ATXR5/6 Forms Alternative Protein Complexes with PCNA and the Nucleosome Core Particle

The proliferating cell nuclear antigen (PCNA) is a sliding clamp associated with DNA polymerases and serves as a binding platform for the recruitment of regulatory proteins linked to DNA damage repair, cell cycle regulation, and epigenetic signaling. The histone H3 lysine-27 (H3K27) mono-methyltransferase Arabidopsis trithorax-related protein 5/6 (ATXR5/6) associates with PCNA, and this interaction has been proposed to act as a key determinant controlling the reestablishment of H3K27 mono-methylation following replication. In this study, we provide biochemical evidence showing that PCNA inhibits ATXR6 enzymatic activity. The structure of the ATXR6 PCNA-interacting peptide (PIP) in complex with PCNA indicates that a trio of hydrophobic residues contributes to the binding of the enzyme to the sliding clamp. Finally, despite the presence of three PIP binding clefts, only two molecules of ATXR6 bind to PCNA likely enabling the recruitment of a third protein to the sliding clamp. Collectively, these results rule out the model wherein PCNA-bound ATXR6 actively reestablishes H3K27 mono-methylation following DNA replication and provides insights into the role of ATXR6 PIP motif in its interaction with PCNA.     

Interaction between PCNA and diubiquitinated Mcm10 is essential for cell growth in budding yeast

The minichromosome maintenance protein 10 (Mcm10) is an evolutionarily conserved factor that is essential for replication initiation and elongation. Mcm10 is part of the eukaryotic replication fork and interacts with a variety of proteins, including the Mcm2-7 helicase and DNA polymerase alpha/primase complexes. A motif search revealed a match to the proliferating cell nuclear antigen (PCNA)-interacting protein (PIP) box in Mcm10. Here, we demonstrate a direct interaction between Mcm10 and PCNA that is alleviated by mutations in conserved residues of the PIP box. Interestingly, only the diubiquitinated form of Mcm10 binds to PCNA. Diubiquitination of Mcm10 is cell cycle regulated; it first appears in late G(1) and persists throughout S phase. During this time, diubiquitinated Mcm10 is associated with chromatin, suggesting a direct role in DNA replication. Surprisingly, a Y245A substitution in the PIP box of Mcm10 that inhibits the interaction with PCNA abolishes cell proliferation. This severe-growth phenotype, which has not been observed for analogous mutations in other PCNA-interacting proteins, is rescued by a compensatory mutation in PCNA that restores interaction with Mcm10-Y245A. Taken together, our results suggest that diubiquitinated Mcm10 interacts with PCNA to facilitate an essential step in DNA elongation.     

Crystal structures of the Arabidopsis thaliana proliferating cell nuclear antigen 1 and 2 proteins complexed with the human p21 C‐terminal segment

The proliferating cell nuclear antigen (PCNA) is well recognized as one of the essential cellular components of the DNA replication machinery in all eukaryotic organisms. Despite their prominent importance, very little biochemical and structural information about plant PCNAs is available, in comparison with that obtained from other eukaryotic organisms. We have determined the atomic resolution crystal structures of the two distinct Arabidopsis thaliana PCNAs (AtPCNA), both complexed with the C‐terminal segment of human p21. Both AtPCNAs form homotrimeric ring structures, which are essentially identical to each other, including the major contacts with the p21 peptide. The structure of the amino‐terminal half of the p21 peptide, containing the typical PIP box sequence, is remarkably similar to those observed in the previously reported crystal structures of the human and archaeal PCNA‐PIP box complexes. Meanwhile, the carboxy‐terminal halves of the p21 peptide in the plant PCNA complexes are bound to the protein in a unique manner, most probably because of crystal packing effects. A surface plasmon resonance analysis revealed high affinity between each AtPCNA and the C‐terminal fragment of human p21. This result strongly suggests that the interaction is functionally significant, although no plant homologs of p21 have been identified yet. We also discovered that AtPCNA1 and AtPCNA2 form heterotrimers, implying that hetero‐PCNA rings may play critical roles in cellular signal transduction, particularly in DNA repair.     

Direct role for proliferating cell nuclear antigen in substrate recognition by the E3 ubiquitin ligase CRL4Cdt2

The E3 ubiquitin ligase Cullin-ring ligase 4-Cdt2 (CRL4(Cdt2)) is emerging as an important cell cycle regulator that targets numerous proteins for destruction in S phase and after DNA damage, including Cdt1, p21, and Set8. CRL4(Cdt2) substrates contain a "PIP degron," which consists of a canonical proliferating cell nuclear antigen (PCNA) interaction motif (PIP box) and an adjacent basic amino acid. Substrates use their PIP box to form a binary complex with PCNA on chromatin and the basic residue to recruit CRL4(Cdt2) for substrate ubiquitylation. Using Xenopus egg extracts, we identify an acidic residue in PCNA that is essential to support destruction of all CRL4(Cdt2) substrates. This PCNA residue, which adjoins the basic amino acid of the bound PIP degron, is dispensable for substrate binding to PCNA but essential for CRL4(Cdt2) recruitment to chromatin. Our data show that the interaction of CRL4(Cdt2) with substrates requires molecular determinants not only in the substrate degron but also on PCNA. The results illustrate a potentially general mechanism by which E3 ligases can couple ubiquitylation to the formation of protein-protein interactions.     

A Conserved Sequence Extending Motif III of the Motor Domain in the Snf2-Family DNA Translocase Rad54 Is Critical for ATPase Activity

Rad54 is a dsDNA-dependent ATPase that translocates on duplex DNA. Its ATPase function is essential for homologous recombination, a pathway critical for meiotic chromosome segregation, repair of complex DNA damage, and recovery of stalled or broken replication forks. In recombination, Rad54 cooperates with Rad51 protein and is required to dissociate Rad51 from heteroduplex DNA to allow access by DNA polymerases for recombination-associated DNA synthesis. Sequence analysis revealed that Rad54 contains a perfect match to the consensus PIP box sequence, a widely spread PCNA interaction motif. Indeed, Rad54 interacts directly with PCNA, but this interaction is not mediated by the Rad54 PIP box-like sequence. This sequence is located as an extension of motif III of the Rad54 motor domain and is essential for full Rad54 ATPase activity. Mutations in this motif render Rad54 non-functional in vivo and severely compromise its activities in vitro. Further analysis demonstrated that such mutations affect dsDNA binding, consistent with the location of this sequence motif on the surface of the cleft formed by two RecA-like domains, which likely forms the dsDNA binding site of Rad54. Our study identified a novel sequence motif critical for Rad54 function and showed that even perfect matches to the PIP box consensus may not necessarily identify PCNA interaction sites.     

Different types of interaction between PCNA and PIP boxes contribute to distinct cellular functions of Y-family DNA polymerases

Translesion DNA synthesis (TLS) by the Y-family DNA polymerases Polη, Polι and Polκ, mediated via interaction with proliferating cell nuclear antigen (PCNA), is a crucial pathway that protects human cells against DNA damage. We report that Polη has three PCNA-interacting protein (PIP) boxes (PIP1, 2, 3) that contribute differentially to two distinct functions, stimulation of DNA synthesis and promotion of PCNA ubiquitination. The latter function is strongly associated with formation of nuclear Polη foci, which co-localize with PCNA. We also show that Polκ has two functionally distinct PIP boxes, like Polη, whereas Polι has a single PIP box involved in stimulation of DNA synthesis. All three polymerases were additionally stimulated by mono-ubiquitinated PCNA in vitro. The three PIP boxes and a ubiquitin-binding zinc-finger of Polη exert redundant and additive effects in vivo via distinct molecular mechanisms. These findings provide an integrated picture of the orchestration of TLS polymerases.     

Chromatin Assembly Factor I Mutants Defective for PCNA Binding Require Asf1/Hir Proteins for Silencing

Chromatin assembly factor I (CAF-I) is a conserved histone H3/H4 deposition complex. Saccharomyces cerevisiae mutants lacking CAF-I subunit genes (CAC1 to CAC3) display reduced heterochromatic gene silencing. In a screen for silencing-impaired cac1 alleles, we isolated a mutation that reduced binding to the Cac3p subunit and another that impaired binding to the DNA replication protein PCNA. Surprisingly, mutations in Cac1p that abolished PCNA binding resulted in very minor telomeric silencing defects but caused silencing to be largely dependent on Hir proteins and Asf1p, which together comprise an alternative silencing pathway. Consistent with these phenotypes, mutant CAF-I complexes defective for PCNA binding displayed reduced nucleosome assembly activity in vitro but were stimulated by Asf1p-histone complexes. Furthermore, these mutant CAF-I complexes displayed a reduced preference for depositing histones onto newly replicated DNA. We also observed a weak interaction between Asf1p and Cac2p in vitro, and we hypothesize that this interaction underlies the functional synergy between these histone deposition proteins.     

Polyphosphoinositide micelles and polyphosphoinositide-containing vesicles dissociate endogenous gelsolin-actin complexes and promote actin assembly from the fast-growing end of actin filaments blocked by gelsolin

The Ca2+-activated actin-binding protein gelsolin regulates actin filament length by severing preformed filaments and by binding actin monomers, stabilizing nuclei for their assembly into filaments. Gelsolin binds to phosphatidylinositol 4,5-bisphosphate (PIP2), with consequent inhibition of its filament severing activity and dissociation of EGTA-resistant complexes made with rabbit macrophage or human plasma gelsolin and rabbit muscle actin. This study provides evidence for an interaction of gelsolin with phosphatidylinositol monophosphate (PIP) as well as PIP2 and further describes their effects on gelsolin's function. Both phosphoinositides completely dissociate EGTA-insensitive rabbit macrophage cytoplasmic gelsolin-actin complexes and inhibit gelsolin's severing activity. The magnitude of inhibition depends strongly on the physical state of the phosphoinositides, being maximal in preparations that contain small micelles of either purified PIP or PIP2. Aggregation of PIP or PIP2 micelles by divalent cations or insufficient sonication or their incorporation into vesicles containing other phospholipids decreases but does not eliminate the inhibitory properties of the polyphosphoinositides. The presence of gelsolin partly inhibits the divalent cation-induced aggregation of PIP2 micelles. PIP2 in combination with EGTA inactivates gelsolin molecules that block the fast-growing end of actin filaments, thereby accelerating actin polymerization. Regulation of gelsolin by the intracellular messengers Ca2+ and polyphosphoinositides allows for the formation of several different gelsolin-actin intermediates with distinct functional properties that may be involved in changes in the state of cytoplasmic actin following cell stimulation.     

Interaction of transcriptional intermediary factor 2 nuclear receptor box peptides with the coactivator binding site of estrogen receptor alpha

The activation function 2/ligand-dependent interaction between nuclear receptors and their coregulators is mediated by a short consensus motif, the so-called nuclear receptor (NR) box. Nuclear receptors exhibit distinct preferences for such motifs depending both on the bound ligand and on the NR box sequence. To better understand the structural basis of motif recognition, we characterized the interaction between estrogen receptor alpha and the NR box regions of the p160 coactivator TIF2. We have determined the crystal structures of complexes between the ligand-binding domain of estrogen receptor alpha and 12-mer peptides from the Box B2 and Box B3 regions of TIF2. Surprisingly, the Box B3 module displays an unexpected binding mode that is distinct from the canonical LXXLL interaction observed in other ligand-binding domain/NR box crystal structures. The peptide is shifted along the coactivator binding site in such a way that the interaction motif becomes LXXYL rather than the classical LXXLL. However, analysis of the binding properties of wild type NR box peptides, as well as mutant peptides designed to probe the Box B3 orientation, suggests that the Box B3 peptide primarily adopts the "classical" LXXLL orientation in solution. These results highlight the potential difficulties in interpretation of protein-protein interactions based on co-crystal structures using short peptide motifs.     

Multiple elements in the eIF4G1 N-terminus promote assembly of eIF4G1•PABP mRNPs in vivo

eIF4G is the scaffold subunit of the eIF4F complex, whose binding domains for eIF4E and poly(A)-binding protein (PABP) are thought to enhance formation of activated eIF4F•mRNA•PABP complexes competent to recruit 43S pre-initiation complexes. We found that the RNA-binding region (RNA1) in the N-terminal domain (NTD) of yeast eIF4G1 can functionally substitute for the PABP-binding segment to rescue the function of an eIF4G1-459 mutant impaired for eIF4E binding. Assaying RNA-dependent PABP–eIF4G association in cell extracts suggests that RNA1, the PABP-binding domain, and two conserved elements (Box1 and Box2) between these segments have overlapping functions in forming native eIF4G•mRNA•PABP complexes. In vitro experiments confirm the role of RNA1 in stabilizing eIF4G–mRNA association, and further indicate that RNA1 and Box1 promote PABP binding, in addition to RNA binding, by the eIF4G1 NTD. Our findings indicate that PABP–eIF4G association is only one of several interactions that stabilize eIF4F•mRNA complexes, and emphasize that closed-loop mRNP formation via PABP–eIF4G interaction is non-essential in vivo. Interestingly, two other RNA-binding regions in eIF4G1 have critical functions downstream of eIF4F•mRNA assembly.     

A conserved proliferating cell nuclear antigen-interacting protein sequence in Chk1 is required for checkpoint function

Human checkpoint kinase 1 (Chk1) is an essential kinase required for cell cycle checkpoints and for coordination of DNA synthesis. To gain insight into the mechanisms by which Chk1 carries out these functions, we used mass spectrometry to identify previously uncharacterized interacting partners of Chk1. We describe a novel interaction between Chk1 and proliferating cell nuclear antigen (PCNA), an essential component of the replication machinery. Binding between Chk1 and PCNA was reduced in the presence of hydroxyurea, suggesting that the interaction is regulated by replication stress. A highly conserved PCNA-interacting protein (PIP) box motif was identified in Chk1. The intact PIP box is required for efficient DNA damage-induced phosphorylation and release of activated Chk1 from chromatin. We find that the PIP box of Chk1 is crucial for Chk1-mediated S-M and G(2)-M checkpoint responses. In addition, we show that mutations in the PIP box of Chk1 lead to decreased rates of replication fork progression and increased aberrant replication. These findings suggest an additional mechanism by which essential components of the DNA replication machinery interact with the replication checkpoint apparatus.     

Electrostatic interaction of internal Mg2+ with membrane PIP2 Seen with KCNQ K+ channels

Activity of KCNQ (Kv7) channels requires binding of phosphatidylinositol 4,5-bisphosphate (PIP(2)) from the plasma membrane. We give evidence that Mg(2+) and polyamines weaken the KCNQ channel-phospholipid interaction. Lowering internal Mg(2+) augmented inward and outward KCNQ currents symmetrically, and raising Mg(2+) reduced currents symmetrically. Polyvalent organic cations added to the pipette solution had similar effects. Their potency sequence followed the number of positive charges: putrescine (+2) < spermidine (+3) < spermine (+4) < neomycin (+6) < polylysine (>+6). The inhibitory effects of Mg(2+) were reversible with sequential whole-cell patching. Internal tetraethylammonium ion (TEA) gave classical voltage-dependent block of the pore with changes of the time course of K(+) currents. The effect of polyvalent cations was simpler, symmetric, and without changes of current time course. Overexpression of phosphatidylinositol 4-phosphate 5-kinase Igamma to accelerate synthesis of PIP(2) attenuated the sensitivity to polyvalent cations. We suggest that Mg(2+) and other polycations reduce the currents by electrostatic binding to the negative charges of PIP(2), competitively reducing the amount of free PIP(2) available for interaction with channels. The dose-response curves could be modeled by a competition model that reduces the pool of free PIP(2). This mechanism is likely to modulate many other PIP(2)-dependent ion channels and cellular processes.     

Phosphoinositide-binding peptides derived from the sequences of gelsolin and villin.

The polyphosphoinositides phosphatidylinositol 4-monophosphate (PIP) and phosphatidylinositol 4,5-bisphosphate (PIP2) inactivate the actin filament-severing proteins villin and gelsolin and dissociate them from monomeric and polymeric actin. A potential polyphosphoinositide- (PPI) binding site of human plasma gelsolin regulating filament severing has been localized to the region between residues 150-169 and to the corresponding region in villin which occurs in the second of six homologous domains present in both proteins. Synthetic peptides based on these sequences bind tightly to both PIP and PIP2, in either micelles or bilayer vesicles, compete with gelsolin for binding to PPIs, and dissociate gelsolin-PIP2 complexes, restoring severing activity to the protein. These peptides also bind with moderate affinity to F-actin, suggesting that inactivation of the severing function of the intact proteins by PPIs results from competition between actin and PPIs for a critical binding site on gelsolin-villin. The PPI-binding peptides contain numerous basic amino acids, but their effects on PPIs are far greater than those of Arg or Lys oligomers, a highly basic peptide derived from the calmodulin-binding site of myristoylated, alanine-rich kinase C substrate protein, or the 5-kDa actin-binding protein thymosin beta-4, suggesting that specific aspects of the primary and secondary structure of these basic peptides are important for their interaction with the acidic headgroups of PPIs. In addition to elucidating the structure of PIP2-binding sites in gelsolin, the results describe a sensitive assay for phosphoinositide-binding molecules based on their ability to prevent inhibition of gelsolin function.     

HDM2 ERKs PCNA

In this issue, a study by Groehler and Lannigan (2010. J. Cell Biol. doi:10.1083/jcb.201002124) sheds light on the regulation of proliferating cell nuclear antigen (PCNA) turnover and how it is counteracted by the small chromatin-bound kinase ERK8 (extracellular signal-regulated kinase 8). Importantly, inactivation of ERK8 results in genome instability and is associated with cell transformation.Almost 30 yr ago, proliferating cell nuclear antigen (PCNA) was first identified in dividing cells using sera derived from patients suffering from systemic lupus erythematosus (Takasaki et al., 1981). A few years later, the “mother” of all cancer markers had been associated with DNA synthesis (Madsen and Celis, 1985), but it wasn’t until 1988 that Bauer and Burgers (1988) and Prelich and Stillman (1988) discovered that the homotrimeric clamp served as a processivity factor for DNA polymerases. In 1992, Shivji et al. (1992) showed that PCNA was required for DNA repair, and 10 yr later, it was identified as a target of ubiquitin and SUMO (small ubiquitin-like modifier) conjugation after exposure to ultraviolet light (Hoege et al., 2002). For a protein that has been in the spotlight of modern biochemistry, it is quite remarkable that almost nothing is known about its normal cellular turnover.Insight into this process comes now from the study of an unlikely regulator. In this issue, Groehler and Lannigan (2010) demonstrate that the relatively poorly characterized ERK8 (extracellular signal-regulated kinase 8) takes center stage in the regulation of PCNA stability in primary mammary epithelial cells. The ERK family of kinases belongs to the mitogen-activated protein kinase superfamily and carries a Thr-Glu-Tyr (T-E-Y) activation motif that needs to be phosphorylated to enable kinase activity (Abe et al., 2002). Interestingly, ERK8 also needs to bind to chromatin to become active. The authors identified a highly conserved PXXXP motif in the C-terminal half of ERK8 that appeared to confer autoinhibition, an activity which is relieved upon chromatin binding. Relatively close by, in the middle of ERK8, resides a PCNA-interacting peptide (PIP) box required for the interaction with PCNA (Warbrick, 1998). Curiously, only the chromatin-bound fraction of ERK8 bound to the chromatin-bound fraction of PCNA. However, a functional PIP box was not required for ERK8 to associate with nuclear DNA in the cell. These results argue that ERK8 is not anchored to chromatin by PCNA but associates with it independently. Moreover, they strongly suggest that ERK8’s PIP box binds to PCNA only when the kinase is associated with chromatin. Importantly, overexpression of an ERK8 PIP box mutant resulted in destabilization of PCNA. The effect on PCNA stability seemed to be highly specific, as depletion of ERK8 caused codepletion of PCNA but did not lead to a decrease in steady-state levels of a variety of other cell cycle regulators.Why is the interaction with PCNA confined to chromatin? The reason is likely due to the fact that ERK8’s PIP box is buried in the middle of the protein. Most PCNA-interacting proteins carry their PIP box either at the N or C terminus (Vivona and Kelman, 2003). One other well-studied example for a protein with an internal PIP box is the essential replication factor MCM10 (minichromosome maintenance protein 10). MCM10 undergoes cell cycle–regulated modification, which probably induces a conformational switch that is necessary for the PIP box–mediated interaction with PCNA (Das-Bradoo et al., 2006). In the same vein, it is conceivable that chromatin association and the accompanying relief of autoinhibition of ERK8 cause the middle portion of the kinase to change its configuration, thereby assuming a functional PIP box domain that can be recognized by PCNA. In situations in which the rapid unloading of PCNA is required, regulation of ERK8 may be the most effective way to dispose of chromatin-bound PCNA, which is known to have an exceedingly low exchange rate (Sporbert et al., 2002). Despite the fact that interaction with ERK8 is necessary to stabilize chromatin-bound PCNA, it remains unclear whether PCNA is a direct target of ERK8-mediated phosphorylation.The next goal of Groehler and Lannigan (2010) was to dissect the mechanism underlying the ERK8-regulated degradation of PCNA. Based on the consideration that physical contact between the kinase and PCNA was an integral part of the protection, they hypothesized that ERK8 might compete with an E3 ubiquitin ligase that may target PCNA via its own PIP box. This turned out to be a smart guess because the only candidate to test was the E3 ligase HDM2, the human homologue of murine double minute 2 (Momand et al., 1992). In a set of well-controlled experiments, the authors not only demonstrate that HDM2 interacts directly with and degrades PCNA when ERK8 is absent, but they also exclude indirect effects by p53 and retinoblastoma (Rb) on this process. p53 is a direct target of HDM2 and is stabilized when their interaction is inhibited (Tao and Levine, 1999). Elevated levels of p53 trigger cell cycle arrest concomitant with hypophosphorylation of Rb, but none of these changes affect the stability of PCNA. It is not hard to imagine that the loss of chromatin-bound PCNA has severe consequences for the functionality of DNA replication and repair, resulting in chromosome breakage. The authors argued that a similar level of genome instability should be visible in ERK8-depleted cells. This was indeed the case as visualized by the accumulation of γ-H2AX foci and broken DNA (Rogakou et al., 1998). Importantly, Groehler and Lannigan (2010) observed similar effects in the ERK8 PIP box mutant, further lending credence to their model. It is worthwhile pointing out that the turnover of PCNA expands the spectrum of replication factors whose degradation is tightly linked to chromatin. CDT1, a member of the prereplication complex (Cook, 2009), is rapidly degraded in the face of DNA damage. Its degradation occurs exclusively on the chromatin-associated fraction of the protein pool and is dependent on CDT1 binding to PCNA (Arias and Walter, 2005; Hu and Xiong, 2006; Senga et al., 2006).An important question that this study raises is of course to what extent, if at all, is PCNA turnover deregulated in cancer cells? The commonly high levels of PCNA in transformed cells would be most compatible with a deregulation of ERK8 and/or HDM2 to provide a significant growth advantage. Indeed, the authors show in the last part of their study that in at least two transformed cell lines, PCNA is rendered inert to the presence of ERK8. They speculate that the underlying reason is a defect in HDM2, and although this is the most likely explanation, it still needs to be validated. It will be interesting to see how common the misregulation of PCNA turnover is in cancer tissues. At this point, it is intriguing to envision a dynamic scenario in which a two-step mechanism facilitates cell transformation (Fig. 1). Initially, deregulation of ERK8 may cause PCNA levels to decrease. This would contribute to genome instability and the accumulation of new mutations, including those affecting proper function of HDM2. In step two, deregulation of HDM2 may turn things around and result in an increase of PCNA, supporting rapid proliferation.Open in a separate windowFigure 1.Role of ERK8 in maintaining genome stability. (A) In normal cells, chromatin-bound ERK8 interacts with the chromatin fraction of PCNA, which resides at the replication fork (here just shown at the leading strand for simplicity). ERK8 binding inhibits the E3 ubiquitin ligase HDM2 from interacting with PCNA. (B) In cancer cells, inactivation of ERK8 enables HDM2 to interact with and ubiquitinate PCNA, targeting it for degradation. A decrease in PCNA levels causes an increase in DNA damage, resulting in the accumulation of new mutations. These new mutations may render HDM2 nonfunctional (rectangular form), which ultimately results in an increase of PCNA stability and facilitates cell proliferation. The homotrimeric PCNA structure (Protein Data Bank ID 2OD8) was generated using the Chimera software program (Pettersen et al., 2004).     

Targeting Holliday junctions by origin DNA-binding protein of herpes simplex virus type 1

In the present paper, the interactions of the origin binding protein (OBP) of herpes simplex virus type 1 (HSV1) with synthetic four-way Holliday junctions (HJs) were studied using electrophoresis mobility shift assay and the FRET method and compared with the interactions of the protein with duplex and single-stranded DNAs. It has been found that OBP exhibits a strong preference for binding to four-way and three-way DNA junctions and possesses much lower affinities to duplex and single-stranded DNAs. The protein forms three types of complexes with HJs. It forms complexes I and II which are reminiscent of the tetramer and octamer complexes with four-way junction of HJ-specific protein RuvA of Escherichia coli. The binding approaches saturation level when two OBP dimers are bound per junction. In the presence of Mg²⁺ ions (≥2 mM) OBP also interacts with HJ in the stacked arm form (complex III). In the presence of 5 mM ATP and 10 mM Mg²⁺ ions OBP catalyzes processing of the HJ in which one of the annealed oligonucleotides has a 3′-terminal tail containing 20 unpaired thymine residues. The observed preference of OBP for binding to the four-way DNA junctions provides a basis for suggestion that OBP induces large DNA structural changes upon binding to Box I and Box II sites in OriS. These changes involve the bending and partial melting of the DNA at A+T-rich spacer and also include the formation of HJ containing Box I and Box II inverted repeats and flanking DNA sequences.     

PCNA dependent cellular activities tolerate dramatic perturbations in PCNA client interactions

Proliferating cell nuclear antigen (PCNA) is an essential cofactor for DNA replication and repair, recruiting multiple proteins to their sites of action. We examined the effects of the PCNA^S228I mutation that causes PCNA-associated DNA repair disorder (PARD). Cells from individuals affected by PARD are sensitive to the PCNA inhibitors T3 and T2AA, showing that the S228I mutation has consequences for undamaged cells. Analysis of the binding between PCNA and PCNA-interacting proteins (PIPs) shows that the S228I change dramatically impairs the majority of these interactions, including that of Cdt1, DNMT1, PolD3^p66 and PolD4^p12. In contrast p21 largely retains the ability to bind PCNA^S228I. This property is conferred by the p21 PIP box sequence itself, which is both necessary and sufficient for PCNA^S228I binding. Ubiquitination of PCNA is unaffected by the S228I change, which indirectly alters the structure of the inter-domain connecting loop. Despite the dramatic in vitro effects of the PARD mutation on PIP-degron binding, there are only minor alterations to the stability of p21 and Cdt1 in cells from affected individuals. Overall our data suggests that reduced affinity of PCNA^S228I for specific clients causes subtle cellular defects in undamaged cells which likely contribute to the etiology of PARD.     

PCNA activates the Holliday junction endonuclease Hjc

The resolving enzyme Hjc, which cleaves Holliday junctions with a high degree of structural specificity, is conserved in all archaea. Like RuvC in Escherichia coli, Hjc functions in the related processes of homologous recombination and double-strand break repair. In bacteria, the RuvAB complex binds Holliday junctions and catalyses ATP-dependent branch migration, but the equivalent proteins in archaea and eukarya are unknown. Here, we demonstrate that Hjc from Sulfolobus solfataricus forms a physical interaction with the sliding clamp PCNA via a C-terminal PCNA-interacting peptide (PIP) motif in Hjc. PCNA stimulates the Holliday junction cleavage activity of Hjc in vitro, and deletion of the PIP motif abrogates this effect. This is the first report of a functional interaction between a sliding clamp and a junction-resolving enzyme, and raises the possibility that PCNA could recruit a variety of different proteins to act on Holliday junctions in vivo.