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Loop II of DNA polymerase beta (pol beta) consists of 14 amino acid residues and is highly flexible and solvent exposed. Previous research from our laboratory has shown that this loop is important for polymerase activity and fidelity. In the study presented here, we demonstrate that a shortened five amino acid residue loop compromises the fidelity of pol beta. This five-residue loop, termed ENEYP, induces one base frameshift errors and A-C transversions within a specific sequence context. We demonstrate that ENEYP misincorporates dGTP opposite template A at higher efficiencies than wild-type pol beta. The kinetic basis for misincorporation is a defect in discrimination of the correct from incorrect dNTP substrate at the level of ground-state binding. Our results are consistent with the idea that loop II of pol beta functions to maintain accurate DNA synthesis by a direct or indirect influence on the nucleotide binding pocket.

The accurate replication and transmission of genetic information is critical in the life of an organism. During its entire lifespan, the genetic information is constantly under attack from endogenous and exogenous sources of damage. To ensure that the content of its genetic information is faithfully preserved for synthesis and transmission, eukaryotic cells have developed a complex system of genomic quality control. Key players in this process are DNA polymerases, the enzymes responsible for synthesizing the DNA, because errors introduced into the genome by polymerase can result in mutations. We use DNA polymerase beta (pol beta) as a model system to investigate mechanisms of preserving fidelity during nucleotide incorporation. In the study described here, we characterized the role that loop II of pol beta plays in maintaining the activity and fidelity of pol beta. We report here that the absence or shortening of loop II compromises the catalytic activity of pol beta. Our data also show that loop variants of a specific length have a lower fidelity when compared to the wild-type polymerase. Taken together, our results indicate that loop II is important for the catalytic activity and fidelity of pol beta.

Gap junctional communication modulates intercellular calcium signaling in many cell types. We have investigated whether gap junctional communication modulates calcium oscillatory behavior of cells responding to an agonist. Extracellular UTP induced calcium oscillations in 70% of HeLa cells cultured in monolayer, and neighboring cells oscillated independently of each other. In HeLa cell transfectants expressing connexin43 (HeLa/Cx43), extracellular UTP induced calcium transients, but calcium oscillations occurred in only 10% of cells. Inhibition of gap junctional communication with anandamide in HeLa/Cx43 transfectants substantially restored oscillations (55% of cells). In HeLa/Cx45 transfectants, UTP initiated calcium oscillations similar to those seen in HeLa cells (63% of cells), but HeLa/Cx46 transfectants demonstrated calcium oscillations that were dampened compared to those of the parental HeLa cells, and occurred in only 40% of cells. These experiments demonstrate that gap junctional communication modulates calcium oscillatory behavior in cell monolayers, presumably by allowing cells to share a small molecule such as inositol trisphosphate. These studies suggest that gap junctional communication may alter the nature of signals induced by calcium mobilizing agonists in a connexin-dependent fashion by modulating calcium oscillatory behavior.

Osteoclasts are multinucleated giant cells of the mononuclear phagocyte lineage that are responsible for bone resorption in vivo. Osteoprotegerin ligand (OPGL) has recently been identified as an important differentiation and activation factor for osteoclasts (Kong et al. [1999] Nature 397:315-323). We and others have demonstrated P2-mediated responses in mononuclear phagocytes and ATP-mediated effects on osteoclasts have also been described (Yu and Ferrier [1994] Cell Signal 6:905-914). The goal of these studies is to identify the role of P2 receptor activation in osteoclast formation and function. In particular, we assessed the role of P2 receptors in intercellular calcium signaling among macrophages and osteoclasts and between osteoblasts and osteoclasts, and studied the role of P2X 7 in OPGL-mediated osteoclast formation. We found that mechanically induced intercellular calcium signaling occurred between osteoblasts and osteoclasts, among J774 macrophage-like cells, and among osteoclasts, but not among bone marrow-derived macrophages. Interestingly, different P2 receptors seemed to be responsible for the propagation of intercellular calcium signals among different cells. In our studies of giant cell formation, we confirmed that expression of P2X 7 enhances giant cell formation in J774 macrophages and in HEK cells. In contrast, RAW cell clones that were selected for resistance to ATP permeabilization were able to form giant cells in response to OPGL, suggesting that the pathways for giant cell and osteoclast may be different.

Insulin-mediated increases in cytosolic calcium are synchronized among the cells in a pancreatic islet, and result in pulsatile secretion of insulin. Pancreatic beta cells express the gap junction protein connexin43 and are functionally coupled, making gap junctional communication a likely mechanism for the synchronization of calcium transients among islet cells. To define the mechanism by which pancreatic islet cells coordinate calcium responses, we studied mechanically-induced intercellular calcium waves in the communication-deficient rat insulinoma cell line RINm5f, and in RINm5f cells transfected with the gap junction protein connexin43. Both RINm5f and RINm5f cells transfected with connexin43 propagated calcium waves that required release of calcium from intracellular stores, did not involve gap junctional communication, and appeared to be mediated by autocrine activity of secreted ATP acting on P2U purinergic receptors. Connexin43 transfectants also propagated calcium waves that required gap junctional communication and influx of extracellular calcium through voltage-gated calcium channels. Gap junction-dependent intercellular calcium waves were inhibited by preventing plasma membrane depolarization. These studies demonstrate two distinct pathways by which insulin-secreting cells can coordinate cytosolic calcium rises, and show that it is by ionic traffic that gap junctions synchronize calcium-dependent events in these cells.