Repair complexes of FEN1 endonuclease, DNA, and Rad9-Hus1-Rad1 are distinguished from their PCNA counterparts by functionally important stability

Processivity clamps such as proliferating cell nuclear antigen (PCNA) and the checkpoint sliding clamp Rad9/Rad1/Hus1 (9-1-1) act as versatile scaffolds in the coordinated recruitment of proteins involved in DNA replication, cell-cycle control, and DNA repair. Association and handoff of DNA-editing enzymes, such as flap endonuclease 1 (FEN1), with sliding clamps are key processes in biology, which are incompletely understood from a mechanistic point of view. We have used an integrative computational and experimental approach to define the assemblies of FEN1 with double-flap DNA substrates and either proliferating cell nuclear antigen or the checkpoint sliding clamp 9-1-1. Fully atomistic models of these two ternary complexes were developed and refined through extensive molecular dynamics simulations to expose their conformational dynamics. Clustering analysis revealed the most dominant conformations accessible to the complexes. The cluster centroids were subsequently used in conjunction with single-particle electron microscopy data to obtain a 3D EM reconstruction of the human 9-1-1/FEN1/DNA assembly at 18-Å resolution. Comparing the structures of the complexes revealed key differences in the orientation and interactions of FEN1 and double-flap DNA with the two clamps that are consistent with their respective functions in providing inherent flexibility for lagging strand DNA replication or inherent stability for DNA repair.Flap endonuclease 1 (FEN1) belongs to a class of essential nucleases (the FEN1 5′ nuclease superfamily) present in all domains of life (1). FEN1 catalyzes the endonucleolytic cleavage of bifurcated DNA or RNA structures known as 5′ flaps. These 5′ flaps are generated during lagging strand DNA synthesis or during long-patch base excision repair. The FEN1 substrates are in fact double-flap DNA (dfDNA) with DNA on the opposite side of the 5′ flap, forming a single nucleotide 3′ flap when bound to the enzyme (2, 3). By removing the 5′ ssDNA or RNA flap from such substrates, FEN1 produces a single nicked product that could be sealed by the subsequent action of a DNA ligase (4). Consistent with its crucial role in DNA replication and repair, FEN1 is highly expressed in all proliferative tissues, and its activity is key for the maintenance of genomic integrity (5). FEN1 has been identified as a cancer susceptibility gene, and mutations in it have been linked to a number of genetic diseases, such as EM map myotonic dystrophy, Huntington disease, several ataxias, fragile X syndrome, and cancer (6–10).The nuclease activity of FEN1 can be stimulated by association with processivity clamps such as proliferating cell nuclear antigen (PCNA), which encircle DNA at sites of replication and repair (11–13). PCNA is a recognized master coordinator of cellular responses to DNA damage and interacts with numerous DNA repair and cell-cycle control proteins. In this capacity, PCNA serves not only as a mobile platform for the attachment of these proteins to DNA but, importantly, plays an active role in the recruitment and release of these crucial participants at the replication fork (14, 15). The dominant mode of interaction for many of these factors is through attachment to the interdomain connector (IDC) loop of PCNA and the PCNA C terminus (2, 13, 16). The trimeric PCNA ring can provide, at most, three binding sites for replication and repair factors. The crystal structure of human FEN1 with PCNA indeed revealed three FEN1 enzymes bound to the sliding clamp in different orientations (16). Additionally, a biochemical study of the Sulfolobus solfataricus proteins supported the idea that distinct protein partners such as DNA polymerase, FEN1, and DNA ligase could simultaneously associate with PCNA (17). The competition among proteins to simultaneously bind to the surface of PCNA as well as to their common DNA substrate has led to the notion of conformational switching and handoffs of repair intermediates (2, 17); these are key processes in PCNA biology, which are incompletely understood from a mechanistic perspective.In addition to PCNA, FEN1 is known to associate with the alternative checkpoint clamp Rad9-Rad1-Hus1 (9-1-1 complex). Whereas PCNA is comprised of three identical subunits, 9-1-1 is a heterotrimeric sliding clamp (18–20). This fact reflects the different protein partners the two clamps engage and the distinct roles these complexes play in coordinating DNA processing. In contrast to PCNA, the 9-1-1 complex is thought to serve as a recruitment platform to bring checkpoint effector kinases to sites of DNA damage, thus activating checkpoint control, and also functions to stabilize stalled replication forks that have encountered DNA lesions (21–23). It has also been demonstrated that 9-1-1 interacts with and stimulates enzymes involved in base excision repair (BER), such as NEIL1, MYH, TDG, FEN1, and DNA Ligase I, thus potentially linking BER activities to checkpoint coordination (24–27).In view of their crucial involvement in replication and repair, a detailed structural comparison of the ternary PCNA/FEN1/DNA and 9-1-1/FEN1/DNA complexes would be of great value. Though structural snapshots are available for the individual components of such assemblies (PCNA [Protein Data Bank (PDB) ID code: 1VYM], human 9-1-1 [3GGR]) (Fig. S1) and for two binary complexes [FEN1/DNA (3Q8L) and FEN1/PCNA (1UL1)] (3, 16, 18–20, 28), the larger ternary assemblies present extreme challenges to molecular crystallography (MX). Here we report models for the ternary PCNA/FEN1/DNA and 9-1-1/FEN1/DNA assemblies, which were constructed by combining all available high-resolution MX data for the individual components and subassemblies. The models were refined by multinanosecond atomistic molecular dynamics (MD) simulations. Single-particle electron microscopy (EM) of negatively stained samples indicated that the structure defined by the 9-1-1/FEN1/DNA model exists in solution. Subsequently, the computational model was integrated with the EM data resulting in a 3D reconstruction for the ternary assembly determined at 18-Å resolution. Finally, we present a detailed comparative analysis of the two ternary complexes.     

Ubiquitinated proliferating cell nuclear antigen activates translesion DNA polymerases eta and REV1

In response to DNA damage, the Rad6/Rad18 ubiquitin-conjugating complex monoubiquitinates the replication clamp proliferating cell nuclear antigen (PCNA) at Lys-164. Although ubiquitination of PCNA is recognized as an essential step in initiating postreplication repair, the mechanistic relevance of this modification has remained elusive. Here, we describe a robust in vitro system that ubiquitinates yeast PCNA specifically on Lys-164. Significantly, only those PCNA clamps that are appropriately loaded around effector DNA by its loader, replication factor C, are ubiquitinated. This observation suggests that, in vitro, only PCNA present at stalled replication forks is ubiquitinated. Ubiquitinated PCNA displays the same replicative functions as unmodified PCNA. These functions include loading onto DNA by replication factor C, as well as Okazaki fragment synthesis and maturation by the PCNA-coordinated actions of DNA polymerase delta, the flap endonuclease FEN1, and DNA ligase I. However, whereas the activity of DNA polymerase zeta remains unaffected by ubiquitination of PCNA, ubiquitinated PCNA specifically activates two key enzymes in translesion synthesis: DNA polymerase eta, the yeast Xeroderma pigmentosum ortholog, and Rev1, a deoxycytidyl transferase that functions in organizing the mutagenic DNA replication machinery. We propose that ubiquitination of PCNA increases its functionality as a sliding clamp to promote mutagenic DNA replication.     

An 8-oxo-guanine repair pathway coordinated by MUTYH glycosylase and DNA polymerase λ

Reactive oxygen species (ROS) interact with DNA, frequently generating highly mutagenic 7,8-dihydro-8-oxoguanine (8-oxo-G) lesions. Replicative DNA polymerases (pols) often misincorporate adenine opposite 8-oxo-G. The subsequent repair mechanism allowing the removal of adenine and formation of C:8-oxo-G base pair is essential to prevent C:G to A:T transversion mutations. Here, we show by immunofluorescence experiments, in cells exposed to ROS, the involvement of MutY glycosylase homologue (MUTYH) and DNA pol λ in the repair of A:8-oxo-G mispairs. We observe specific recruitment of MUTYH, DNA pol λ, proliferating cell nuclear antigen (PCNA), flap endonuclease 1 (FEN1) and DNA ligases I and III from human cell extracts to A:8-oxo-G DNA, but not to undamaged DNA. Using purified human proteins and a DNA template, we reconstitute the full pathway for the faithful repair of A:8-oxo-G mispairs involving MUTYH, DNA pol λ, FEN1, and DNA ligase I. These results reveal a cellular response pathway to ROS, important to sustain genomic stability and modulate carcinogenesis.     

Proliferating cell nuclear antigen (PCNA)-associated KIAA0101/PAF15 protein is a cell cycle-regulated anaphase-promoting complex/cyclosome substrate

The anaphase-promoting complex/cyclosome (APC/C) is a cell cycle-regulated E3 ubiquitin ligase that controls the degradation of substrate proteins at mitotic exit and throughout the G1 phase. We have identified an APC/C substrate and cell cycle-regulated protein, KIAA0101/PAF15. PAF15 protein levels peak in the G2/M phase of the cell cycle and drop rapidly at mitotic exit in an APC/C- and KEN-box-dependent fashion. PAF15 associates with proliferating cell nuclear antigen (PCNA), and depletion of PAF15 decreases the number of cells in S phase, suggesting a role for it in cell cycle regulation. Following irradiation, PAF15 colocalized with γH2AX foci at sites of DNA damage through its interaction with PCNA. Finally, PAF15 depletion led to an increase in homologous recombination-mediated DNA repair, and overexpression caused sensitivity to UV-induced DNA damage. We conclude that PAF15 is an APC/C-regulated protein involved in both cell cycle progression and the DNA damage response.     

Dual inactivation of Hus1 and p53 in the mouse mammary gland results in accumulation of damaged cells and impaired tissue regeneration

In response to DNA damage, checkpoint proteins halt cell cycle progression and promote repair or apoptosis, thereby preventing mutation accumulation and suppressing tumor development. The DNA damage checkpoint protein Hus1 associates with Rad9 and Rad1 to form the 9-1-1 complex, which localizes to DNA lesions and promotes DNA damage signaling and repair. Because complete inactivation of mouse Hus1 results in embryonic lethality, we developed a system for regulated Hus1 inactivation in the mammary gland to examine roles for Hus1 in tissue homeostasis and tumor suppression. Hus1 inactivation in the mammary epithelium resulted in genome damage that induced apoptosis and led to depletion of Hus1-null cells from the mammary gland. Conditional Hus1 knockout females retained grossly normal mammary gland morphology, suggesting compensation by cells that failed to undergo Cre-mediated Hus1 deletion. p53-deficiency delayed the clearance of Hus1-null cells from conditional Hus1 knockout mice and caused the accumulation of damaged, dying cells in the mammary gland. Notably, compensatory responses were impaired following combined Hus1 and p53 loss, resulting in aberrant mammary gland morphology and lactation defects. Overall, these results establish a requirement for Hus1 in the survival and proliferation of mammary epithelium and identify a role for p53 in mammary gland tissue regeneration and homeostasis.     

Role of tyrosyl-DNA phosphodiesterase (TDP1) in mitochondria

Human tyrosyl-DNA phosphodiesterase (TDP1) hydrolyzes the phosphodiester bond at a DNA 3'-end linked to a tyrosyl moiety and has been implicated in the repair of topoisomerase I (Top1)-DNA covalent complexes. TDP1 can also hydrolyze other 3'-end DNA alterations including 3'-phosphoglycolate and 3'-abasic sites, and exhibits 3'-nucleosidase activity indicating it may function as a general 3'-end-processing DNA repair enzyme. Here, using laser confocal microscopy, subcellular fractionation and biochemical analyses we demonstrate that a fraction of the TDP1 encoded by the nuclear TDP1 gene localizes to mitochondria. We also show that mitochondrial base excision repair depends on TDP1 activity and provide evidence that TDP1 is required for efficient repair of oxidative damage in mitochondrial DNA. Together, our findings provide evidence for TDP1 as a novel mitochondrial enzyme.     

p21Cip1/Waf1 disrupts the recruitment of human Fen1 by proliferating-cell nuclear antigen into the DNA replication complex.

Fen1 or maturation factor 1 is a 5'-3' exonuclease essential for the degradation of the RNA primer-DNA junctions at the 5' ends of immature Okazaki fragments prior to their ligation into a continuous DNA strand. The gene is also necessary for repair of damaged DNA in yeast. We report that human proliferating-cell nuclear antigen (PCNA) associates with human Fen1 with a Kd of 60 nM and an apparent stoichiometry of three Fen1 molecules per PCNA trimer. The Fen1-PCNA association is seen in cell extracts without overexpression of either partner and is mediated by a basic region at the C terminus of Fen1. Therefore, the polymerase delta-PCNA-Fen1 complex has all the activities associated with prokaryotic DNA polymerases involved in replication: 5'-3' polymerase, 3'-5' exonuclease, and 5'-3' exonuclease. Although p21, a regulatory protein induced by p53 in response to DNA damage, interacts with PCNA with a comparable Kd (10 nM) and a stoichiometry of three molecules of p21 per PCNA trimer, a p21-PCNA-Fen1 complex is not formed. This mutually exclusive interaction suggests that the conformation of a PCNA trimer switches such that it can either bind p21 or Fen1. Furthermore, overexpression of p21 can disrupt Fen1-PCNA interaction in vivo. Therefore, besides interfering with the processivity of polymerase delta-PCNA, p21 also uncouples Fen1 from the PCNA scaffold.     

Requirement of proliferating cell nuclear antigen in RAD6-dependent postreplicational DNA repair.

The proliferating cell nuclear antigen (PCNA) acts as a processivity factor for replicative DNA polymerases and is essential for DNA replication. In vitro studies have suggested a role for PCNA-in the repair synthesis step of nucleotide excision repair, and PCNA interacts with the cyclin-dependent kinase inhibitor p21. However, because of the lack of genetic evidence, it is not clear which of the DNA repair processes are in fact affected by PCNA in vivo. Here, we describe a PCNA mutation, pol30-46, that confers ultraviolet (UV) sensitivity but has no effect on growth or cell cycle progression, and the mutant pcna interacts normally with DNA polymerase delta and epsilon. Genetic studies indicate that the pol30-46 mutation is specifically defective in RAD6-dependent postreplicational repair of UV damaged DNA, and this mutation impairs the error-free mode of bypass repair. These results implicate a role for PCNA as an intermediary between DNA replication and postreplicational DNA repair.     

Recruitment of DNA methyltransferase I to DNA repair sites

In mammalian cells, the replication of genetic and epigenetic information is directly coupled; however, little is known about the maintenance of epigenetic information in DNA repair. Using a laser microirradiation system to introduce DNA lesions at defined subnuclear sites, we tested whether the major DNA methyltransferase (Dnmt1) or one of the two de novo methyltransferases (Dnmt3a, Dnmt3b) are recruited to sites of DNA repair in vivo. Time lapse microscopy of microirradiated mammalian cells expressing GFP-tagged Dnmt1, Dnmt3a, or Dnmt3b1 together with red fluorescent protein-tagged proliferating cell nuclear antigen (PCNA) revealed that Dnmt1 and PCNA accumulate at DNA damage sites as early as 1 min after irradiation in S and non-S phase cells, whereas recruitment of Dnmt3a and Dnmt3b was not observed. Deletion analysis showed that Dnmt1 recruitment was mediated by the PCNA-binding domain. These data point to a direct role of Dnmt1 in the restoration of epigenetic information during DNA repair.     

Predicted poxvirus FEN1-like nuclease required for homologous recombination,double-strand break repair and full-size genome formation

Poxviruses encode many if not all of the proteins required for viral genome replication in the cytoplasm of the host cell. In this context, we investigated the function of the vaccinia virus G5 protein because it belongs to the FEN1-like family of nucleases and is conserved in all poxviruses. A vaccinia virus G5 deletion mutant was severely impaired, as the yield of infectious virus was reduced by approximately two orders of magnitude. The mutant virions contained an apparently normal complement of proteins but appeared spherical rather than brick-shaped and contained no detectable DNA. The inability of G5 with substitutions of the predicted catalytic aspartates to complement the deletion mutant suggested that G5 functions as a nuclease during viral DNA replication. Although the amount of viral DNA produced in the absence of G5 was similar to that made by wild-type virus, the mean size was approximately one-fourth of the genome length. Experiments with transfected plasmids showed that G5 was required for double-strand break repair by homologous recombination, suggesting a similar role during vaccinia virus genome replication.     

Sequential switching of binding partners on PCNA during in vitro Okazaki fragment maturation

The homotrimeric sliding clamp proliferating cell nuclear antigen (PCNA) mediates Okazaki fragment maturation through tight coordination of the activities of DNA polymerase δ (Pol δ), flap endonuclease 1 (FEN1) and DNA ligase I (Lig1). Little is known regarding the mechanism of partner switching on PCNA and the involvement of PCNA''s three binding sites in coordinating such processes. To shed new light on PCNA-mediated Okazaki fragment maturation, we developed a novel approach for the generation of PCNA heterotrimers containing one or two mutant monomers that are unable to bind and stimulate partners. These heterotrimers maintain the native oligomeric structure of PCNA and exhibit high stability under various conditions. Unexpectedly, we found that PCNA heterotrimers containing only one functional binding site enable Okazaki fragment maturation by efficiently coordinating the activities of Pol δ, FEN1, and Lig1. The efficiency of switching between partners on PCNA was not significantly impaired by limiting the number of available binding sites on the PCNA ring. Our results provide the first direct evidence, to our knowledge, that simultaneous binding of multiple partners to PCNA is unnecessary, and if it occurs, does not provide significant functional advantages for PCNA-mediated Okazaki fragment maturation in vitro. In contrast to the “toolbelt” model, which was demonstrated for bacterial and archaeal sliding clamps, our results suggest a mechanism of sequential switching of partners on the eukaryotic PCNA trimer during DNA replication and repair.Proliferating cell nuclear antigen (PCNA) is a central coordinator of genome duplication and maintenance pathways in eukaryotes (1, 2). A member of the conserved sliding clamp family, PCNA is a homotrimeric ring-shaped protein that encircles DNA and serves as a processivity factor for DNA polymerases and a binding platform for many DNA modifying enzymes. PCNA interacts with partners involved in numerous processes, including DNA replication, recombination and repair, chromatin remodeling, and cell-cycle regulation. PCNA recruits these partners to replication forks or other chromosomal locations, enhances their catalytic activities, and orchestrates their cooperation in multistep enzymatic processes. Because most partners interact with the same binding site on PCNA, competition for binding must be tightly regulated during complex PCNA-mediated processes. The switching of partners on the PCNA platform has been shown to be crucial for the proper progression of multiple DNA replication and repair pathways, such as lagging strand replication, translesion synthesis, and mismatch repair (1). In recent years, several regulatory mechanisms, mostly involving posttranslational modifications of PCNA by ubiquitin or small ubiquitin-like modifier, have been shown to affect partner switching on PCNA by favoring the recruitment of specific partners (3–5).Despite extensive research into the regulation of PCNA-mediated processes, very little is known regarding how PCNA coordinates the activity of several enzymes during sequential processes. Two simple models have been proposed to explain this coordination (1, 2, 6, 7). The first model assumes highly dynamic partner switching on PCNA due to sequential binding and release events on the same or different PCNA monomers (Fig. 1, Upper). This model predicts that a single functional binding site on the PCNA trimer should be sufficient for the coordination of the entire process. In contrast, the second model assumes simultaneous binding of two or three partners to different monomers on the PCNA trimer (Fig. 1, Lower). In this case, the partners are stably associated with PCNA, which acts as a “toolbelt” throughout the process. According to this model, only PCNA trimers with two or three functional binding sites would be able to coordinate the process.Open in a separate windowFig. 1.Two possible models describing PCNA-mediated Okazaki fragment maturation. (Upper) A dynamic model in which Pol δ, FEN1, and Lig1 are bound and released from PCNA in a sequential manner. (Lower) The toolbelt model in which the three enzymes are simultaneously bound to PCNA using all available PCNA binding sites. The red segments represent the RNA primers; glowing circles represent enzymes currently active on the substrate.One of the best studied examples of such a multipartner PCNA-mediated process is the synthesis and maturation of Okazaki fragments during lagging strand DNA replication. This process involves the sequential activity of three PCNA binding partners—DNA polymerase δ (Pol δ), flap endonuclease 1 (FEN1), and DNA ligase I (Lig1), which mediate DNA synthesis, flap cleavage, and ligation, respectively (7–9). This is a fast and efficient process that is estimated to take place ∼100,000 times during each yeast cell division with a low tolerance for errors (8). The enzymes involved must cooperate through PCNA in a tightly regulated manner, acting sequentially on the same substrate while repeatedly exchanging access to it (Fig. 1). In particular, removal of the initiator RNA requires several rapid iterative switches between Pol δ and FEN1 (7). This PCNA-dependent cooperation is particularly important to ensure that flaps will not become too long for processing by this short-flap pathway (7, 10, 11).To directly examine the mechanism of partner switching on PCNA and the functional significance of its homotrimeric structure, we developed a novel approach for the generation of PCNA heterotrimers that contain both wild-type (WT) and mutant monomers that are unable to bind different partners. We used these heterotrimers to determine whether simultaneous binding of more than one partner to a PCNA trimer is necessary to coordinate PCNA-mediated nick translation and Okazaki fragment maturation. Contrary to the toolbelt model, our findings indicate that simultaneous binding is not required, and sequential switching of partners on a single monomer of PCNA is sufficient to coordinate Okazaki fragment maturation. Our findings suggest that PCNA can efficiently orchestrate complex processes by regulating sequential binding and release events of several partners without binding them simultaneously.     

SUMO-1 conjugation to topoisomerase I: A possible repair response to topoisomerase-mediated DNA damage

Ubiquitin/26S proteasome-dependent degradation of topoisomerase I (TOP1) has been suggested to be a unique repair response to TOP1-mediated DNA damage. In the current study, we show that treatment of mammalian cells or yeast cells expressing human DNA TOP1 with camptothecin (CPT) induces covalent modification of the TOP1 by SUMO-1/Smt3p, a ubiquitin-like protein. This conclusion is based on the following observations: (i) Mammalian DNA TOP1 conjugates induced by CPT were cross-reactive with SUMO-1/Smt3p-specific antibodies both in yeast expressing human DNA TOP1 as well as mammalian cells. (ii) The formation of TOP1 conjugates was shown to be dependent on UBC9, the E2 enzyme for SUMO-1/Smt3p. (iii) TOP1 physically interacts with UBC9. (iv) Ubc9 mutant yeast cells expressing human DNA TOP1 was hypersensitive to CPT, suggesting that UBC9/SUMO-1 may be involved in the repair of TOP1-mediated DNA damage.     

Extrahelical (CAG)/(CTG) triplet repeat elements support proliferating cell nuclear antigen loading and MutLα endonuclease activation

MutLα endonuclease can be activated on covalently continuous DNA that contains a MutSα- or MutSβ-recognizable lesion and a helix perturbation that supports proliferating cell nuclear antigen (PCNA) loading by replication factor C, providing a potential mechanism for triggering mismatch repair on nonreplicating DNA. Because mouse models for somatic expansion of disease-associated (CAG)_n/(CTG)_n triplet repeat sequences have implicated both MutSβ and MutLα and have suggested that expansions can occur in the absence of replication, we have asked whether an extrahelical (CAG)_n or (CTG)_n element is sufficient to trigger MutLα activation. (CAG)_n and (CTG)_n extrusions in relaxed closed circular DNA do in fact support MutSβ-, replication factor C-, and PCNA-dependent activation of MutLα endonuclease, which can incise either DNA strand. Extrahelical elements of two or three repeat units are the preferred substrates for MutLα activation, and extrusions of this size also serve as moderately effective sites for loading the PCNA clamp. Relaxed heteroduplex DNA containing a two or three-repeat unit extrusion also triggers MutSβ- and MutLα-endonuclease-dependent mismatch repair in nuclear extracts of human cells. This reaction occurs without obvious strand bias at about 10% the rate of that observed with otherwise identical nicked heteroduplex DNA. These findings provide a mechanism for initiation of triplet repeat processing in nonreplicating DNA that is consistent with several features of the model of Gomes-Pereira et al. [Gomes-Pereira M, Fortune MT, Ingram L, McAbney JP, Monckton DG (2004) Hum Mol Genet 13(16):1815–1825]. They may also have implications for triplet repeat processing at a replication fork.     

Differential Effects of Ethanol on Insulin-Signaling Through the Insulin Receptor Substrate-1

Insulin stimulation increases cell proliferation and energy metabolism by activating the insulin receptor substrate I (IRS-1)-signaling pathways. This downstream signaling is mediated by interactions of specific tyrosyl phosphorylated (PY) IRS-1 motifs with SH2-containing molecules such as growth-factor receptor-bound protein 2 (Grb2) and Syp. Ethanol inhibits insulin-stimulated tyrosyl phosphorylation of IRS-1 and DNA synthesis. This study explores the roles of the Grb2- and Syp-binding motifs of IRS-1 in relation to the inhibitory effects of ethanol on insulin-stimulated DNA synthesis, proliferating cell nuclear antigen (PCNA) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) expression, and activation of mitogen-activated protein kinase (MAPK), which is known to be essential for cell proliferation. NIH3T3 cells were stably transfected with wild-type IRS-1, or IRS-1 mutated at the Grb2 (IRS-1deltaGrb2), Syp (IRS-1deltaSyp), or Grb2 and Syp (IRS-1deltaGrb2deltaSyp)- binding sites. Cells transfected with IRS-1 had increased levels of DNA synthesis, PCNA, GAPDH, and activated MAPK. The IRS-1deltaGrb2 transfectants were highly responsive to insulin stimulation, achieving levels of GAPDH, PCNA, and activated MAPK that were higher than control. In contrast, the IRS-1deltaSyp and IRS-1deltaGrb2deltaSyp transfectants had reduced levels of DNA synthesis, PCNA, and activated MAPK. Ethanol exposure decreased insulin-stimulated DNA synthesis, PCNA, GAPDH, and activated MAPK levels in all clones, but the wild-type IRS-1 transfectants were relatively resistant, and the IRS-1deltaGrb2 transfectants were extraordinarily sensitive to these inhibitory effects of ethanol. The findings suggest that insulin-stimulated DNA synthesis and PCNA expression are mediated through the Syp-binding domain, whereas GAPDH expression and MAPK activation are modulated through both the Grb2 and Syp motifs of IRS-1. In addition, ethanol exposure may preferentially inhibit downstream signaling that requires interaction between Syp and PY-IRS-1.     

The human Bloom syndrome gene suppresses the DNA replication and repair defects of yeast dna2 mutants

Bloom syndrome is a disorder of profound and early cancer predisposition in which cells become hypermutable, exhibit high frequency of sister chromatid exchanges, and show increased micronuclei. BLM, the gene mutated in Bloom syndrome, has been cloned previously, and the BLM protein is a member of the RecQ family of DNA helicases. Many lines of evidence suggest that BLM is involved either directly in DNA replication or in surveillance during DNA replication, but its specific roles remain unknown. Here we show that hBLM can suppress both the temperature-sensitive growth defect and the DNA damage sensitivity of the yeast DNA replication mutant dna2-1. The dna2-1 mutant is defective in a helicase-nuclease that is required either to coordinate with the crucial Saccharomyces cerevisiae (sc) FEN1 nuclease in Okazaki fragment maturation or to compensate for scFEN1 when its activity is impaired. We show that human BLM interacts with both scDna2 and scFEN1 by using coimmunoprecipitation from yeast extracts, suggesting that human BLM participates in the same steps of DNA replication or repair as scFEN1 and scDna2.     

Cdk-interacting protein 1 directly binds with proliferating cell nuclear antigen and inhibits DNA replication catalyzed by the DNA polymerase delta holoenzyme.

Cdk-interacting protein 1 (Cip1) is a p53-regulated 21-kDa protein that inhibits several members of the cyclin-dependent kinase (CDK) family. It was initially observed in complexes containing CDK4, cyclin D, and proliferating cell nuclear antigen (PCNA). PCNA, in conjunction with activator 1, acts as a processivity factor for eukaryotic DNA polymerase (pol) delta, and these three proteins constitute the pol delta holoenzyme. In this report, we demonstrate that Cip1 can also directly inhibit DNA synthesis in vitro by binding to PCNA. Cip1 efficiently inhibits simian virus 40 replication dependent upon pol alpha, activator 1, PCNA, and pol delta, and this inhibition can be overcome by additional PCNA. Simian virus 40 DNA replication, catalyzed solely by high levels of pol alpha-primase complex, is unaffected by Cip1. Using the surface plasmon resonance technique, a direct physical interaction of PCNA and Cip1 was detected. We have observed that Cip1 efficiently inhibits synthesis of long (7.2 kb) but not short (10 nt) templates, suggesting that its association with PCNA is likely to impair the processive movement of pol delta during DNA chain elongation, as opposed to blocking assembly of the pol delta holoenzyme. The implications of the Cip1-PCNA interaction with respect to regulation of DNA synthesis, cell cycle checkpoint control, and DNA repair are discussed.     

The C-terminal region of Schizosaccaromyces pombe proliferating cell nuclear antigen is essential for DNA polymerase activity

Proliferating cell nuclear antigen (PCNA), the processivity factor (sliding clamp) of DNA polymerases (Pols), plays essential roles in DNA metabolism. In this report, we examined the functional role of the C-terminal region of Schizosaccaromyces pombe PCNA both in vitro and in vivo. The deletion or Ala substitution of the last 9 aa (252-260A), as well as Ala replacement of only 4 aa (252-255A) at the C terminus, failed to substitute for the wild-type PCNA protein for cell growth in S. pombe. Two other PCNA mutant proteins, A251V and K253E, exhibited cold-sensitive phenotypes. Several yeast strains harboring mutations, including those at the acidic C-terminal region, showed elevated sensitivity to DNA damage. The ability of the mutant PCNA proteins to stimulate DNA synthesis by Poldelta and Polepsilon also was studied in vitro. The mutant proteins that did not support cell growth and a mutant protein containing a single amino acid substitution at position 252, where Pro is replaced by Ala, stimulated Poldelta and Polepsilon activities poorly. All mutant PCNA proteins, however, were assembled around DNA by the clamp loader, replication factor C, efficiently. Thus, the C-terminal region of PCNA is important for interactions with both Poldelta and Polepsilon and for cell survival after DNA damage. The C terminus of sliding clamps from other organisms has been shown to be important for clamp loading as well as polymerase interactions. The relationship between the conserved sequence in this region in different organisms is discussed.