PCNA Binding Research Articles

Kinetic analysis of PCNA clamp binding and release in the clamp loading reaction catalyzed by Saccharomyces cerevisiae replication factor C

DNA polymerases require a sliding clamp to achieve processive DNA synthesis. The toroidal clamps are loaded onto DNA by clamp loaders, members of the AAA+family of ATPases. These enzymes utilize the energy of ATP binding and hydrolysis to perform a variety of cellular functions. In this study, a clamp loader-clamp binding assay was developed to measure the rates of ATP-dependent clamp binding and ATP-hydrolysis-dependent clamp release for the Saccharomyces cerevisiae clamp loader (RFC) and clamp (PCNA). Pre-steady-state kinetics of PCNA binding showed that although ATP binding to RFC increases affinity for PCNA, ATP binding rates and ATP-dependent conformational changes in RFC are fast relative to PCNA binding rates. Interestingly, RFC binds PCNA faster than the Escherichia coli γ complex clamp loader binds the β-clamp. In the process of loading clamps on DNA, RFC maintains contact with PCNA while PCNA closes, as the observed rate of PCNA closing is faster than the rate of PCNA release, precluding the possibility of an open clamp dissociating from DNA. Rates of clamp closing and release are not dependent on the rate of the DNA binding step and are also slower than reported rates of ATP hydrolysis, showing that these rates reflect unique intramolecular reaction steps in the clamp loading pathway.

Flexibility of PCNA-protein interface accommodates differential binding partners.

The expanding roles of PCNA in functional assembly of DNA replication and repair complexes motivated investigation of the structural and dynamic properties guiding specificity of PCNA-protein interactions. A series of biochemical and computational analyses were combined to evaluate the PIP Box recognition features impacting complex formation. The results indicate subtle differences in topological and molecular descriptors distinguishing both affinity and stoichiometry of binding among PCNA-peptide complexes through cooperative effects. These features were validated using peptide mimics of p85α and Akt, two previously unreported PCNA binding partners. This study characterizes for the first time a reverse PIP Box interaction with PCNA. Small molecule ligand binding at the PIP Box interaction site confirmed the adaptive nature of the protein in dictating overall shape and implicates allosterism in transmitting biological effects.

The ADP-ribosyltransferase PARP10/ARTD10 Interacts with Proliferating Cell Nuclear Antigen (PCNA) and Is Required for DNA Damage Tolerance

All cells rely on genomic stability mechanisms to protect against DNA alterations. PCNA is a master regulator of DNA replication and S-phase-coupled repair. PCNA post-translational modifications by ubiquitination and SUMOylation dictate how cells stabilize and re-start replication forks stalled at sites of damaged DNA. PCNA mono-ubiquitination recruits low fidelity DNA polymerases to promote error-prone replication across DNA lesions. Here, we identify the mono-ADP-ribosyltransferase PARP10/ARTD10 as a novel PCNA binding partner. PARP10 knockdown results in genomic instability and DNA damage hypersensitivity. Importantly, we show that PARP10 binding to PCNA is required for translesion DNA synthesis. Our work identifies a novel PCNA-linked mechanism for genome protection, centered on post-translational modification by mono-ADP-ribosylation.

The PCNA binding domain of Rad2p plays a role in mutagenesis by modulating the cell cycle in response to DNA damage

The xeroderma pigmentosum group G (XPG) gene, encoding an essential element in nucleotide excision repair (NER), has a proliferating cell nuclear antigen-binding domain (PCNA-BD) at its C-terminal region. However, the role of this domain is controversial because its presence does not affect NER. Using yeast RAD2, a homolog of human XPG, we show that Rad2p interacts with PCNA through its PCNA-BD and the PCNA-BD of Rad2p plays a role in UV-induced mutagenesis. While a mutation of Rad2p endonuclease activity alone causes dramatically increased mutation rates and UV sensitivity, as well as growth retardation after UV irradiation, a mutation of the Rad2p PCNA-BD in the same mutant causes dramatically decreased mutation rates, reduced UV sensitivity and increased growth rate after UV irradiation. After UV irradiation, large-budded cells of Rad2p endonuclease defective mutants wane due to a mutation of the Rad2p PCNA-BD. Besides, the Rad2p PCNA-BD mutant protein exhibits alleviated PCNA-binding efficiency. These results show a hitherto unsuspected role of the Rad2p PCNA-BD that controls mutagenesis via cell cycle modulation together with PCNA. Furthermore, the high mutation rate of cells with other NER gene mutations was also decreased by the mutation of the Rad2p PCNA-BD, which indicates that the Rad2p-PCNA interaction might be responsible for mutagenesis control in the general NER pathway. Our results suggest that the drastically increased incidence of skin cancer in xeroderma pigmentosum patients could arise from the synergistic effects between cell cycle arrest due to the XPG-PCNA interaction and the accumulation of damaged DNA via defects in DNA damage repair.

Degradation of p12 Subunit by CRL4Cdt2 E3 Ligase Inhibits Fork Progression after DNA Damage

After acute DNA damage, the cell arrests S-phase progression by inhibiting origin initiation and fork progression to repair damaged DNA. The intra-S-phase checkpoint kinase Chk1 phosphorylates Cdc25A to target the latter for degradation by CRL1(β-TrCP) and so inhibit origin firing. The mechanism for inhibiting fork progression, however, has not been identified. Here, we show that degradation of p12, the fourth subunit of DNA polymerase δ, is critical for inhibiting fork progression. CRL4(Cdt2) is an E3 ligase that ubiquitinates and degrades p12 after UV treatment. Cells expressing a stable form of p12 exhibit UV-resistant DNA synthesis. DNA fiber assay and alkaline-sucrose gradient assay demonstrate that the impairment of fork progression after DNA damage requires p12 degradation. These results suggest that ubiquitination of p12 through CRL4(Cdt2) and subsequent degradation form one mechanism by which a cell responds to DNA damage to inhibit fork progression.

Abstract 1281: POLD3 is required for DNA damage response to endogenous and exogenous DNA damage in human cells.

Abstract Methyl methanesulfonate (MMS) induces mutations in a nonlinear (hockey-stick-shaped) dose-response curve in DT40 cells, about 55% of which are located at A:T sites with a high frequency of A to T mutations. We hypothesized that these transversion mutations are due to base excision repair (BER) intermediates, such as abasic sites and single strand breaks derived from N3-methyladenine. One candidate polymerase to bypass abasic sites is polymerase delta (POLD). POLD is responsible for the lagging strand during DNA replication. POLD is referred to as a high fidelity DNA polymerase because of its ability to discriminate deoxynucleotides and its 3’->5’ exo-nuclease activity. One of the POLD subunits, POLD3, is important to recruit PCNA binding to the POLD core enzyme and is implied to have critical roles for translesion DNA synthesis. In yeast, cells deficient in POLD3 homologous gene POL32 are sensitive to DNA damaging agents and resistant to mutations caused by mutagens, such as MMS and UV. Here we generated POLD3 knock-down (KD) cells via lentivirus based shRNA in three different human cell lines. All POLD3 KD cells grow slower than the control cells, suggesting POLD3 function tolerates endogenous DNA lesions. Furthermore, we found that POLD3 KD cells were sensitive to various genotoxic agents including MMS and potassium chromate. To further investigate the role of POLD3 in the DNA damage response, we introduced a series of deletion mutations of POLD3 in POLD3 deficient cells. We found that both the POLD2 binding domain and the middle linker domain are critical for the activity of POLD3 while the C-terminal PCNA binding domain is dispensable. While there is little homology between human POLD3 and yeast POL32, function of these two proteins appears to be quite similar in structure biology analysis. Our results also suggest that POLD3 plays important roles to bypass DNA lesions caused by endogenous and various exogenous genotoxic agents. Citation Format: Xu Tian, James Swenberg, Jun Nakamura. POLD3 is required for DNA damage response to endogenous and exogenous DNA damage in human cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1281. doi:10.1158/1538-7445.AM2013-1281

Systems-wide analysis of ubiquitylation dynamics reveals a key role for PAF15 ubiquitylation in DNA-damage bypass

Protein ubiquitylation has emerged as a key regulatory mechanism in DNA-damage signalling and repair pathways. We report a proteome-wide, site-specific survey of ubiquitylation changes after ultraviolet irradiation, identifying numerous upregulated and downregulated ubiquitylation sites on known components of DNA-damage signalling, as well as on proteins not previously implicated in this process. Our results uncover a critical role for PCNA-associated factor PAF15 (p15(PAF)/KIAA0101) ubiquitylation during DNA replication. During unperturbed S phase, chromatin-associated PAF15 is modified by double mono-ubiquitylation of Lys 15 and 24 templated through PCNA binding. Replication blocks trigger rapid, proteasome-dependent removal of Lys 15/24-ubiquitylated PAF15 from PCNA, facilitating bypass of replication-fork-blocking lesions by allowing recruitment of translesion DNA synthesis polymerase polη to mono-ubiquitylated PCNA at stalled replisomes. Our findings demonstrate widespread involvement of ubiquitin signalling in genotoxic-stress responses and identify a critical function for dynamic PAF15 ubiquitylation in safeguarding genome integrity when DNA replication is challenged.

Molecular Cloning, Expression, and Characterization of Starfish DNA (Cytosine-5)-methyltransferases

To determine whether and if so how a DNA methylation-dependent epigenetic mechanism for transcriptional gene silencing functions in Echinoderms, we cloned and sequenced dnmt1 and dnmt3 cDNAs of the starfish Asterina pectinifera. Since the Strongylocentrotus purpuratus genome has only two loci of DNA (cytosine-5)-methyltransferase genes encoding Dnmt1 and Dnmt3, they might constitute a sufficient set of dnmt genes in Echinoderms. The starfish Dnmt3 whose cDNA we cloned showed highest homology to a mammalian Dnmt3a2 splicing variant. Essentially all the characteristic motifs and sequences of the mammalian counterparts were found in the starfish Dnmts as well, except that a typical PCNA binding domain motif was lacking in the starfish Dnmt1. RT-PCR analysis indicated that the dnmt1 mRNA exists in both ovary and oocytes, but its levels in other tissues were very low or almost negligible. In contrast, the dnmt3 mRNA was detected only in the ovary, and not at all in the oocytes. The size of a dnmt1 transcript was about 6.5 kb on Northern blot analysis. On heterologous expression, the starfish Dnmt1 protein was expressed in insect cells in catalytically active form.

Mutations in the PCNA-binding domain of CDKN1C cause IMAGe syndrome

IMAGe syndrome (intrauterine growth restriction, metaphyseal dysplasia, adrenal hypoplasia congenita and genital anomalies) is an undergrowth developmental disorder with life-threatening consequences. An identity-by-descent analysis in a family with IMAGe syndrome identified a 17.2-Mb locus on chromosome 11p15 that segregated in the affected family members. Targeted exon array capture of the disease locus, followed by high-throughput genomic sequencing and validation by dideoxy sequencing, identified missense mutations in the imprinted gene CDKN1C (also known as P57KIP2) in two familial and four unrelated patients. A familial analysis showed an imprinted mode of inheritance in which only maternal transmission of the mutation resulted in IMAGe syndrome. CDKN1C inhibits cell-cycle progression, and we found that targeted expression of IMAGe-associated CDKN1C mutations in Drosophila caused severe eye growth defects compared to wild-type CDKN1C, suggesting a gain-of-function mechanism. All IMAGe-associated mutations clustered in the PCNA-binding domain of CDKN1C and resulted in loss of PCNA binding, distinguishing them from the mutations of CDKN1C that cause Beckwith-Wiedemann syndrome, an overgrowth syndrome.

Overcoming natural replication barriers: differential helicase requirements

DNA sequences that form secondary structures or bind protein complexes are known barriers to replication and potential inducers of genome instability. In order to determine which helicases facilitate DNA replication across these barriers, we analyzed fork progression through them in wild-type and mutant yeast cells, using 2-dimensional gel-electrophoretic analysis of the replication intermediates. We show that the Srs2 protein facilitates replication of hairpin-forming CGG/CCG repeats and prevents chromosome fragility at the repeat, whereas it does not affect replication of G-quadruplex forming sequences or a protein-bound repeat. Srs2 helicase activity is required for hairpin unwinding and fork progression. Also, the PCNA binding domain of Srs2 is required for its in vivo role of replication through hairpins. In contrast, the absence of Sgs1 or Pif1 helicases did not inhibit replication through structural barriers, though Pif1 did facilitate replication of a telomeric protein barrier. Interestingly, replication through a protein barrier but not a DNA structure barrier was modulated by nucleotide pool levels, illuminating a different mechanism by which cells can regulate fork progression through protein-mediated stall sites. Our analyses reveal fundamental differences in the replication of DNA structural versus protein barriers, with Srs2 helicase activity exclusively required for fork progression through hairpin structures.

Abstract 5361: DNA damage by platinum-based drugs induces p21-dependent recruitment of PCNA as a homotrimer into the inhibited G1-phase CDK complex

Abstract DNA damage results in activation of cell cycle checkpoints and cell cycle arrest as a result of inhibition of cyclin-dependent kinases (CDK). In G1 phase, checkpoint activation is often associated with inhibition of CDK4/cyclin D1 and CDK2/cyclin E activities, usually by p21 in a p53-dependent manner. Our investigations in A2780 ovarian tumor cells with cisplatin and 1R,2R-diaminocyclohexane-(trans-diacetato)(dichloro)-platinum(IV) (DAP), a platinum analog that circumvents cisplatin resistance, demonstrated that both agents activated the p53-p21 pathway, which inhibited G1-phase CDK4 and CDK2 kinases, beginning at ∼18 h after drug treatment. However, biochemical analysis of the inhibited complex demonstrated an increase in size by ∼100-150 kDa, which was not consistent with binding of p21 alone and the anticipated stoichiometry of 1 for CDK:p21. In order to account for the p21-dependent increase in the size of the CDK complex, we undertook large-scale immunoprecipitation with antibodies to cyclin D (for the CDK4 complex) or cyclin E (for the CDK2 complex) following DAP treatment, and analyzed the inhibited complex by mass spectrometry for additional recruited proteins. This analysis revealed only one additional protein, namely PCNA, in the inhibited CDK2 and CDK4 complex. As confirmation of p21-dependence, immunodepletion of p21 in boiled cell lysates from DAP-treated cells abolished the binding of PCNA to the CDK complex in a cell-free binding assay. Similar p27 depletion did not prevent the binding of PCNA to the complex. However, PCNA binding with p21 at a stocihiometry of 1 was insufficient to account for the increase in molecular size of the inhibited CDK complex, and it was likely that PCNA was present in its trimeric state. This was confirmed by employing mutant PCNA (Y114A) which could not form the homotrimer and, consequently, was unable to bind to p21 and become recruited into the CDK complex. Moreover, inhibiting the binding of PCNA to p21 did not prevent p21 from inhibiting CDK2, and this indicated that PCNA is not vital for p21-dependent inhibition of kinase activity. On the other hand, knockdown of PCNA by siRNA in A2780 cells potentiated the cytotoxicity of DAP, whereas p21 knockout in HCT-116(p21-/-) cells decreased the sensitivity to this agent. Taken together, our data demonstrate that p21-dependent recruitment of PCNA as a homotrimer substantially increases the molecular size of the CDK complex, but whereas PCNA recruitment does not contribute to p21-dependent inhibition of the kinase, it may attenuate the effect of p21 on drug-induced cytotoxicity. (Supported by NCI RO1 CA127263). Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5361. doi:10.1158/1538-7445.AM2011-5361

PARG is recruited to DNA damage sites through poly(ADP-ribose)- and PCNA-dependent mechanisms

Post-translational poly(ADP-ribosyl)ation has diverse essential functions in the cellular response to DNA damage as it contributes to avid DNA damage detection and assembly of the cellular repair machinery but extensive modification eventually also induces cell death. While there are 17 human poly(ADP-ribose) polymerase (PARP) genes, there is only one poly(ADP-ribose) glycohydrolase (PARG) gene encoding several PARG isoforms located in different subcellular compartments. To investigate the recruitment of PARG isoforms to DNA repair sites we locally introduced DNA damage by laser microirradiation. All PARG isoforms were recruited to DNA damage sites except for a mitochondrial localized PARG fragment. Using PARP knock out cells and PARP inhibitors, we showed that PARG recruitment was only partially dependent on PARP-1 and PAR synthesis, indicating a second, PAR-independent recruitment mechanism. We found that PARG interacts with PCNA, mapped a PCNA binding site and showed that binding to PCNA contributes to PARG recruitment to DNA damage sites. This dual recruitment mode of the only nuclear PARG via the versatile loading platform PCNA and by a PAR dependent mechanism likely contributes to the dynamic regulation of this posttranslational modification and ensures the tight control of the switch between efficient DNA repair and cell death.

Mapping the FEN1 interaction domain with hTERT

The activity of telomerase in cancer cells is tightly regulated by numerous proteins including DNA replication factors. However, it is unclear how replication proteins regulate telomerase action in higher eukaryotic cells. Previously we have demonstrated that the multifunctional DNA replication and repair protein flap endonuclease 1 (FEN1) is in complex with telomerase and may regulate telomerase activity in mammalian cells. In this study, we further analyzed the nature of this association. Our results show that FEN1 and telomerase association occurs throughout the S phase, with the maximum association in the mid S phase. We further mapped the physical domains in FEN1 required for this association and found that the C-terminus and the nuclease domain of FEN1 are involved in this interaction, whereas the PCNA binding ability of FEN1 is dispensable for the interaction. These results provide insights into the nature of possible protein–protein associations that telomerase participates in for maintaining functional telomeres.

PCNA directs type 2 RNase H activity on DNA replication and repair substrates

Ribonuclease H2 is the major nuclear enzyme degrading cellular RNA/DNA hybrids in eukaryotes and the sole nuclease known to be able to hydrolyze ribonucleotides misincorporated during genomic replication. Mutation in RNASEH2 causes Aicardi–Goutières syndrome, an auto-inflammatory disorder that may arise from nucleic acid byproducts generated during DNA replication. Here, we report the crystal structures of Archaeoglobus fulgidus RNase HII in complex with PCNA, and human PCNA bound to a C-terminal peptide of RNASEH2B. In the archaeal structure, three binding modes are observed as the enzyme rotates about a flexible hinge while anchored to PCNA by its PIP-box motif. PCNA binding promotes RNase HII activity in a hinge-dependent manner. It enhances both cleavage of ribonucleotides misincorporated in DNA duplexes, and the comprehensive hydrolysis of RNA primers formed during Okazaki fragment maturation. In addition, PCNA imposes strand specificity on enzyme function, and by localizing RNase H2 and not RNase H1 to nuclear replication foci in vivo it ensures that RNase H2 is the dominant RNase H activity during nuclear replication. Our findings provide insights into how type 2 RNase H activity is directed during genome replication and repair, and suggest a mechanism by which RNase H2 may suppress generation of immunostimulatory nucleic acids.

Bi-directional routing of DNA mismatch repair protein human exonuclease 1 to replication foci and DNA double strand breaks

Human exonuclease 1 (hEXO1) is implicated in DNA metabolism, including replication, recombination and repair, substantiated by its interactions with PCNA, DNA helicases BLM and WRN, and several DNA mismatch repair (MMR) proteins. We investigated the sub-nuclear localization of hEXO1 during S-phase progression and in response to laser-induced DNA double strand breaks (DSBs). We show that hEXO1 and PCNA co-localize in replication foci. This apparent interaction is sustained throughout S-phase. We also demonstrate that hEXO1 is rapidly recruited to DNA DSBs. We have identified a PCNA interacting protein (PIP-box) region on hEXO1 located in its COOH-terminal ( 788QIKLNELW 795). This motif is essential for PCNA binding and co-localization during S-phase. Recruitment of hEXO1 to DNA DSB sites is dependent on the MMR protein hMLH1. We show that two distinct hMLH1 interaction regions of hEXO1 (residues 390–490 and 787–846) are required to direct the protein to the DNA damage site. Our results reveal that protein domains in hEXO1 in conjunction with specific protein interactions control bi-directional routing of hEXO1 between on-going DNA replication and repair processes in living cells.

Role of Amblyomin-X on microcirculatory vessels, endothelial cell migration and tumor growth

Role of Amblyomin-X on microcirculatory vessels, endothelial cell migration and tumor growth

Abstract 485: Role of DNA methyltransferase I (DNMT1) in the DNA damage response

Abstract DNA methylation is one of the major epigenetic modifications regulating biological processes such as normal development, chromosomal stability, and gene regulation. Aberrant hypermethylation of promoter CpG islands is an important mechanism for the inactivation of tumor suppressor genes and is mediated by a family of DNA methyltransferases (DNMTs). The loss of DNMTs results in genome-wide demethylation which causes alterations in gene expression. Recent studies showed that DNMT1, which maintains DNA methylation during DNA replication, localizes to UV laser-induced DNA damage areas. The aim of this study was to understand the mechanism of the recruitment of DNMT1 to areas of DNA damage and to investigate the function of DNMT1 in DNA damage repair pathways. Our preliminary results demonstrate that DNMT1 and PCNA localize to 800 nm laser-induced DNA damage, which induces DNA double strand breaks, by live cell imaging, but not other DNMTs like DNMT3A and DNMT3B. Catalytic subunit mutated (Cys1229Ala) DNMT1 also showed localization to DNA damage, but its recruitment was slow compared to wild type DNMT1. DNMT1 is targeted to sites of DNA replication via its PCNA binding domain (PBD) during S phase. To test whether PCNA is responsible for the recruitment of DNMT1 during DNA repair, we deleted the PBD (DNMT1 ΔPBD) and measured its localization. Unlike wild type DNMT1, DNMT1 ΔPBD remained diffuse and showed no accumulation at irradiated sites. 5-aza-2’-deoxycytidine (5-azadC) treatment, a drug which incorporates into DNA and covalently traps the DNMTs on DNA, results in the inhibition of DNMT1 recruitment to DNA damage areas, without affecting PCNA recruitment. To further investigate the mechanism of DNMT1 accumulation to DNA damage sites, we used drugs that inhibit known DNA damage repair proteins. After transfection of DNMT1 or PCNA, cells were treated with NU7026 (DNA-PKcs inhibitor), UCN-01 (CHK1 inhibitor) or caffeine (ATM and ATR inhibitor). DNMT1 and PCNA accumulation were not affected by NU7026 treatment, but UCN-01 and caffeine significantly inhibited both DNMT1 and PCNA accumulation to the irradiated region, indicating CHK1 activation is required for DNMT1/PCNA accumulation. Using immunofluorescence analyses after laser irradiation, we observed DNMT1 accumulation at DNA damage sites up to 2 hr after irradiation. Unlike DNMT1, PCNA accumulation was observed up to 6 hr after DNA damage in both S phase and non-S phase cells. To understand the role of DNMT1 in the DNA damage repair response, we used HCT116 cells deficient in DNMT1. Under normal conditions, γH2AX and phospho-ATM (pATM) levels, markers of DNA double strand breaks, were significantly increased in this mutant cell line. However, their recruitment to DNA damage sites was not changed compared to parental HCT116. Taken together our data suggest that CHK1 regulates the recruitment of DNMT1 and PCNA to DNA damage sites and DNMT1 is also involved in the regulation of DNA damage repair. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 485.

Docking of a Specialized PIP Box onto Chromatin-Bound PCNA Creates a Degron for the Ubiquitin Ligase CRL4Cdt2

Substrates of the E3 ubiquitin ligase CRL4(Cdt2), including Cdt1 and p21, contain a PCNA-binding motif called a PIP box. Upon binding of the PIP box to PCNA on chromatin, CRL4(Cdt2) is recruited and the substrate is ubiquitylated. Importantly, a PIP box cannot be sufficient for destruction, as most PIP box proteins are stable. Using Xenopus egg extracts, we identify two sequence elements in CRL4(Cdt2) substrates that promote their proteolysis: a specialized PIP box that confers exceptionally efficient PCNA binding and a basic amino acid 4 residues downstream of the PIP box, which recruits CRL4(Cdt2) to the substrate-PCNA complex. We also identify two mechanisms that couple CRL4(Cdt2)-dependent proteolysis to the chromatin-bound form of PCNA, ensuring that this proteolysis pathway is active only in S phase or after DNA damage. Thus, CRL4(Cdt2) recognizes an unusual degron, which is assembled specifically on chromatin via the binding of a specialized PIP box to PCNA.

The ATPase Reaction of S. cerevisiae RFC Reveals a Finely Choreographed Mechanism for Loading PCNA Clamps on DNA

Circular clamp proteins enhance the processivity of DNA replication and participate in numerous other DNA metabolic and cellular processes. Replication factor C (RFC) loads PCNA clamps onto DNA, and our analysis of S. cerevisiae RFC ATPase kinetics reveals new information on the mechanism whereby energy from ATP binding and hydrolysis applied to this task. We find that ATP binding drives a slow, rate-limiting change in RFC conformation that is necessary for DNA binding, which in turn causes rapid ATP hydrolysis, DNA dissociation and catalytic turnover. PCNA accelerates this slow step, leading to formation of an RFC-ATP-PCNA(open) intermediate that allows DNA entry into the clamp. Thus, the conformational change serves as a checkpoint in favor of PCNA binding and opening, before DNA binds RFC and triggers ATP hydrolysis to end the reaction. The data showcase the remarkable choreography of the reaction components that results in circular clamps linked topologically to DNA.

A Vanishing Act, Explained

In a paper in this issue of Developmental Cell, Shibutani et al. (2008) uncover the mechanism that underlies tightly regulated S-phase degradation of Drosophila E2F1 during development. They show that dE2F1 is degraded by the Cul4(Cdt2) ubiquitin ligase in a manner that resembles the DNA replication-dependent turnover of Cdt1.