Reconstitution of an ATM-dependent checkpoint that inhibits chromosomal DNA replication following DNA damage.

Cell cycle checkpoints lead to the inhibition of cell cycle progression following DNA damage. A cell-free system derived from Xenopus eggs has been established that reconstitutes the checkpoint pathway inhibiting DNA replication initiation. DNA containing double-strand breaks inhibits replication initiation in a dose-dependent manner. Upon checkpoint activation, a prereplicative complex is assembled that contains ORC, Cdc6, Cdc7, and MCM proteins but lacks Cdc45. The checkpoint is ATM dependent. Cdk2/CyclinE acts downstream of ATM and is downregulated by Cdk2 phosphorylation on tyrosine 15. Cdk2AF/CyclinE is refractory to checkpoint signaling, and Cdc25A overrides the checkpoint and restores DNA replication. This report provides the description of a DNA damage checkpoint pathway that prevents the onset of S phase independently of the transcriptional function of p53 in a vertebrate organism.

The intrinsic DNA helicase activity of Methanobacterium thermoautotrophicum delta H minichromosome maintenance protein.

Minichromosome maintenance proteins (MCMs) form a family of conserved molecules that are essential for initiation of DNA replication. All eukaryotes contain six orthologous MCM proteins that function as heteromultimeric complexes. The sequencing of the complete genomes of several archaebacteria has shown that MCM proteins are also present in archaea. The archaea Methanobacterium thermoautotrophicum contains a single MCM-related sequence. Here we report on the expression and purification of the recombinant M. thermoautotrophicum MCM protein (MtMCM) in both Escherichia coli and baculovirus-infected cells. We show that purified MtMCM protein assembles in large macromolecular complexes consistent in size with being double hexamers. We demonstrate that MtMCM contains helicase activity that preferentially uses dATP and DNA-dependent dATPase and ATPase activities. The intrinsic helicase activity of MtMCM is abolished when a conserved lysine in the helicase domain I/nucleotide binding site is mutated. MtMCM helicase unwinds DNA duplexes in a 3' --> 5' direction and can unwind up to 500 base pairs in vitro. The kinetics, processivity, and directionality of MtMCM support its role as a replicative helicase in M. thermoautotrophicum. This strongly suggests that this function is conserved for MCM proteins in eukaryotes where a replicative helicase has yet to be identified.

DNA replication in vertebrates requires a homolog of the Cdc7 protein kinase.

CDC7 is an essential gene required for DNA replication in Saccharomyces cerevisiae. Cdc7p homologs have recently been identified in vertebrates, but their role in DNA replication has not yet been addressed. Here we show that antibodies to the Xenopus laevis homolog, xCdc7, interfere with DNA replication in vivo in developing embryos and in vitro in cycling egg extracts. We also demonstrate cell cycle-dependent association of xCdc7 with the Mcm complex, which binds to replication origins and also is required for DNA synthesis. Taken together, these data indicate that the function of xCdc7 is conserved from fungi to vertebrates. xCdc7 protein accumulates after stimulation of resting oocytes with progesterone, suggesting a molecular explanation for previous observations that the development of the capacity for DNA replication requires protein synthesis late in meiosis I.

Two tandem and independent sub-activation domains in the amino terminus of p53 require the adaptor complex for activity.

The ability of p53 to function as a tumor suppressor is linked to its function as a transcriptional activator, since p53 mutants that do not transactivate are unable to suppress tumor cell growth. Previous studies identified an activation domain in the amino terminal 40 residues of the protein, a region that binds to several general transcription factors and to some oncogene products. For example, mdm-2, a cellular oncoprotein, binds to this region and represses p53 transactivation. Here we describe a new activation domain within the amino terminus of p53 that maps between amino acids 40-83, and whose residues trp-53 and phe-54 are critical for function both in yeast and in mammalian cells. In vivo studies in yeast show that the new activation subdomain, unlike the previously described, is mdm-2 independent. Both p53 activation subdomains (1-40 and 40-83) require the yeast adaptor complex ADA2/ADA3/GCN5 for transcriptional activation. Moreover, since activation by p53 requires GCN5's enzymatic histone acetyltransferase domain, p53 may regulate gene expression by influencing chromatin modification.

Identification of human proteins functionally conserved with the yeast putative adaptors ADA2 and GCN5.

Transcriptional adaptor proteins are required for full function of higher eukaryotic acidic activators in the yeast Saccharomyces cerevisiae, suggesting that this pathway of activation is evolutionarily conserved. Consistent with this view, we have identified possible human homologs of yeast ADA2 (yADA2) and yeast GCN5 (yGCN5), components of a putative adaptor complex. While there is overall sequence similarity between the yeast and human proteins, perhaps more significant is conservation of key sequence features with other known adaptors. We show several functional similarities between the human and yeast adaptors. First, as shown for yADA2 and yGCN5, human ADA2 (hADA2) and human GCN5 (hGCN5) interacted in vivo in a yeast two-hybrid assay. Moreover, hGCN5 interacted with yADA2 in this assay, suggesting that the human proteins form similar complexes. Second, both yADA2 and hADA2 contain cryptic activation domains. Third, hGCN5 and yGCN5 had similar stabilizing effects on yADA2 in vivo. Furthermore, the region of yADA2 that interacted with yGCN5 mapped to the amino terminus of yADA2, which is highly conserved in hADA2. Most striking, is the behavior of the human proteins in human cells. First, GAL4-hADA2 activated transcription in HeLa cells, and second, either hADA2 or hGCN5 augmented GAL4-VP16 activation. These data indicated that the human proteins correspond to functional homologs of the yeast adaptors, suggesting that these cofactors play a key role in transcriptional activation.

Renal revascularization to preserve and restore renal function.

A total of 97 patients underwent 107 renal revascularization procedures for restoration and preservation of renal function. Of the 4 groups of high risk surgical patients that emerged an overall successful outcome was achieved in 83%, with a 6% mortality rate and an 11% morbidity rate. Renal revascularization for restoration and preservation of renal function can be performed safely with good results. The preoperative serum creatinine level was not predictive of the surgical outcome. Alternative bypass procedures are preferred.

Changing concepts in surgical management of renovascular hypertension.

As newer surgical techniques and concepts have emerged, including revascularization of the totally occluded renal artery and alternatives to aortorenal bypass (hepatic, splenic, or iliac artery to renal artery grafts), our patient population has changed. Patients with diffuse atherosclerotic disease, bilateral renal artery stenosis, totally occluded renal arteries, and azotemia are being referred for renal revascularization, thereby changing the indications for operation and the results that can be anticipated. Although our results in patients operated on solely for uncontrollable hypertension or renal failure have been successful, much work needs to be done to improve the results obtained when patients have a combination of uncontrollable hypertension and renal failure.

Renal revascularization in the azotemic hypertensive patient resistant to therapy.

We undertook this study to assess the frequency of renovascular hypertension in patients with azotemia and hypertension refractory to drug therapy and to determine the effects of renal revascularization on blood pressure and renal function in these subjects. Thirty-nine of 106 consecutive patients admitted for diagnostic evaluation of severe hypertension proved to have renovascular hypertension. Of 21 hypertensive patients with renal insufficiency, 10 appeared to have renovascular hypertension with either bilateral atherosclerotic renovascular disease or unilateral renal arterial stenosis in a solitary functioning kidney. Medical therapy in the hospital often induced further deterioration of renal function despite enhanced blood-pressure control. However, surgical revascularization or percutaneous transluminal angioplasty produced improvement or stabilization of renal function and control of blood pressure in all patients with azotemia who were treated in this manner, despite longstanding hypertension. The benefits of therapy have persisted for 10 to 42 months of follow-up. These studies indicate that refractory hypertension in association with renal insufficiency is a relatively common clinical presentation for renovascular hypertension and bilateral renal-artery disease. Diagnostic evaluation and consideration of renal revascularization appear warranted in such patients, both for the control of the hypertension and for improvement in renal function.

Cutaneous effects of pulsed nitrogen gas laser irradiation.

The effects of pulsed nitrogen gas laser emission (337.1 nm wavelength) were studied on human skin. The laser provides high-intensity monochromatic UVA radiation and can elicit delayed erythema in an actual exposure time of about 1 msec (105,000 pulses, each lasting 10 nsec, delivered over 210 sec). The effects of nitrogen laser irradiation were compared clinically and histologically with conventional erythemogenic UVA and UVB exposures from xenon arc or mercury arc lamps and were found to be similar in many respects. The minimal erythema dose is comparable to that obtained using more conventional continuous light sources which have more than 100 times lower intensity. A phototoxicity comparison of oral and topically applied psoralens is presented, indicating that the laser may prove useful in comparing photosenitizing capacity of certain compounds.