Regulation Of Endonuclease Activity Research Articles

Order out of disorder: Regulation of endonuclease activity during eukaryotic mismatch repair.

Order out of disorder: Regulation of endonuclease activity during eukaryotic mismatch repair.

Differential activation of the influenza virus polymerase via template RNA binding

Primary transcripts synthesized by the influenza virus polymerase contain the capped 5' ends of eukaryotic mRNAs. These sequences are derived from host mRNA and scavenged by the viral polymerase as a prerequisite to transcription. The first step in this reaction is the specific binding of the viral polymerase to the cap structure of the host RNA. The role that template RNA plays in this RNA binding reaction was examined in quantitative capped mRNA binding and endonuclease assays. Capped RNA binding was shown to be a template-dependent property of the influenza virus polymerase. Addition of only the 5' end of viral RNA stimulates capped mRNA binding by the viral polymerase, but endonuclease activity requires the addition of the 3' end. The addition of template RNA corresponding to the positive-sense complementary RNA replicative intermediate was also able to stimulate capped mRNA binding but was not able to efficiently activate the viral endonuclease. Thus, regulation of endonuclease activity by the influenza virus polymerase can be dependent on template RNA binding.

Follicular dendritic cells inhibit apoptosis in human B lymphocytes by a rapid and irreversible blockade of preexisting endonuclease.

During germinal center reactions, a minority of B lymphocytes are selected after successful binding to follicular dendritic cells (FDCs). The majority of the B cells, however, die by apoptosis. One of the characteristics of apoptosis is rapid fragmentation of DNA by an endogenous endonuclease. The regulation of apoptosis and endonuclease activity in germinal center (GC) B cells is largely unknown. In this study we have investigated the induction and inhibition of endonuclease activity in GC B cells. We also investigated the role of FDCs, surface Ig (sIg), sIgM, CD21, CD22 CD40, and intracellular Zn2+ in the regulation of endonuclease activity. We have found that DNA fragmentation in GC B cells is caused by a preexisting endonuclease very similar to NUC-18 (an 18-kD endonuclease identified in rat thymocytes). Endonuclease activity in GC B cells appears to be rapidly and irreversibly blocked after interaction with FDCs, but not after cross-linkage of sIg, sIgM, CD21, CD22, or CD40. Addition of soluble CD40-human IgM fusion protein (sCD40) to FDC-B cell cultures also did not interfere with FDC-mediated B cell rescue. Chelation of intracellular Zn2+ during FDC-B cell cultures resulted in abrogated B cell rescue. These data suggest that FDCs inhibit apoptosis in GC B cells by a rapid inactivation of preexisting endonuclease using a mechanism distinct from CD40 ligation.

Estrogens inhibit and androgens enhance ovarian granulosa cell apoptosis.

Apoptotic cell death has recently been suggested to be the underlying mechanism of ovarian follicle atresia. To study the regulation of follicle cell apoptosis by sex steroids, we have analyzed ovarian DNA fragmentation, the hallmark of apoptosis, in rats treated with estrogens and androgens. Immature rats were hypophysectomized and implanted with diethylstilbestrol (DES) capsules. Two days later, DES implants were removed in some animals, followed by treatment with estrogens with or without androgens. The extent of ovarian apoptotic DNA fragmentation was analyzed by autoradiography of size-fractionated DNA labeled at 3'-ends by [32P]dideoxy-ATP. After DES withdrawal, ovarian weight decreased and DNA fragmentation increased in a time-dependent manner. In granulosa cells, an increase in apoptotic DNA fragmentation was seen 12 h after withdrawal of DES implants, followed by a 25-fold increase at 48 h. In situ analysis of DNA fragmentation on histological sections of ovaries, using a nonisotopic labeling of DNA by digoxigenin-dideoxy-UTP, also demonstrated that apoptosis induced by DES withdrawal is confined to the granulosa cells in early antral and preantral follicles. No increase in DNA breakdown was detected in thecal cells and interstitial tissues or granulosa cells of primordial and primary follicles. In contrast, replacement with DES (0.5 mg twice daily) or estradiol benzoate (3 mg daily) completely prevented the observed ovarian weight loss and increases in granulosa cell apoptosis. Treatment with estradiol benzoate (0.003-3 mg/day) dose dependently suppressed the apoptosis seen 2 days after removal of DES implants. Furthermore, the antiatretogenic effect of estrogen was blocked by treatment with testosterone (0.5 mg twice daily), which increased ovarian apoptotic DNA fragmentation and decreased ovarian weight in DES-treated animals in a time-dependent manner. Also, in situ examination showed that androgen treatment increased apoptosis in the granulosa cells in a subpopulation of early antral and preantral follicles. The specificity of testosterone action was further demonstrated by the lack of effect of progesterone and cortisol on ovarian apoptosis. These data suggest that sex steroids play an important role in the regulation of ovarian apoptotic cell death, with estrogens preventing apoptosis and androgens antagonizing the effect of estrogens. These data provide the basis for future studies on the role of sex steroid hormones in follicular atresia and the regulation of endonuclease activity by steroid hormones.

Ca 2+-dependent mechanisms of cytotoxicity and programmed cell death

There is increasing evidence that the calcium ion plays a critical role in both toxic cell killing and programmed cell death. Thus, in a variety of experimental systems a perturbation of intracellular Ca 2+ homeostasis due to increased Ca 2+ influx and/or inhibition of Ca 2+ extrusion has been found to be an early event in the development of cell injury. It is clear that sustained increases in intracellular Ca 2+ can activate cytotoxic mechanisms which result in perturbations of cellular structure and function. For example, the stimulation of Ca 2+-dependent proteases can result in a disruption of cytoskeletal organization and the formation of surface protrusions (blebs) and Ca 2+ -mediated phospholipase activation can result in an impairment of mitochondrial function with collapse of membrane potential and cessation of ATP synthesis. The activation of a Ca 2+, Mg 2+-dependent nuclear endonuclease is associated with chromatin cleavage and appears to play a crucial role in programmed cell death (apoptosis) in the immune system and other tissues. There is also recent evidence that this process may be responsible for the immunotoxicity of dioxins and organotin compounds and involved in the killing of adenocarcinoma cells by tumor necrosis factor alpha. Although calcium ions appear to be required for endonuclease activity during apoptosis, this process is also influenced by other factors, e.g. protein kinase C activity, intracellular polyamine and Zn 2+ levels, chromatin structure, etc. Thus, the regulation of endonuclease activity under both physiological and toxicological conditions appears to be complex and to involve multiple factors.