Aquaporin-2 in the ��-omics�� Era

Vasopressin controls renal water excretion largely through actions to regulate the water channel aquaporin-2 in collecting duct principal cells. Our knowledge of the mechanisms involved has increased markedly in recent years with the advent of methods for large-scale systems-level profiling such as protein mass spectrometry, yeast two-hybrid analysis, and oligonucleotide microarrays. Here we review this progress.Regulation of water excretion by the kidney is one of the most visible aspects of everyday physiology. An outdoor tennis game on a hot summer day can result in substantial water losses by sweating, and the kidneys respond by reducing water excretion. In contrast, excessive intake of water, a frequent occurrence in everyday life, results in excretion of copious amounts of clear urine. These responses serve to exact tight control on the tonicity of body fluids, maintaining serum osmolality in the range of 290–294 mosmol/kg of H₂O through the regulated return of water from the pro-urine in the renal collecting ducts to the bloodstream.The importance of this process is highlighted when the regulation fails. For example, polyuria (rapid uncontrolled excretion of water) is a sometimes devastating consequence of lithium therapy for bipolar disorder. On the other side of the coin are water balance disorders that result from excessive renal water retention causing systemic hypo-osmolality or hyponatremia. Hyponatremia due to excessive water retention can be seen with severe congestive heart failure, hepatic cirrhosis, and the syndrome of inappropriate antidiuresis.The chief regulator of water excretion is the peptide hormone AVP,² whereas the chief molecular target for regulation is the water channel AQP2. In this minireview, we describe new progress in the understanding of the molecular mechanisms involved in regulation of AQP2 by AVP in collecting duct cells, with emphasis on new information derived from “systems-level” approaches involving large-scale profiling and screening techniques such as oligonucleotide arrays, protein mass spectrometry, and yeast two-hybrid analysis. Most of the progress with these techniques is in the identification of individual molecules involved in AVP signaling and binding interactions with AQP2. Additional related issues are addressed in several recent reviews (1–4).     

Differential regulation of cell death programs in males and females by Poly (ADP-Ribose) Polymerase-1 and 17 β estradiol

Cell death can be divided into the anti-inflammatory process of apoptosis and the pro-inflammatory process of necrosis. Necrosis, as apoptosis, is a regulated form of cell death, and Poly-(ADP-Ribose) Polymerase-1 (PARP-1) and Receptor-Interacting Protein (RIP) 1/3 are major mediators. We previously showed that absence or inhibition of PARP-1 protects mice from nephritis, however only the male mice. We therefore hypothesized that there is an inherent difference in the cell death program between the sexes. We show here that in an immune-mediated nephritis model, female mice show increased apoptosis compared to male mice. Treatment of the male mice with estrogens induced apoptosis to levels similar to that in female mice and inhibited necrosis. Although PARP-1 was activated in both male and female mice, PARP-1 inhibition reduced necrosis only in the male mice. We also show that deletion of RIP-3 did not have a sex bias. We demonstrate here that male and female mice are prone to different types of cell death. Our data also suggest that estrogens and PARP-1 are two of the mediators of the sex-bias in cell death. We therefore propose that targeting cell death based on sex will lead to tailored and better treatments for each gender.     

Identification of the Lipopolysaccharide Core of Yersinia pestis and Yersinia pseudotuberculosis as the Receptor for Bacteriophage ��A1122

φA1122 is a T7-related bacteriophage infecting most isolates of Yersinia pestis, the etiologic agent of plague, and used by the CDC in the identification of Y. pestis. φA1122 infects Y. pestis grown both at 20°C and at 37°C. Wild-type Yersinia pseudotuberculosis strains are also infected but only when grown at 37°C. Since Y. pestis expresses rough lipopolysaccharide (LPS) missing the O-polysaccharide (O-PS) and expression of Y. pseudotuberculosis O-PS is largely suppressed at temperatures above 30°C, it has been assumed that the phage receptor is rough LPS. We present here several lines of evidence to support this. First, a rough derivative of Y. pseudotuberculosis was also φA1122 sensitive when grown at 22°C. Second, periodate treatment of bacteria, but not proteinase K treatment, inhibited the phage binding. Third, spontaneous φA1122 receptor mutants of Y. pestis and rough Y. pseudotuberculosis could not be isolated, indicating that the receptor was essential for bacterial growth under the applied experimental conditions. Fourth, heterologous expression of the Yersinia enterocolitica O:3 LPS outer core hexasaccharide in both Y. pestis and rough Y. pseudotuberculosis effectively blocked the phage adsorption. Fifth, a gradual truncation of the core oligosaccharide into the Hep/Glc (l-glycero-d-manno-heptose/d-glucopyranose)-Kdo/Ko (3-deoxy-d-manno-oct-2-ulopyranosonic acid/d-glycero-d-talo-oct-2-ulopyranosonic acid) region in a series of LPS mutants was accompanied by a decrease in phage adsorption, and finally, a waaA mutant expressing only lipid A, i.e., also missing the Kdo/Ko region, was fully φA1122 resistant. Our data thus conclusively demonstrated that the φA1122 receptor is the Hep/Glc-Kdo/Ko region of the LPS core, a common structure in Y. pestis and Y. pseudotuberculosis.     

SDF2L1, a Component of the Endoplasmic Reticulum Chaperone Complex, Differentially Interacts with ��-, ��-, and ��-Defensin Propeptides

Mammalian defensins are cationic antimicrobial peptides that play a central role in host innate immunity and as regulators of acquired immunity. In animals, three structural defensin subfamilies, designated as α, β, and θ, have been characterized, each possessing a distinctive tridisulfide motif. Mature α- and β-defensins are produced by simple proteolytic processing of their prepropeptide precursors. In contrast, the macrocyclic θ-defensins are formed by the head-to-tail splicing of nonapeptides excised from a pair of prepropeptide precursors. Thus, elucidation of the θ-defensin biosynthetic pathway provides an opportunity to identify novel factors involved in this unique process. We incorporated the θ-defensin precursor, proRTD1a, into a bait construct for a yeast two-hybrid screen that identified rhesus macaque stromal cell-derived factor 2-like protein 1 (SDF2L1), as an interactor. SDF2L1 is a component of the endoplasmic reticulum (ER) chaperone complex, which we found to also interact with α- and β-defensins. However, analysis of the SDF2L1 domain requirements for binding of representative α-, β-, and θ-defensins revealed that α- and β-defensins bind SDF2L1 similarly, but differently from the interactions that mediate binding of SDF2L1 to pro-θ-defensins. Thus, SDF2L1 is a factor involved in processing and/or sorting of all three defensin subfamilies.Mammalian defensins are tridisulfide-containing antimicrobial peptides that contribute to innate immunity in all species studied to date. Defensins are comprised of three structural subfamilies: the α-, β-, and θ-defensins (1). α- and β-Defensins are peptides of about 29–45-amino acid residues with similar three-dimensional structures. Despite their similar tertiary conformations, the disulfide motifs of α- and β-defensins differ. Expression of human α-defensins is tissue-specific. Four myeloid α-defensins (HNP1–4) are expressed predominantly by neutrophils and monocytes wherein they are packaged in granules, while two enteric α-defensins (HD-5 and HD-6) are expressed at high levels in Paneth cells of the small intestine. Myeloid α-defensins constitute about 5% of the protein mass of human neutrophils. HNPs are discharged into the phagosome during phagocytic ingestion of microbial particles. HD-5 and HD-6 are produced and stored as propeptides in Paneth cell granules and are processed extracellularly by intestinal trypsin (2). β-Defensins are produced primarily by various epithelia (e.g. skin, urogenital tract, airway) and are secreted by the producing cells in their mature forms. In contrast to pro-α-defensins, which contain a conserved prosegment of ∼40 amino acids, the prosegments in β-defensins vary in length and sequence. θ-Defensins are found only in Old World monkeys and orangutans and are the only known circular peptides in animals. These 18-residue macrocyclic peptides are formed by ligation of two nonamer sequences excised from two precursor polypeptides, which are truncated versions of ancestral α-defensins. Like myeloid α-defensins, θ-defensins are stored primarily in neutrophil and monocyte granules (3).Numerous laboratories have demonstrated that the antimicrobial properties of defensins derive from their ability to bind and disrupt target cell membranes (4), and studies have shown defensins to be active against Gram-positive and -negative bacteria (5), viruses (6–9), fungi (10, 11), and parasites such as Giardia lamblia (12). Defensins also play a regulatory role in acquired immunity as they are known to chemoattract T lymphocytes, monocytes, and immature dendritic cells (13, 14), act as adjuvants, stimulate B cell responses, and up-regulate proliferation and cytokine production by spleen cells and T helper cells (15, 16).Defensins are produced as pre-propeptides and undergo post-translational processing to form the mature peptides. While much has been learned about regulation of defensin expression, little is known about the factors involved in their biosynthesis. Valore and Ganz (17) investigated the processing of defensins in cultured cells and demonstrated that maturation of HNPs occurs through two proteolytic steps that lead to formation of mature α-defensins, but the proteases involved have yet to be identified. Moreover, there are virtually no published data regarding endoplasmic reticulum (ER)² factors that are responsible for the folding, processing, and sorting steps necessary for defensin maturation and secretion or trafficking to the proper subcellular compartment. It is likely that several chaperones, proteases, and protein-disulfide isomerase (PDI) family proteins are involved. Consistent with this possibility, Gruber et al. (18) recently demonstrated the role of a PDI in biosynthesis of cyclotides, small ∼30-residue macrocyclic peptides produced by plants.The primary structures of α- and θ-defensin precursors are closely related. We therefore undertook studies to identify proteins that interact with representative propeptides of each defensin subfamily with the goal of determining common and unique processes that regulate biosynthesis of α- and θ-defensins. We used two-hybrid analysis to first identify interactors of the θ-defensin precursor, proRTD1a. As described, we identified SDF2L1, a component of the ER-chaperone complex as an interactor, and showed that it also specifically interacts with α- and β-defensins. This suggests that SDF2L1 is involved in the maturation/trafficking of defensins at a step common to all three subfamilies of mammalian defensins.     

Acquired Antibiotic Resistance: Are We Born with It?

The rapid emergence of antibiotic resistance (AR) is a major public health concern. Recent findings on the prevalence of food-borne antibiotic-resistant (ART) commensal bacteria in ready-to-consume food products suggested that daily food consumption likely serves as a major avenue for dissemination of ART bacteria from the food chain to human hosts. To properly assess the impact of various factors, including the food chain, on AR development in hosts, it is important to determine the baseline of ART bacteria in the human gastrointestinal (GI) tract. We thus examined the gut microbiota of 16 infant subjects, from the newborn stage to 1 year of age, who fed on breast milk and/or infant formula during the early stages of development and had no prior exposure to antibiotics. Predominant bacterial populations resistant to several antibiotics and multiple resistance genes were found in the infant GI tracts within the first week of age. Several ART population transitions were also observed in the absence of antibiotic exposure and dietary changes. Representative AR gene pools including tet(M), ermB, sul2, and bla(TEM) were detected in infant subjects. Enterococcus spp., Staphylococcus spp., Klebsiella spp., Streptococcus spp., and Escherichia coli/Shigella spp. were among the identified AR gene carriers. ART bacteria were not detected in the infant formula and infant foods examined, but small numbers of skin-associated ART bacteria were found in certain breast milk samples. The data suggest that the early development of AR in the human gut microbiota is independent of infants' exposure to antibiotics but is likely impacted by exposure to maternal and environmental microbes during and after delivery and that the ART population is significantly amplified within the host even in the absence of antibiotic selective pressure.     

Polo-like Kinase 2 (PLK2) Phosphorylates ��-Synuclein at Serine 129 in Central Nervous System

Several neurological diseases, including Parkinson disease and dementia with Lewy bodies, are characterized by the accumulation of α-synuclein phosphorylated at Ser-129 (p-Ser-129). The kinase or kinases responsible for this phosphorylation have been the subject of intense investigation. Here we submit evidence that polo-like kinase 2 (PLK2, also known as serum-inducible kinase or SNK) is a principle contributor to α-synuclein phosphorylation at Ser-129 in neurons. PLK2 directly phosphorylates α-synuclein at Ser-129 in an in vitro biochemical assay. Inhibitors of PLK kinases inhibited α-synuclein phosphorylation both in primary cortical cell cultures and in mouse brain in vivo. Finally, specific knockdown of PLK2 expression by transduction with short hairpin RNA constructs or by knock-out of the plk2 gene reduced p-Ser-129 levels. These results indicate that PLK2 plays a critical role in α-synuclein phosphorylation in central nervous system.The importance of α-synuclein to the pathogenesis of Parkinson disease (PD)⁴ and the related disorder, dementia with Lewy bodies (DLB), is suggested by its association with Lewy bodies and Lewy neurites, the inclusions that characterize these diseases (1–3), and demonstrated by the existence of mutations that cause syndromes mimicking sporadic PD and DLB (4–6). Furthermore, three separate mutations cause early onset forms of PD and DLB. It is particularly telling that duplications or triplications of the gene (7–9), which increase levels of α-synuclein with no alteration in sequence, also cause PD or DLB.α-Synuclein has been reported to be phosphorylated on serine residues, at Ser-87 and Ser-129 (10), although to date only the Ser-129 phosphorylation has been identified in the central nervous system (11, 12). Phosphorylation at tyrosine residues has been observed by some investigators (13, 14) but not by others (10–12). Phosphorylation at Ser-129 (p-Ser-129) is of particular interest because the majority of synuclein in Lewy bodies contains this modification (15). In addition, p-Ser-129 was found to be the most extensive and consistent modification in a survey of synuclein in Lewy bodies (11). Results have been mixed from studies investigating the function of phosphorylation using S129A and S129D mutations to respectively block and mimic the modification. Although the phosphorylation mimic was associated with pathology in studies in Drosophila (16) and in transgenic mouse models (17, 18), studies using adeno-associated virus vectors to overexpress α-synuclein in rat substantia nigra found an exacerbation of pathology with the S129A mutation, whereas the S129D mutation was benign, if not protective (19). Interpretation of these studies is complicated by a recent study showing that the S129D and S129A mutations themselves have effects on the aggregation properties of α-synuclein independent of their effects on phosphorylation, with the S129A mutation stimulating fibril formation (20). Clearly, determination of the role of p-Ser-129 phosphorylation would be helped by identification of the responsible kinase. In addition, identification will provide a pathologically relevant way to increase phosphorylation in a cell or animal model.Several kinases have been proposed to phosphorylate α-synuclein, including casein kinases 1 and 2 (10, 12, 21) and members of the G-protein-coupled receptor kinase family (22). In this report, we offer evidence that a member of the polo-like kinase (PLK) family, PLK2 (or serum-inducible kinase, SNK), functions as an α-synuclein kinase. The ability of PLK2 to directly phosphorylate α-synuclein at Ser-129 is established by overexpression in cell culture and by in vitro reaction with the purified kinase. We show that PLK2 phosphorylates α-synuclein in cells, including primary neuronal cultures, using a series of kinase inhibitors as well as inhibition of expression with RNA interference. In addition, inhibitor and knock-out studies in mouse brain support a role for PLK2 as an α-synuclein kinase in vivo.     

Learning/Memory Impairment and Reduced Expression of the HNK-1 Carbohydrate in ��4-Galactosyltransferase-II-deficient Mice

The glycosylation of glycoproteins and glycolipids is important for central nervous system development and function. Although the roles of several carbohydrate epitopes in the central nervous system, including polysialic acid, the human natural killer-1 (HNK-1) carbohydrate, α2,3-sialic acid, and oligomannosides, have been investigated, those of the glycan backbone structures, such as Galβ1-4GlcNAc and Galβ1-3GlcNAc, are not fully examined. Here we report the generation of mice deficient in β4-galactosyltransferase-II (β4GalT-II). This galactosyltransferase transfers Gal from UDP-Gal to a nonreducing terminal GlcNAc to synthesize the Gal β1-4GlcNAc structure, and it is strongly expressed in the central nervous system. In behavioral tests, the β4GalT-II^-/- mice showed normal spontaneous activity in a novel environment, but impaired spatial learning/memory and motor coordination/learning. Immunohistochemistry showed that the amount of HNK-1 carbohydrate was markedly decreased in the brain of β4GalT-II^-/- mice, whereas the expression of polysialic acid was not affected. Furthermore, mice deficient in glucuronyltransferase (GlcAT-P), which is responsible for the biosynthesis of the HNK-1 carbohydrate, also showed impaired spatial learning/memory as described in our previous report, although their motor coordination/learning was normal as shown in this study. Histological examination showed abnormal alignment and reduced number of Purkinje cells in the cerebellum of β4GalT-II^-/- mice. These results suggest that the Galβ1-4GlcNAc structure in the HNK-1 carbohydrate is mainly synthesized by β4GalT-II and that the glycans synthesized by β4GalT-II have essential roles in higher brain functions, including some that are HNK-1-dependent and some that are not.The glycosylation of glycoproteins, proteoglycans, and glycolipids is important for their biological activities, stability, transport, and clearance from circulation, and cell-surface glycans participate in cell-cell and cell-extracellular matrix interactions. In the central nervous system, several specific carbohydrate epitopes, including polysialic acid (PSA),³ the human natural killer-1 (HNK-1) carbohydrate, α2,3-sialic acid, and oligomannosides play indispensable roles in neuronal generation, cell migration, axonal outgrowth, and synaptic plasticity (1). Functional analyses of the glycan backbone structures, like lactosamine core (Galβ1-4GlcNAc), neolactosamine core (Galβ1-3GlcNAc), and polylactosamine (Galβ1-4GlcNAcβ1-3) have been carried out using gene-deficient mice in β4-galactosyltransferase-I (β4GalT-I) (2, 3), β4GalT-V (4), β3-N-acetylglucosaminyl-transferase-II (β3GnT-II) (5), β3GnT-III (Core1-β3GnT) (6), β3GnT-V (7), and Core2GnT (8). However, the roles of these glycan backbone structures in the nervous system have not been examined except the olfactory sensory system (9).β4GalTs synthesize the Galβ1-4GlcNAc structure via the β4-galactosylation of glycoproteins and glycolipids; the β4GalTs transfer galactose (Gal) from UDP-Gal to a nonreducing terminal N-acetylglucosamine (GlcNAc) of N- and O-glycans with a β-1,4-linkage. The β4GalT family has seven members (β4GalT-I to VII), of which at least five have similar Galβ1-4GlcNAc-synthesizing activities (10, 11). Each β4GalT has a tissue-specific expression pattern and substrate specificity with overlapping, suggesting each β4GalT has its own biological role as well as redundant functions. β4GalT-I and β4GalT-II share the highest identity (52% at the amino acid level) among the β4GalTs (12), suggesting these two galactosyltransferases can compensate for each other. β4GalT-I is strongly and ubiquitously expressed in various non-neural tissues, whereas β4GalT-II is strongly expressed in neural tissues (13, 14). Indeed, the β4GalT activity in the brain of β4GalT-I-deficient (β4GalT-I^-/-) mice remains as high as 65% of that of wild-type mice, and the expression levels of PSA and the HNK-1 carbohydrate in the brain of these mice are normal (15). These results suggest β4GalTs other than β4GalT-I, like β4GalT-II, are important in the nervous system.Among the β4GalT family members, only β4GalT-I^-/- mice have been examined extensively; this was done by us and another group. We reported that glycans synthesized by β4GalT-I play various roles in epithelial cell growth and differentiation, inflammatory responses, skin wound healing, and IgA nephropathy development (2, 16-18). Another group reported that glycans synthesized by β4GalT-I are involved in anterior pituitary hormone function and in fertilization (3, 19). However, no other nervous system deficits have been reported in these mice, and the role of the β4-galactosylation of glycoproteins and glycolipids in the nervous system has not been fully examined.In this study, we generated β4GalT-II^-/- mice and examined them for behavioral abnormalities and biochemical and histological changes in the central nervous system. β4GalT-II^-/- mice were impaired in spatial learning/memory and motor coordination/learning. The amount of HNK-1 carbohydrate was markedly decreased in the β4GalT-II^-/- brain, but PSA expression was not affected. These results suggest that the Galβ1-4GlcNAc structure in the HNK-1 carbohydrate is mainly synthesized by β4GalT-II and that glycans synthesized by β4GalT-II have essential roles in higher brain functions, including ones that are HNK-1 carbohydrate-dependent and ones that are independent of HNK-1.     

The Block of DNA Polymerase �� Strand Displacement Activity by an Abasic Site Can Be Rescued by the Concerted Action of DNA Polymerase �� and Flap Endonuclease 1

Abasic (AP) sites are very frequent and dangerous DNA lesions. Their ability to block the advancement of a replication fork has been always viewed as a consequence of their inhibitory effect on the DNA synthetic activity of replicative DNA polymerases (DNA pols). Here we show that AP sites can also affect the strand displacement activity of the lagging strand DNA pol δ, thus preventing proper Okazaki fragment maturation. This block can be overcome through a polymerase switch, involving the combined physical and functional interaction of DNA pol β and Flap endonuclease 1. Our data identify a previously unnoticed deleterious effect of the AP site lesion on normal cell metabolism and suggest the existence of a novel repair pathway that might be important in preventing replication fork stalling.Loss of purine and pyrimidine bases is a significant source of DNA damage in prokaryotic and eukaryotic organisms. Abasic (apurinic and apyrimidinic) lesions occur spontaneously in DNA; in eukaryotes it has been estimated that about 10⁴ depurination and 10² depyrimidation events occur per genome per day. An equally important source of abasic DNA lesions results from the action of DNA glycosylases, such as uracil glycosylase, which excises uracil arising primarily from spontaneous deamination of cytosines (1). Although most AP sites are removed by the base excision repair (BER)⁵ pathway, a small fraction of lesions persists, and DNA with AP lesions presents a strong block to DNA synthesis by replicative DNA polymerases (DNA pols) (2, 3). Several studies have been performed to address the effects of AP sites on the template DNA strand on the synthetic activity of a variety of DNA pols. The major replicative enzyme of eukaryotic cells, DNA pol δ, was shown to be able to bypass an AP lesion, but only in the presence of the auxiliary factor proliferating cell nuclear antigen (PCNA) and at a very reduced catalytic efficiency if compared with an undamaged DNA template (4). On the other hand, the family X DNA pols β and λ were shown to bypass an AP site but in a very mutagenic way (5). Recent genetic evidence in Saccharomyces cerevisiae cells showed that DNA pol δ is the enzyme replicating the lagging strand (6). According to the current model for Okazaki fragment synthesis (7–9), the action of DNA pol δ is not only critical for the extension of the newly synthesized Okazaki fragment but also for the displacement of an RNA/DNA segment of about 30 nucleotides on the pre-existing downstream Okazaki fragment to create an intermediate Flap structure that is the target for the subsequent action of the Dna2 endonuclease and the Flap endonuclease 1 (Fen-1). This process has the advantage of removing the entire RNA/DNA hybrid fragment synthesized by the DNA pol α/primase, potentially containing nucleotide misincorporations caused by the lack of a proofreading exonuclease activity of DNA pol α/primase. This results in a more accurate copy synthesized by DNA pol δ. The intrinsic strand displacement activity of DNA pol δ, in conjunction with Fen-1, PCNA, and replication protein A (RP-A), has been also proposed to be essential for the S phase-specific long patch BER pathway (10, 11). Although it is clear that an AP site on the template strand is a strong block for DNA pol δ-dependent synthesis on single-stranded DNA, the functional consequences of such a lesion on the ability of DNA pol δ to carry on strand displacement synthesis have never been investigated so far. Given the high frequency of spontaneous hydrolysis and/or cytidine deamination events, any detrimental effect of an AP site on the strand displacement activity of DNA pol δ might have important consequences both for lagging strand DNA synthesis and for long patch BER. In this work, we addressed this issue by constructing a series of synthetic gapped DNA templates with a single AP site at different positions with respect to the downstream primer to be displaced by DNA pol δ (see Fig. 1A). We show that an AP site immediately upstream of a single- to double-strand DNA junction constitutes a strong block to the strand displacement activity of DNA pol δ, even in the presence of RP-A and PCNA. Such a block could be resolved only through a “polymerase switch” involving the concerted physical and functional interaction of DNA pol β and Fen-1. The closely related DNA pol λ could only partially substitute for DNA pol β. Based on our data, we propose that stalling of a replication fork by an AP site not only is a consequence of its ability to inhibit nucleotide incorporation by the replicative DNA pols but can also stem from its effects on strand displacement during Okazaki fragment maturation. In summary, our data suggest the existence of a novel repair pathway that might be important in preventing replication fork stalling and identify a previously unnoticed deleterious effect of the AP site lesion on normal cell metabolism.Open in a separate windowFIGURE 1.An abasic site immediately upstream of a double-stranded DNA region inhibits the strand displacement activity of DNA polymerase δ. The reactions were performed as described under “Experimental Procedures.” A, schematic representation of the various DNA templates used. The size of the resulting gaps is indicated in nt. The position of the AP site on the 100-mer template strand is indicated relative to the 3′ end. Base pairs in the vicinity of the lesion are indicated by dashes. The size of the gaps (35–38 nt) is consistent with the size of ssDNA covered by a single RP-A molecule, which has to be released during Okazaki fragment synthesis when the DNA pol is approaching the 5′-end of the downstream fragment. When the AP site is covered by the downstream terminator oligonucleotide (Gap-3 and Gap-1 templates) the nucleotide placed on the opposite strand is C to mimic the situation generated by spontaneous loss of a guanine or excision of an oxidized guanine, whereas when the AP site is covered by the primer (nicked AP template), the nucleotide placed on the opposite strand is A to mimic the most frequent incorporation event occurring opposite an AP site. B, human PCNA was titrated in the presence of 15 nm (lanes 2–4 and 10–12) or 30 nm (lanes 6–8 and 14–16) recombinant human four subunit DNA pol δ, on a linear control (lanes 1–8) or a 38-nt gap control (lanes 9–16) template. Lanes 1, 5, 9, and 13, control reactions in the absence of PCNA. C, human PCNA was titrated in the presence of 60 nm DNA pol δ, on a linear AP (lanes 2–4) or 38-nt gap AP (lanes 6–9) template. Lanes 1 and 5, control reactions in the absence of PCNA.