Ezrin is a membrane-cytoskeletal linking protein that is concentrated in actin-rich surface structures. It is closely related to the microvillar proteins radixin and moesin and to the tumor suppressor merlin/schwannomin. Cell extracts contain ezrin dimers and ezrin-moesin heterodimers in addition to monomers. Truncated ezrin fusion proteins were assayed by blot overlay to determine which regions mediate self-association. Here we report that ezrin self-association occurs by head-to-tail joining of distinct N-terminal and C-terminal domains. It is likely that these domains, termed N- and C-ERMADs (ezrin-radixin-moesin association domain), are responsible for homotypic and heterotypic associations among ERM family members. The N-ERMAD of ezrin resided within amino acids 1-296; deletion of 10 additional residues resulted in loss of activity. The C-ERMAD was mapped to the last 107 amino acids of ezrin, residues 479-585. The two residues at the C-terminus were required for activity, and the region from 530-585 was insufficient. The C-ERMAD was masked in the native monomer. Exposure of this domain required unfolding ezrin with sodium dodecyl sulfate or expressing the domain as part of a truncated protein. Intermolecular association could not occur unless the C-ERMAD had been made accessible to its N-terminal partner. It can be inferred that dimerization in vivo requires an activation step that exposes this masked domain. The conformationally inaccessible C-terminal region included the F-actin binding site, suggesting that this activity is likewise regulated by masking.

In mammalian cells, DNA replication occurs at discrete nuclear sites termed replication factories. Here we demonstrate that DNA ligase I and the large subunit of replication factor C (RF-C p140) have a homologous sequence of~20 amino acids at their N-termini that functions as a replication factory targeting sequence (RFTS). This motif consists of two boxes: box 1 contains the sequence IxxFF whereas box 2 is rich in positively charged residues. N-terminal fragments of DNA ligase I and the RF-C large subunit that contain the RFTS both interact with proliferating cell nuclear antigen (PCNA) in vitro. Moreover, the RFTS of DNA ligase I and of the RF-C large subunit is necessary and sufficient for the interaction with PCNA. Both subnuclear targeting and PCNA binding by the DNA ligase I RFTS are abolished by replacement of the adjacent phenylalanine residues within box 1. Since sequences similar to the RFTS/PCNA-binding motif have been identified in other DNA replication enzymes and in p21 CIP1/WAF1 , we propose that, in addition to functioning as a DNA polymerase processivity factor, PCNA plays a central role in the recruitment and stable association of DNA replication proteins at replication factories.

Eukaryotic DNA mismatch repair requires the concerted action of several proteins, including proliferating cell nuclear antigen (PCNA) and heterodimers of MSH2 complexed with either MSH3 or MSH6. Here we report that MSH3 and MSH6, but not MSH2, contain N-terminal sequence motifs characteristic of proteins that bind to PCNA. MSH3 and MSH6 peptides containing these motifs bound PCNA, as did the intact Msh2-Msh6 complex. This binding was strongly reduced when alanine was substituted for conserved residues in the motif. Yeast strains containing alanine substitutions in the PCNA binding motif of Msh6 or Msh3 had elevated mutation rates, indicating that these interactions are important for genome stability. When human MSH3 or MSH6 peptides containing the PCNA binding motif were added to a human cell extract, mismatch repair activity was inhibited at a step preceding DNA resynthesis. Thus, MSH3 and MSH6 interactions with PCNA may facilitate early steps in DNA mismatch repair and may also be important for other roles of these eukaryotic MutS homologs.The mutation rate of an organism is reduced by the ability of the general DNA mismatch repair (MMR) 1 system to correct DNA replication errors. In eukaryotes, MMR is initiated when one of two protein complexes binds to mismatches (reviewed in Refs. 1-3). The MutS␣ heterodimer consists of the MutS homologs MSH2 and MSH6 and is involved in the recognition and repair of base-base and small insertion/deletion mismatches. MutS␤ is comprised of MSH2 and MSH3 and is primarily responsible for binding to and correcting insertion/deletion mutations. Other proteins participate in MMR, including heterodimers of MutL homologs, PCNA, exonuclease I, replication protein A, replication factor C, and DNA polymerase ␦. Working in concert, these proteins complete a DNA excision/resynthesis reaction that specifically corrects errors in the nascent strand (1-3).Although the signal that directs mismatch repair to the newly replicated strand in eukaryotic cells is unknown, repair of mismatched duplexes in extracts of eukaryotic cells can be directed to one strand by a discontinuity in the DNA backbone. We previously suggested that one-strand discontinuity that might serve as a strand discrimination signal is the primer terminus at the replication fork and that PCNA may link DNA replication and mismatch repair to facilitate recognition and repair of errors in the nascent strand (4). PCNA is the essential sliding clamp that topologically encircles DNA and physically associates with DNA polymerase ␦ to enhance its processivity (reviewed in Ref. 5). PCNA is required at an early step in DNA mismatch repair that precedes excision of the mismatch (4), as well as for the DNA resynthesis that follows mismatch excision (6). Yeast PCNA has been suggested to interact with Mlh1 in vivo (4), and it interacts with the Msh2-Msh3 heterodimer in vitro (7). Human PCNA can be co-immunoprecipitated with MSH2, MLH1, and PMS2 (6), and a PCNA affinity column binds MSH2 and MSH6 (8). Yeast strains with certain mutant PCN...

Abstract. Ezrin is a component of the microvillus cytoskeleton of a variety of polarized epithelial cells and is believed to function as a membrane-cytoskeletal linker. In this study, we isolated microvilli from human placental syncytiotrophoblast as a model system for biochemical analysis of ezrin function. In contrast to intestinal microvilli, ezrin is a major protein component of placental microvilli, comprising ~5 % of the total protein mass and present at about one quarter of the molar abundance of actin. Gel filtration and chemical cross-linking studies demonstrated that ezrin exists mainly in the form of noncovalent dimers and higher order oligomers in extracts of placental microvilli. A novel form of ezrin, apparently representing covalently cross-linked adducts, was present as a relatively minor constituent of placental microvilli. Both oligomers and adducts remained associated with the detergent-insoluble cytoskeleton, indicating a tight interaction with actin filaments. Moreover, stimulation of human A431 carcinoma cells with EGF induces the rapid formation of ezrin oligomers in vivo, thus identifying a signal transduction pathway involving ezrin oligomerization coincident with microvillus assembly. In addition to time course studies, experiments with tyrosine kinase and tyrosine phosphatase inhibitors revealed a correlation between the phosphorylation of ezrin on tyrosine and the onset of oligomer formation, consistent with the possibility that phosphorylation of ezrin might be required for the generation of stable oligomers. Based on these observations, a model for the assembly of cell surface structures is proposed.ZRIN was identified over twelve years ago as a minor component of the isolated intestinal microvillus cytoskeleton (Bretscher, 1983), and has since been found in the apical microvilli of a wide variety of polarized epithelial cell types within the body (Hanzel et al

Proliferating cell nuclear antigen (PCNA) is a DNA polymerase accessory factor that is required for DNA replication during S phase of the cell cycle and for resynthesis during nucleotide excision repair of damaged DNA. PCNA binds to flap endonuclease 1 (FEN-1), a structure-specific endonuclease involved in DNA replication. Here we report the direct physical interaction of PCNA with xeroderma pigmentosum (XP) G, a structurespecific repair endonuclease that is homologous to FEN-1. We have identified a 28-amino acid region of human FEN-1 (residues 328 -355) and a 29-amino acid region of human XPG (residues 981-1009) that contains the PCNA binding activity. These regions share key hydrophobic residues with the PCNA-binding domain of the cyclin-dependent kinase inhibitor p21 Waf1/Cip1 , and all three competed with one another for binding to PCNA. A conserved arginine in FEN-1 (Arg 339 ) and XPG (Arg 992 ) was found to be crucial for PCNA binding activity. R992A and R992E mutant forms of XPG failed to fully reconstitute nucleotide excision repair in an in vivo complementation assay. These results raise the possibility of a mechanistic linkage between excision and repair synthesis that is mediated by PCNA.Exposure to UV light causes damage to DNA primarily in the form of cyclobutane pyrimidine dimers and (6-4) photoproducts. These types of DNA lesions, as well as bulky adducts produced by some chemical mutagens, are processed by nucleotide excision repair (NER). 1 The human genetic disorder xeroderma pigmentosum (XP) is the result of defects in this DNA damage repair pathway. Symptoms of XP include extreme sensitivity to sunlight exposure and a greatly elevated risk of skin cancer. In the past few years, much progress has been made in understanding the molecular events associated with NER (1). The DNA-binding protein XPA is involved in damage recognition. In concert with replication protein A, which binds singlestranded DNA, and helicases XPB and XPD, a ϳ27-29-base oligonucleotide segment containing the lesion is excised as the result of dual incision by structure-specific endonucleases XPF-ERCC1 and XPG. The XPF-ERCC1 complex cleaves the damaged strand at a 5Ј site about 23 nucleotides from the lesion, whereas XPG cleaves the strand approximately 5 nucleotides to the 3Ј side of the damage. The resultant gap is filled in by the action of DNA polymerase ␦ or ⑀, and then DNA ligase seals the nick to complete repair. The resynthesis step requires proliferating cell nuclear antigen (PCNA; Refs. 2 and 3), a ring-shaped homotrimeric protein that encircles DNA and acts as a "sliding clamp" that links the polymerase to the DNA template (4). PCNA performs the same essential function in replicative DNA synthesis during S phase of the cell cycle. PCNA requires replication factor C, a primer recognition protein that loads the PCNA trimer onto DNA in an ATP-dependent manner (5-7).XPG is homologous to another structure-specific endonuclease, FEN-1. FEN-1 is involved in Okazaki fragment processing during DNA replication (8), and it i...

The S phase-specific activation of NEIL1 and not of the other DNA glycosylases responsible for repairing oxidatively damaged bases in mammalian genomes and the activation of NEIL1 by proliferating cell nuclear antigen (PCNA) suggested preferential action by NEIL1 in oxidized base repair during DNA replication. Here we show that NEIL1 interacts with flap endonuclease 1 (FEN-1), an essential component of the DNA replication. FEN-1 is present in the NEIL1 immunocomplex isolated from human cell extracts, and the two proteins colocalize in the nucleus. FEN-1 stimulates the activity of NEIL1 in vitro in excising 5-hydroxyuracil from duplex, bubble, forked, and single-stranded DNA substrates by up to 5-fold. The disordered region near the C terminus of NEIL1, which is dispensable for activity, is necessary and sufficient for high affinity binding to FEN-1 (K D Х 0.2 M). The interacting interface of FEN-1 is localized in its disordered C-terminal region uniquely present in mammalian orthologs. Fine structure mapping identified several Lys and Arg residues in this region that form salt bridges with Asp and Glu residues in NEIL1. NEIL1 was previously shown to initiate single nucleotide excision repair, which does not require FEN-1 or PCNA. The present study shows that NEIL1 could also participate in strand displacement repair synthesis (long patch repair (LP-BER)) mediated by FEN-1 and stimulated by PCNA. Interaction between NEIL1 and FEN-1 is essential for efficient NEIL1-initiated LP-BER. These studies strongly implicate NEIL1 in a distinct subpathway of LP-BER in replicating genomes.Reactive oxygen species are the most pervasive genotoxic agents in mammals (and other aerobic organisms), because they are continuously generated in vivo as by-products of respiration. Reactive oxygen species are also produced during various pathophysiologic states and oxidative metabolism of xenobiotics (1-3). Reactive oxygen species have been implicated in the etiology of cancer and various diseases and also in aging (4); these induce a plethora of oxidatively modified bases in DNA, together with strand breaks via oxidation of the deoxyribose (5). Many oxidized bases and abasic (AP)2 sites generated by enzymatic removal of these damaged bases are mutagenic and likely to be responsible for sporadic mutations leading to cancer and other diseases (4, 6). For example, 5-hydroxyuracil (5-OHU; an abundant DNA lesion generated by oxidative deamination of C) behaves like T, and its replication would lead to a G:C 3 A:T mutation. 5-OHU is excised from DNA by all mammalian glycosylases to varying degrees. Oxidized bases (and singlestranded breaks) in DNA are primarily repaired via the base excision repair (BER) pathway, a highly conserved process that is initiated with excision of the damaged base by a DNA glycosylase. Four oxidized base-specific DNA glycosylases, namely OGG1 (8-oxoguanine-DNA glycosylase 1), NTH1 (endonuclease III homolog), and NEIL1 and -2 (endonuclease VIII-like 1 and 2), have so far been characterized in mammalian cells and are...

There are two distinct pathways for the removal of modified DNA bases through base excision repair (BER) in vertebrates. Following 5 incision by AP endonuclease, the pathways diverge as two different excision mechanisms are possible. In short-patch repair, DNA polymerase ␤ accounts for both excision activity and single nucleotide repair synthesis. In long-patch repair, the damage-containing strand is excised by the structure-specific endonuclease FEN-1 and approximately 2-8 nucleotides are incorporated by proliferating cell nuclear antigen (PCNA)-dependent synthesis. PCNA is an accessory factor of DNA polymerases ␦ and ⑀ that is required for DNA replication and repair. PCNA binds to FEN-1 and stimulates its nuclease activity, but the physiological significance of this interaction is unknown. The importance of the PCNA-FEN-1 interaction in BER was investigated. In a reconstituted BER assay system containing FEN-1, omission of PCNA caused the accumulation of pre-excision reaction intermediates which could be converted to completely repaired product by addition of PCNA. When dNTPs were omitted from the reaction to suppress repair synthesis, PCNA was required for the formation of excised reaction intermediates. In contrast, a PCNA mutant that could not bind to FEN-1 was unable to stimulate excision. To further study this effect, a mutant of FEN-1 was identified that retained full nuclease activity but was specifically defective in binding to PCNA. The mutant FEN-1 exhibited one-tenth the specific activity of wild type FEN-1 in the reconstituted BER assay, and this repair defect was due to a kinetic block at the excision step as evidenced by the accumulation of pre-excision intermediates when dNTPs were omitted. These results indicate that PCNA facilitates excision during long-patch BER through its interaction with FEN-1.Damage to DNA bases can occur spontaneously under physiological conditions. Bases may be modified by deamination, or lost entirely due to hydrolysis of the N-glycosidic bond that links them to the sugar-phosphate backbone. Reactive oxygen species that are the by-products of normal cellular respiration also damage DNA. Exogenous agents such as chemical mutagens and ionizing radiation are additional sources of base modification. Aberrant DNA bases generated by such processes can be restored by base excision repair (BER) 1 (reviewed in Refs. 1 and 2). This repair system includes several types of DNA glycosylases that recognize and remove many types of modified bases to leave an apurinic or apyrimidinic site (AP site). Alternatively, an AP site may be the direct result of damage. The AP site must be removed during the course of repair. AP endonuclease initiates the process by hydrolyzing the phosphodiester bond immediately to the 5Ј side of the abasic site. This enzyme is capable of recognizing various classes of abasic lesions (3). After this incision has been made, there are two mechanistically distinct methods for AP site removal that distinguish short-patch BER from long-patch BER in higher eukaryote...

Fen1/Rad27 nuclease activity, which is important in DNA metabolism, is stimulated by proliferating cell nuclear antigen (PCNA) in vitro. The in vivo role of the PCNA interaction was investigated in the yeast Rad27. A nuclease-defective rad27 mutation had a dominant-negative effect that was suppressed by a mutation in the PCNA binding site, thereby demonstrating the importance of the Rad27-PCNA interaction. The PCNA-binding defect alone had little effect on mutation, recombination, and the methyl methanesulfonate (MMS) response in repair-competent cells, but it greatly amplified the MMS sensitivity of a rad51 mutant. Furthermore, the PCNA binding mutation resulted in lethality when combined with a homozygous or even a heterozygous pol3-01 mutation in the 335 exonuclease domain of DNA polymerase ␦. These results suggest that phenotypically mild polymorphisms in DNA metabolic proteins can have dramatic consequences when combined.