, the two phenylalanines in positions seven and eight in the PIP1 motif were mutated to alanine residues (*PIP1, Figure 4A). Previous studies have established that the double phenylalanine to alanine mutation in canonical PIP motifs inhibits binding to PCNA [22,23,24,25]. Figure 4B and 4C show dimensionless Kratky analysis of the wildtype and mutant PIP-PCNA complexes. Unexpectedly, the mutant protein complex exhibited the same overall conformation as the PIP-bound PCNA, as observed in Figure 4C where overlay of the wildtype and PIP1 mutant SAXS data show no significant differences. These results suggest an interaction may still be present in the complex, and residues within the CAF-1 peptide but outside the PIP1 motif are interacting with PCNA.
We used AlphaFold3 to identify possible interactions between PCNA and CAF-1 residues adjacent to the canonical PIP1 residues. Interestingly, AlphaFold predicted a region immediately C-terminal to the PIP1 motif forms an anti-parallel β-sheet with the IDCL of PCNA. As many as eight additional amino acids in CAF-1 are predicted to make polar contacts with the IDCL of PCNA (Figure 5). Therefore, based on both experimental and predicted results, we hypothesized the PIP1 motif of CAF-1 is an extended PIP motif, and requires amino acids C-terminal to the motif itself to bind PCNA with higher affinity.

3.4. Affinity Between the Extended PIP1 Motif and PCNA

To determine whether residues downstream of the PIP1 motif of CAF-1 interact with PCNA and measure the affinity of the extended PIP1 motif-PCNA complex, we carried out SPR experiments with PCNA and various length peptides containing the PIP1 motif of CAF-1 (residues 10 – 27, residues 10 – 32, and residues 10 – 40). To ensure the presence of a negative charge at the C-terminus of each peptide did not interfere with the interaction with PCNA, each peptide was synthesized with an amide group at the C-terminus, which more closely mimics a peptide bond.
Compared to the peptide used in initial SPR experiments (Figure 3), approximately a two-fold increase in affinity for PCNA was observed when only one additional residue was added to the C-terminus (residues 10 – 27) (Figure 6 and Table 2). Importantly, the peptide used in Figure 3 contained a carboxyl group at the C-terminus, so it is possible this negative charge interfered with a more robust interaction between PCNA and the extended residues. It is unlikely the addition of the two upstream amino acids, LQ, contributed to the increased affinity for two reasons. First, to our knowledge, no previous studies have shown interactions between PIP motifs and PCNA more than a few residues upstream of the PIP motif sequence. Second, the AlphaFold predicted structure of the PIP1 motif of CAF-1 bound to PCNA did not predict any interactions between the LQ of this sequence and PCNA. The affinity increased by approximately four-fold when an additional six amino acids were added to the C-terminus of the original Cac1 peptide (residues 10 – 32). However, no further increase in affinity was observed when an additional 14 residues were added to the C-terminus of the PIP1 motif (residues 10 – 40), suggesting the peptide containing amino acids 10 – 32 of Cac1 is sufficient for the interaction with PCNA.
Evaluation of the kinetics for each extended CAF-1 peptide binding to PCNA fit best to a 1:1 binding model. As with the peptide containing only the PIP1 sequence, the association and dissociation rate constants were near the lower and upper detection limits of the instrument, respectively (Table 2). However, the quality of the fit for most experiments resulted in χ2 values which were less than 0.08% of the Rmax, supporting the likelihood that kinetic values obtained are close to the true parameters. Importantly, the dissociation constants for the kinetics and the steady state analyses were also in close agreement with each other for each experiment (71 μM, 36 μM, and 30 μM for the PIP1 (10 – 27), PIP1 (10 – 32), and PIP1 (10 – 40) peptides, respectively).
Overall, results of these binding studies suggest eight residues C-terminal to the PIP1 motif of CAF-1 contribute to and are sufficient for an interaction with PCNA. The presence of these residues increase the affinity of the PIP1 for PCNA and may also result in a modest, ~two-fold faster association with PCNA compared to the PIP1 motif alone. Therefore, we propose this motif represents a novel, extended PIP motif in CAF-1. For clarity, we will refer to the PIP1 sequence alone as “PIP1” and the extended PIP1 motif (containing the additional predicted β-region residues) as “PIP1β” throughout the remainder of this manuscript.

3.5. The Extended PIP1 of CAF-1 Mediates Multivalent Binding to PCNA

Based on the AlphaFold predicted structure, three amino acids – T27, T28, and V29 – are likely important for formation of the β-sheet with the IDCL of PCNA (Figure 5C). We aimed to determine the contribution of each region of the PIP1β motif to the interaction with PCNA. To do this, we performed protein-protein binding studies with PCNA and CAF-1 peptides containing mutations in either the PIP1 motif (containing the F22A/F23A double substitution), the β-region (containing a T27A/T28A/V29A triple substitution) or in both regions simultaneously (containing F22A/F23A/T27A/T28A/V29A substitutions) (Figure 7A).
We first performed SPR using these constructs. However, only the peptide with substitutions in the β−region (*β) provided a measurable affinity and kinetic parameters for binding PCNA, with Kd = 94 ± 6 μM, k1 = 1.1 X 104 ± 1.4 M-1sec-1, and k-1 = 0.70 ± 0.02 sec-1 (Figure 7B – 7D), an affinity intermediate to what was observed for the two PIP1 motif peptides containing a partial β-region (the PIP1 (12 – 26) and PIP1 (10 – 27) peptides) and association kinetics similar to the PIP1 (10 – 27) peptide. This suggests the β-sheet–IDCL interaction alone is too weak for SPR to reliably measure binding kinetics or affinity. Thus, we switched to nano differential scanning fluorimetry (nanoDSF) to qualitatively measure the contribution of each interaction relative to each other (Figure 7E). Wildtype or mutant PIP1 peptide was incubated with PCNA and the melting temperature (Tm) of the complex was measured compared to PCNA alone (ΔTm). Larger increases in ΔTm indicate greater stabilization of the PCNA–peptide complex and are therefore consistent with higher peptide affinity for PCNA.
As expected, the wildtype CAF-1 peptide bound PCNA with the highest affinity (Figure 7E and Table 3). The peptide containing only a functional PIP1 motif (*β) bound with the second-highest affinity, followed by the peptide containing only a functional β-region interaction (*PIP1). This suggests the PIP sequence portion of the PIP1β motif of CAF-1 binds PCNA tighter than the β-region does. Finally, the CAF-1 peptide containing mutations within both the PIP1 and β-region (*PIP1+*β) shows almost no change in melting temperature compared to PCNA alone, suggesting both regions of CAF-1 are required for the full, extensive interaction with PCNA.
To determine whether the overall conformation of the PIP1β-PCNA complex changes in the presence and absence of each interacting region, we carried out SAXS using similar PIP-PCNA fusion proteins as shown in Figure 4. For this, we generated fusion proteins containing the single β−region and double PIP1+β−region substitutions in CAF-1 (Figure 8A). The top panels in Figure 8B are the same data presented in Figure 4B and 4C, for easier comparison. Dimensionless Kratky analysis showed that a single mutation of either the PIP1 or β-region alone (*PIP1 or *β) did not appreciably alter this conformational landscape, as these plots superimpose well onto the plot for wildtype protein. In contrast, simultaneous mutation of both the PIP1 and β-region (*PIP1+*β) show subtle differences in the intermediate and high qRg ranges compared to the wildtype complex (bottom right panel in Figure 8B). Notably, the secondary peak is disrupted and the scattering intensity has a very broad plateau at higher values of qRg, indicating increased conformational flexibility and disorder. This suggests simultaneous loss of both the PIP1 and β-region interactions reduces the degree to which CAF-1 engages PCNA in this construct, where either no or fewer interactions are occurring between the two proteins. It is important to note the structure of PCNA does not change substantially upon binding PIP motifs [7,26,27], so a drastic change was not expected. Together, results demonstrate the PIP1β motif of CAF-1 mediates multivalent binding to PCNA, where the PIP1 and extended β-region residues combined have a greater affinity for PCNA than either motif alone.

3.6. The PIP1β Motif and the PIP2 Motif are Both Required for CAF-1 Function In Vivo

Finally, we set out to determine the importance of the PIP1 motif in the function of CAF-1 in gene silencing in vivo. We measured silencing by Cac1 at the HMR locus in Saccharomyces cerevisiae, where the gene for GFP was inserted in place of the HMR locus and the gene for endogenous Cac1 was removed (cac1Δ). Because CAF-1-dependent silencing was disrupted in these cells, GFP was expressed. Quantification of GFP expression was determined using flow cytometry. A plasmid containing the gene for wildtype Cac1 expression was transformed into these cells, which rescued GFP silencing (Figure 9, compare “WT Cac1” and “Empty Vector” columns).
To understand the significance of the PIP1 motif or the PIP1β motif sequences of CAF-1 in these cells, we generated plasmids with substitutions in the Cac1 gene: 1) in the PIP1 motif sequence alone (*PIP1; FF22/23AA), 2) in the β-region alone (*β; TTV27-29AAA), 3) in the PIP1β (*PIP1+*β; FF22/23AA and TTV27-29AAA), 4) in the PIP2 motif alone (*PIP2; FF233/234AA), 5) in the PIP1 and PIP2 motifs (*PIP1+*PIP2; FF22/23AA and FF233/234AA), and 6) in the PIP1β and PIP2 motifs (*PIP1+*β+*PIP2; FF22/23AA, TTV27-29AAA, and FF233/234AA). These plasmids were transformed into the cac1Δ cells and levels of GFP silencing was observed. Results showed none of the single mutations inhibited CAF-1 activity (*PIP1, *β, or *PIP2). Similar to what has been observed in previous experiments, the PIP2 motif mutant showed a slight decrease in gene silencing compared to wildtype [13]. Cells containing plasmid with the PIP1β mutation (*PIP1+*β) also did not exhibit any defect in activity. However, when both the PIP1 and PIP2 motifs or the PIP1β and the PIP2 motifs of Cac1 were mutated simultaneously, gene silencing was disrupted to a degree comparable to that observed in the complete absence of CAF-1. This suggests the PIP1 or PIP1β and the PIP2 motifs together are essential for CAF-1 function in vivo, while the individual PIP motifs are not required for gene silencing at the HMR locus.

4. Discussion

In this study, we identified a second PIP motif in yeast CAF-1. This motif exists near the N-terminus of the Cac1 subunit in an intrinsically disordered region. We designated this new PIP motif as PIP1, as the previously known PIP motif (designated as “PIP2”) is located downstream of the PIP1 motif. CAF-1 represents one of only a few known yeast proteins that contain multiple PIP motifs, most notably DNA polymerase η (pol η) [17]. As with pol η, the two PIP motifs in CAF-1 bind PCNA with substantially different affinities and kinetics, where the motifs with weaker affinity (the PIP1 of CAF-1 and the PIP2 of pol η, respectively) also bind PCNA with faster kinetics. This suggests the two motifs in each protein carry out differing roles in pol η and CAF-1 during DNA replication and nucleosome assembly, respectively.
We also determined the novel PIP1 motif of CAF-1 is not a standard PIP motif, but is instead an extended PIP motif that makes substantial interactions with the IDCL of PCNA. AlphaFold predicts a β-sheet is formed between these additional residues of the PIP1 motif and PCNA, and we therefore designated the full-length PIP1 motif as the PIP1β motif. We determined this motif binds PCNA with an affinity of approximately 35 μM. There are two other PCNA-binding proteins that interact substantially with the IDCL of PCNA to form β-sheets, p21 and FEN1. The extended PIP motifs of p21 and FEN1 bind PCNA in the mid-nanomolar (0.08 – 0.3 μM) and mid-micromolar range (10 - 60 μM), respectively [15,28,29]. In the case of p21, it has been suggested that these extensive interactions are responsible for the high affinity interaction with PCNA relative to other PIP motifs [4,7]. Although the additional interactions increase the affinity of these proteins to PCNA compared to the PIP motif alone, extended PIP motifs do not always appear to increase affinity to a range higher than other canonical, non-extended PIP motifs. Instead, this suggests the extended motifs may be important for PCNA selectivity of binding partners at the replication fork during DNA metabolic processes.
Two PIP motifs exist in human CAF-1, hPIP1 and hPIP2. These two motifs exist in locations within the p150 subunit that are similar to the locations of the yeast PIP1 and PIP2 in Cac1 (Figure 2). Beyond this, there are little similarities between the yeast PIP1 and hPIP1 motifs and the yeast PIP2 and hPIP2 motifs. The hPIP1 sequence (QARLFF) is more similar to the yeast PIP2 sequence (QSRIGNFF), whereas the hPIP2 sequence (KAEITRFF) is closer in sequence to the yeast PIP1 (KKGILSFF). We determined the yeast PIP1 motif binds PCNA with approximately 10-fold lower affinity than the yeast PIP2 motif, despite the presence of an extended yeast PIP1β sequence. We also show the yeast PIP1β motif binds PCNA via a transient, one-step binding mechanism, compared to the slower, two-step yeast PIP2-PCNA binding mechanism. In contrast, hPIP1 does not appear to interact with the IDCL of PCNA [30], but it has a higher affinity for PCNA than the hPIP2 motif. No precise Kd values or kinetics have been determined for either hPIP1 or hPIP2, but studies show the hPIP2-PCNA interaction is transient [16]. Together, this demonstrates the yeast PIP1β−PCNA interaction is more similar to the hPIP2-PCNA interaction, and the yeast PIP2-PCNA is more similar to the hPIP1-PCNA interaction. In terms of function, hPIP1 has been shown to be dispensable for nucleosome assembly in vivo while hPIP2 is critical for nucleosome assembly and targeting of CAF-1 to the replication fork [16,31]. In contrast, the yeast PIP1β motif is dispensable for gene silencing, whereas mutation of the yeast PIP2 motif has a modest effect. Though the presence of at least one PIP motif is essential for gene silencing in yeast. Overall, the two PIP motifs in CAF-1 have differing functions from each other in both human and yeast. Moreover, data suggest the PCNA-binding activity of hPIP1 is more similar to, but still distinct from that of yeast PIP2, and the same is true for the hPIP2 and yeast PIP1β. However, the PIP2 motif appears to be more significant for the function and/or recruitment of CAF-1 in nucleosome assembly in both organisms. More studies are needed to both understand these differences and to parse out the roles each motif plays in regulating the activity of CAF-1.
Based on our in vivo and kinetics studies of the PIP1β-PCNA and PIP2-PCNA interactions, we propose the PIP2 motif performs the predominant role in recruiting yeast CAF-1 to replication forks. We propose that PIP2 binding to PCNA increases the local concentration of PIP1β, enabling it to bind a separate subunit of PCNA and triggering a conformational change across the CAF-1 complex that drives histone deposition onto nascent DNA behind the replication fork (Figure 10). The extended, β-sheet formed between the PIP1β and PCNA might be necessary for histone deposition, as additional contacts with the IDCL could help stabilize the transition of CAF-1 to the back of PCNA when nucleosome formation is required. Since both PIP motifs are located within disordered regions of the protein, this would allow flexibility for CAF-1 to transition from the front to the back of PCNA. Alternatively, binding of PIP1β to PCNA may prevent other proteins from being recruited to the DNA. After histone deposition, CAF-1 may be released or the PIP2 may hold the PCNA-CAF-1 complex together for the next round of nucleosome assembly as the replication fork proceeds. This process would not interfere with DNA replication, as at least one subunit of PCNA would still be available to bind DNA polymerases.
It is interesting that our in vivo results show one PIP motif can rescue the activity of CAF-1 in the absence of the other PIP motif (Figure 9). CAF-1 is believed to function as a dimer (requiring two heterotrimers) at the replication fork [32,33]. It is therefore possible that a CAF-1 complex containing a non-functional PIP motif can still be recruited to PCNA as long as two functional PIP motifs are present in the entire complex. Here, the handoff of CAF-1 from the front to the back surface of PCNA via binding two different subunits of the PCNA trimer is still feasible.
Previous studies have identified and determined the structure of a mutant form of PCNA that does not bind CAF-1 and is defective in gene silencing, L126A/I128A [9,11]. This mutation does not appear to affect other cellular processes, suggesting the defect in interaction is specific to CAF-1. L126 and I128 both exist in the IDCL of PCNA. Because both the PIP1 and PIP2 motifs of CAF-1 interact more extensively with the IDCL than canonical PIP motifs, the L126A/I128A mutation likely hinders both motifs from binding. This would explain why the L126A/I128A mutant PCNA protein is still able to bind other PIP-containing proteins but not CAF-1.

5. Conclusions

It is unclear how PCNA selectively recruits CAF-1 to replication forks, especially given the large number of PIP-containing proteins competing for access to PCNA. Our studies suggest the presence of drastically different PIP motifs —including differences in sequence, length, binding kinetics, affinities, and secondary structure formation —may aid PCNA in the temporal and spatial selection of CAF-1 during replication-coupled nucleosome assembly. More broadly, it is likely that these same principles govern other cellular processes that require temporal and spatial discrimination by PCNA.

Supplementary Materials

The following supporting information can be downloaded at the website of this paper posted on Preprints.org, Figure S1: Characterization of synthesized peptides; Table S1: Sequences and sources of peptides used in binding studies.

Author Contributions

Conceptualization, L.M.D.; formal analysis, I.H., I.M.D., S.A.L., I.L.W., A.E.A., L.M.D.; investigation, I.H., I.M.D., S.A.L., I.L.W., C.E.C., T.J.S., A.E.A.; resources, J.W.C.; writing—original draft preparation, L.M.D.; writing—review and editing, I.H., I.M.D., S.A.L., I.L.W., T.J.S., A.E.A., J.W.C., L.M.D; supervision, J.W.C., L.M.D, funding acquisition, J.W.C, L.M.D. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by the National Science Foundation (2047553 to L.M.D), the National Institutes of Health (P20GM103427 to L.M.D.), the National Institute of General Medical Sciences (R35 GM142784 to J.W.C.) and the Nebraska Center for Integrated Biomolecular Communication (National Institute of General Medical Sciences P20 GM113126). The APC was funded by the National Science Foundation (2047553 to L.M.D).

Institutional Review Board Statement

Not applicable.

Data Availability Statement

All data generated for this study are included in this article. .

Acknowledgments

We would like to thank Dr. Bruce Stillman at Cold Spring Harbor Laboratory for providing original yeast strains and plasmids for the gene silencing assays. We thank Dr. Maxwell Watkins at BioCAT for assistance and support with the SAXS experiments. We thank Mason Perry and the Flow Cytometry Shared Resource at Creighton University for support with flow cytometry experiments. We thank Dr. M. Todd Washington at the University of Iowa for valuable discussions. This research was performed on APS beam time award(s) (DOI(s): https://doi.org/10.46936/APS-194303/60016966) from the Advanced Photon Source, a U.S. Department of Energy (DOE) Office of Science user facility operated for the DOE Office of Science by Argonne National Laboratory under Contract No. DE-AC02-06CH11357. BioCAT was supported by grant P30 GM138395 from the National Institute of General Medical Sciences of the National Institutes of Health. Use of the Pilatus 3 1M detector was provided by grant 1S10OD018090 from the National Institute of General Medical Sciences. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Science Foundation or the National Institutes of Health.

Conflicts of Interest

The authors declare no conflicts of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results.

Abbreviations

The following abbreviations are used in this manuscript:
PCNA Proliferating cell nuclear antigen
CAF-1 Chromatin assembly factor 1
PIP PCNA-interacting protein
IDCL Interdomain connecting loop
IDR Intrinsically disordered region
SPR Surface plasmon resonance
SAXS Small-angle X-ray scattering
DSF Differential scanning fluorimetry
GFP Green fluorescent protein

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Figure 1. Structure of the known PIP motif of CAF-1 bound to PCNA (PDB 8THW). (A) Three PIP motifs (pink) bound to one PCNA trimer. Each subunit of PCNA is represented by a different color (purple, green, yellow). (B) Close up of one PIP motif of CAF-1 (pink) bound to PCNA (light blue). Polar interactions (black dashed lines) between the residues upstream of the PIP motif bound to the C-terminus (residue F254) of PCNA and the residues downstream of the PIP motif bound to the IDCL (residues F125 and K127) of PCNA.
Figure 1. Structure of the known PIP motif of CAF-1 bound to PCNA (PDB 8THW). (A) Three PIP motifs (pink) bound to one PCNA trimer. Each subunit of PCNA is represented by a different color (purple, green, yellow). (B) Close up of one PIP motif of CAF-1 (pink) bound to PCNA (light blue). Polar interactions (black dashed lines) between the residues upstream of the PIP motif bound to the C-terminus (residue F254) of PCNA and the residues downstream of the PIP motif bound to the IDCL (residues F125 and K127) of PCNA.
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Figure 2. Locations and sequences of PIP motifs in yeast and human Cac1. (A) Sequences of the PIP1 and PIP2 motifs in yeast (Sc) and human (Hs) with the consensus sequence of PIP motifs shown. Dashes indicate gaps in sequence and an “x” indicates any amino acid. Disorder probability prediction plot obtained using the AIUPred prediction tool [38,39] of the Cac1 subunits of CAF-1 in yeast (B) or human (C). Locations of the PIP1 and PIP2 motifs in each protein are indicated. Values below 0.5 (indicated by the horizontal line) are predicted to be structured and values above 0.5 are predicted to be disordered.
Figure 2. Locations and sequences of PIP motifs in yeast and human Cac1. (A) Sequences of the PIP1 and PIP2 motifs in yeast (Sc) and human (Hs) with the consensus sequence of PIP motifs shown. Dashes indicate gaps in sequence and an “x” indicates any amino acid. Disorder probability prediction plot obtained using the AIUPred prediction tool [38,39] of the Cac1 subunits of CAF-1 in yeast (B) or human (C). Locations of the PIP1 and PIP2 motifs in each protein are indicated. Values below 0.5 (indicated by the horizontal line) are predicted to be structured and values above 0.5 are predicted to be disordered.
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Figure 3. SPR binding studies of the CAF-1 PIP1 motif and PCNA. (A) SPR of the isolated CAF-1 PIP1 peptide (residues 12-26; DDTKKKGILSFFQNT) binding to immobilized PCNA. Concentrations of the PIP1 peptide at increasing concentrations were injected onto PCNA captured on the chip. Signal from injection of buffer only (no PIP1 peptide) was subtracted from all data. Resulting curves were fit to a one-step binding equation (BIAevaluation software; black lines). (B) Responses at equilibrium for each concentration of PIP1 peptide were plotted and fit to a non-linear regression to obtain the steady state affinity of the interaction.
Figure 3. SPR binding studies of the CAF-1 PIP1 motif and PCNA. (A) SPR of the isolated CAF-1 PIP1 peptide (residues 12-26; DDTKKKGILSFFQNT) binding to immobilized PCNA. Concentrations of the PIP1 peptide at increasing concentrations were injected onto PCNA captured on the chip. Signal from injection of buffer only (no PIP1 peptide) was subtracted from all data. Resulting curves were fit to a one-step binding equation (BIAevaluation software; black lines). (B) Responses at equilibrium for each concentration of PIP1 peptide were plotted and fit to a non-linear regression to obtain the steady state affinity of the interaction.
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Figure 4. SAXS analysis of the PIP1 motif of CAF-1 bound to PCNA. (A) Linear representation of the PCNA-CAF-1 fusion proteins generated for SAXS experiments. A sequence containing the PIP1 motif of CAF-1 is shown in red, the five amino acid linker is shown in grey, and the PCNA monomer is shown in light purple. The wildtype and PIP1 mutant sequences are indicated, with the mutated residues bolded and underlined. (B) Dimensionless Kratky plot for the wildtype PCNA-CAF-1 fusion protein. (C) Kratky plot for the mutant PCNA-CAF-1 fusion protein overlayed with the wildtype protein.
Figure 4. SAXS analysis of the PIP1 motif of CAF-1 bound to PCNA. (A) Linear representation of the PCNA-CAF-1 fusion proteins generated for SAXS experiments. A sequence containing the PIP1 motif of CAF-1 is shown in red, the five amino acid linker is shown in grey, and the PCNA monomer is shown in light purple. The wildtype and PIP1 mutant sequences are indicated, with the mutated residues bolded and underlined. (B) Dimensionless Kratky plot for the wildtype PCNA-CAF-1 fusion protein. (C) Kratky plot for the mutant PCNA-CAF-1 fusion protein overlayed with the wildtype protein.
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Figure 5. Predicted structure of the extended PIP1 motif of CAF-1 bound to PCNA by AlphaFold. (A) Sequence of the Cac1 peptide used for prediction, where the PIP1 motif is italicized and the additional residues predicted to interact with PCNA are underlined. (B) Structure of the extended PIP1 motif (pink) bound to PCNA (light blue), as predicted by AlphaFold3. The IDCL of PCNA and the β-sheet formed between the additional residues of the PIP1 motif and PCNA are indicated. (C) Predicted polar contacts between the residues C-terminal to the PIP1 motif and PCNA, indicated as dashed lines. (D) Close up view of the β-sheet formed between the PIP1 motif and PCNA. Predicted polar contacts between the “TTV” residues of Cac1 and the IDCL residues of PCNA are shown as dashed lines.
Figure 5. Predicted structure of the extended PIP1 motif of CAF-1 bound to PCNA by AlphaFold. (A) Sequence of the Cac1 peptide used for prediction, where the PIP1 motif is italicized and the additional residues predicted to interact with PCNA are underlined. (B) Structure of the extended PIP1 motif (pink) bound to PCNA (light blue), as predicted by AlphaFold3. The IDCL of PCNA and the β-sheet formed between the additional residues of the PIP1 motif and PCNA are indicated. (C) Predicted polar contacts between the residues C-terminal to the PIP1 motif and PCNA, indicated as dashed lines. (D) Close up view of the β-sheet formed between the PIP1 motif and PCNA. Predicted polar contacts between the “TTV” residues of Cac1 and the IDCL residues of PCNA are shown as dashed lines.
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Figure 6. SPR binding studies of various length PIP1 peptides of CAF-1 and PCNA. Results of the PIP1 (10 – 27) peptide (B), the PIP1 (10 – 32) peptide (C) or the PIP1 (10 – 40) peptide (D) binding to immobilized PCNA. Left: the PIP1 peptides were injected at increasing concentrations onto PCNA captured on the chip. Signal from injection of buffer only (no PIP1 peptide) was subtracted from all data. Resulting curves were fit to a one-step binding equation (BIAevaluation software; black lines). Right: responses at equilibrium for each concentration of PIP1 peptide were plotted and fit to a non-linear regression to obtain the steady state affinity of the interaction. CAF-1 peptide sequences used are indicated in each panel, where amino acids in the PIP1 motif are highlighted in pink and the extended residues predicted to bind to PCNA via AlphaFold are highlighted in blue.
Figure 6. SPR binding studies of various length PIP1 peptides of CAF-1 and PCNA. Results of the PIP1 (10 – 27) peptide (B), the PIP1 (10 – 32) peptide (C) or the PIP1 (10 – 40) peptide (D) binding to immobilized PCNA. Left: the PIP1 peptides were injected at increasing concentrations onto PCNA captured on the chip. Signal from injection of buffer only (no PIP1 peptide) was subtracted from all data. Resulting curves were fit to a one-step binding equation (BIAevaluation software; black lines). Right: responses at equilibrium for each concentration of PIP1 peptide were plotted and fit to a non-linear regression to obtain the steady state affinity of the interaction. CAF-1 peptide sequences used are indicated in each panel, where amino acids in the PIP1 motif are highlighted in pink and the extended residues predicted to bind to PCNA via AlphaFold are highlighted in blue.
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Figure 7. The extended PIP1β of CAF-1 is required for the full PIP-PCNA interaction. (A) Wildtype and mutant PIP1 peptides used for SPR and nanoDSF experiments. Mutated residues in the PIP1 or β-region are highlighted in red. (B) SPR results of the *β mutant PIP1 peptide binding to immobilized PCNA. (C) SPR results of the *PIP1 mutant peptide binding to immobilized PCNA. No binding was observed. (D) SPR results of the *PIP1+*β mutant peptide binding to immobilized PCNA. No binding was observed. (E) First derivative plots of the thermal denaturation of PCNA alone (blue) or in the presence of wildtype or mutant PIP1 peptide (red) using nanoDSF. The difference between the melting temperature (Tm) of PCNA with and without peptide is indicated by the line through the inflection point of each curve.
Figure 7. The extended PIP1β of CAF-1 is required for the full PIP-PCNA interaction. (A) Wildtype and mutant PIP1 peptides used for SPR and nanoDSF experiments. Mutated residues in the PIP1 or β-region are highlighted in red. (B) SPR results of the *β mutant PIP1 peptide binding to immobilized PCNA. (C) SPR results of the *PIP1 mutant peptide binding to immobilized PCNA. No binding was observed. (D) SPR results of the *PIP1+*β mutant peptide binding to immobilized PCNA. No binding was observed. (E) First derivative plots of the thermal denaturation of PCNA alone (blue) or in the presence of wildtype or mutant PIP1 peptide (red) using nanoDSF. The difference between the melting temperature (Tm) of PCNA with and without peptide is indicated by the line through the inflection point of each curve.
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Figure 8. SAXS analysis of mutant PIP1 motif of CAF-1 bound to PCNA. (A) Linear representation of the PCNA-CAF-1 fusion proteins generated for SAXS experiments. The wildtype and PIP1 mutant sequences are indicated, with the mutated residues bolded and underlined. (B) Dimensionless Kratky plots for each of the mutant PCNA-CAF-1 fusion proteins overlayed with the wildtype protein.
Figure 8. SAXS analysis of mutant PIP1 motif of CAF-1 bound to PCNA. (A) Linear representation of the PCNA-CAF-1 fusion proteins generated for SAXS experiments. The wildtype and PIP1 mutant sequences are indicated, with the mutated residues bolded and underlined. (B) Dimensionless Kratky plots for each of the mutant PCNA-CAF-1 fusion proteins overlayed with the wildtype protein.
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Figure 9. Effects of mutant CAF-1 proteins on gene silencing. Silencing of GFP by wildtype (WT) or mutant Cac1 proteins was measured by flow cytometry. GFP-expressing yeast cells containing the pRS313 vector only (empty vector) was used as a control. Data are presented as mean ± standard deviation of n ≥ 3 independent experiments. Significance was determined using an unpaired t-test. **p < 0.05, n.s., not significant. Note that all samples with percentage of cells expressing GFP above 20% (the first three columns) were not significantly different from each other, and all samples with percentage of cells expressing GFP below 10% (the last five columns, excluding the *PIP2 sample) were also not significantly different from each other. These comparisons were not included in the figure to avoid visual clutter and maintain readability.
Figure 9. Effects of mutant CAF-1 proteins on gene silencing. Silencing of GFP by wildtype (WT) or mutant Cac1 proteins was measured by flow cytometry. GFP-expressing yeast cells containing the pRS313 vector only (empty vector) was used as a control. Data are presented as mean ± standard deviation of n ≥ 3 independent experiments. Significance was determined using an unpaired t-test. **p < 0.05, n.s., not significant. Note that all samples with percentage of cells expressing GFP above 20% (the first three columns) were not significantly different from each other, and all samples with percentage of cells expressing GFP below 10% (the last five columns, excluding the *PIP2 sample) were also not significantly different from each other. These comparisons were not included in the figure to avoid visual clutter and maintain readability.
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Table 1. Binding kinetics and affinities of the PIP1 and PIP2 motifs for PCNA.
Table 1. Binding kinetics and affinities of the PIP1 and PIP2 motifs for PCNA.
CAF-1 peptide k1
(M−1sec−1)		 k-1
(sec−1)		 k2
(M−1sec−1)		 k-2
(sec−1)		 Kd
(μM)
PIP1 9.4 × 103 1.1 N/A N/A 120 ± 10
PIP2a 1.4 X 105 7.4 X 10−1 6.2 X 10−4 1.7 X 10−3 4
N/A = not applicable aValues previously determined [13].
Table 2. Binding kinetics and affinities of PIP1 peptides for PCNA.
Table 2. Binding kinetics and affinities of PIP1 peptides for PCNA.
CAF-1 peptide k1
(M−1sec−1)		 k-1
(sec−1)		 Kd
(μM)
PIP1 (10-27) 1.0 X 104 ± 1.6 X 103 0.60 ± 0.02 69 ± 2
PIP1 (10-32) 2.2 X 104 ± 1.9 X 103 0.97 ± 0.07 34 ± 2
PIP1 (10-40) 1.9 X 104 ± 8.6 X 102 0.54 ± 0.06 32 ± 2
Table 3. Melting temperatures of PCNA bound to PIP1 peptides.
Table 3. Melting temperatures of PCNA bound to PIP1 peptides.
CAF-1 peptide ΔTm/°C
WT 1.12 ± 0.17
*PIP1 0.54 ± 0.18
*β 0.89 ± 0.14
*PIP1 + *β 0.13 ± 0.15
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