Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “CLAMPS”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 559 records · Page 31Linked to original sources
PubMed1998

Safety of selective vascular clamping for major hepatectomies.

BACKGROUND: Although hepatic vascular clampings are widely used during major hepatic resections, they may not always be necessary. Selective vascular clamping, which only controls the afferent blood flow of the resected liver, could be a valuable alternative, provided that blood loss is not increased because the opposite liver remains perfused. STUDY DESIGN: The aim of the study was to assess the safety of selective vascular clamping in 43 patients who underwent 36 right hepatectomies and 7 left hepatectomies for lesions located peripherally within the liver. Blood transfusions, hepatic tests, morbidity, mortality, and hospital stay were evaluated. RESULTS: Selective vascular clamping was efficient in 34 of the 43 attempts (79%), but bleeding from the contralateral liver required conversion to portal triad damping in 9 patients (21%). Median blood transfusions were 0 units (range 0 to 4 U), and 28 patients (65%) did not require transfusions. Postoperative laboratory tests showed that larger changes occurred at day 1 and tended to return to preoperative values at the end of the first postoperative week. Median time of hospitalization was 10 days (range 7 to 28 days). Postoperative course was uneventful in 35 patients (81%). Nonlethal complications occurred in 7 patients (16.3%). One patient (2%) with massive hepatic steatosis died of liver failure after right hepatectomy. CONCLUSIONS: Selective vascular clamping is a safe alternative to total inflow occlusion for major hepatectomies applicable in 80% of selected patients with peripheral liver tumors.

Adult↗
PubMed2000

Characterization of the "helix clamp" motif of HIV-1 reverse transcriptase using MALDI-TOF MS and surface plasmon resonance.

A helix-turn-helix motif in the crystal structure of human immunodeficiency virus type 1 reverse transcriptase (HIV-1 RT) was proposed to be a conserved nucleic acid binding domain among several nucleotide polymerizing enzymes (Hermann, T.; Meier, T.; Götte, M.; Heumann, H. Nucleic Acids Res. 1994, 22, 4625-4633). The sequence of this domain is homologous to 259KLVGKL-(X)16KLLR284 of HIV-1 RT, which acts as a "helix clamp" grasping the template-primer (T-P) complex. We characterized the helix clamp motif using MALDI-TOF MS and surface plasmon resonance (BIAcore). Our studies showed that the "helix clamp" has a nucleic acid binding function that may not be sequence specific. This evidence suggests that ionic interactions between the helix clamp and oligonucleotide backbone are not solely responsible for binding. Secondary and tertiary structures of the protein may also play a significant role in nucleic acid binding. The association and dissociation constants, ka and kd, for the binding of single-stranded oligonucleotide to the helix clamp were determined to be 7.03 x 10(3) M(-1) s(-1) and 1.22 x 10(3) s(-1), respectively.

Amino Acid Sequence↗
PubMed2000

Microdialysis of the spinal cord during thoracic aortic cross-clamping in a porcine model.

OBJECTIVE: Utilising microdialysis to measure the changes of glucose, lactate, pyruvate and glutamate levels in the spinal cord after cross-clamping of the thoracic aorta in an established porcine model to monitor the degree of ischaemia. DESIGN: Experimental study with a porcine model. SETTING: University Hospital, Trondheim. SUBJECTS: Six pigs. MAIN OUTCOME MEASURE: Lactate, pyruvate and glutamate concentrations in the microdialysis perfusate from the spinal cord. RESULTS: A significant increase of the lactate-pyruvate ratio during the last 30 min of the 1 h clamping period, with a maximum increase of 169% from the basal value the last 10 min before declamping. No evident change in this ratio between the clamping and the reperfusion period. No significant change in glutamate levels during clamping or reperfusion period. CONCLUSION: Microdialysis reflects the ischaemic state of the spinal cord during cross-clamping of the thoracic aorta in pigs, and is well suited to study each phenomena.

Animals↗
PubMed1999

Endogenous renal nitric oxide metabolism following experimental infrarenal aortic cross-clamp-induced ischaemia-reperfusion injury.

BACKGROUND: Abdominal aortic surgery is associated with marked changes in renal haemodynamics. The aim of this study was to investigate the influence of infrarenal cross-clamping on glomerular filtration rate and endogenous renal nitric oxide metabolism. METHODS: Groups of male Wistar rats were subjected to infrarenal aortic cross-clamping followed by reperfusion. Animals were allowed to recover after a left nephrectomy. The glomerular filtration rate of the remaining kidney was measured on the second and seventh day after the procedure before the animal was killed and the remaining kidney harvested. Total nitric oxide synthase (NOS) activity and expression of inducible NOS (iNOS) was determined in renal tissue following 1 h and 7 days of reperfusion. RESULTS: Glomerular filtration rate was impaired on the second and seventh day after operation in all animals subjected to lower torso ischaemia compared with controls (P < 0.05). Renal NOS activity was increased at 1 h and 7 days in animals subjected to infrarenal cross-clamping compared to controls (P < 0.01). iNOS was detected in renal tissue of animals subjected to infrarenal aortic cross-clamping on the seventh day after operation. CONCLUSION: Infrarenal aortic cross-clamping is associated with impairment of renal function in the early postoperative period. There is an increase in endogenous renal nitric oxide metabolism with iNOS expression. Presented in part to the Surgical Research Society, Dublin, Ireland, July 1998

Animals↗
PubMed1999

The use of the hypoglycaemic clamp in the assessment of pituitary function.

OBJECTIVE: The standard dynamic test used to diagnose hypopituitarism is the insulin tolerance test (ITT), in which insulin-induced secretion of ACTH, GH and cortisol is measured. However, because of differences in insulin sensitivity some patients fail achieve sufficient hypoglycaemia to assess pituitary function and colleagues experience severe hypoglycaemia and are at risk for cardiac dysrhythmia, seizure or coma. This risk may be particularly pertinent in the evaluation of older adults. We hypothesized that the hypoglycaemic clamp may be useful in assessing pituitary function in some patients. PATIENTS AND MEASUREMENTS: Twenty-one normal subjects (14 old [50-76 years] and 7 young [18-36 years]) and 7 hypopituitary subjects were studied. A clamp study was performed in which insulin infusion was given at 2 mU/kg/min and increased to 4 mU/kg/min if the target glucose concentration was not reached after 40 min. Dextrose was infused as needed to clamp the plasma glucose concentration at 2.2 mmol/l for 30 min. On a separate day, 7 young controls also underwent an ITT in which 0.15 U/kg insulin was administered as a bolus intravenous injection at time 0. In both studies, baseline values were taken at - 10, - 5 and 0 min. Samples were then collected every 5 min for plasma glucose and every 10 min for insulin, ACTH, cortisol and GH. RESULTS: ACTH and GH secretion during each test were similar in younger controls (P = NS) but cortisol secretion was lower during ITT (P < 0.01 vs. clamp). Hypopituitary subjects had significantly less ACTH, cortisol and GH secretion than controls of all ages (P < 0.001 for all). Peak GH secretion was significantly lower in the old controls than in young controls (22 +/- 12 vs. 48 +/- 26 mU/l, respectively; P < 0.01) but significantly higher than the hypopituitary subjects (2 +/- 2 mu/l; P < 0.001). CONCLUSION: These data demonstrate that the hypoglycaemic clamp can be used in the assessment of pituitary function and suggest that this technique may be particularly beneficial in the evaluation of GH deficiency in older adults who may not tolerate the ITT.

Adrenocorticotropic Hormone↗
PubMed2003

Circulating RBC volume, measured with biotinylated RBCs, is superior to the Hct to document the hematologic effects of delayed versus immediate umbilical cord clamping in preterm neonates.

BACKGROUND: One problem assessing the hematologic physiology of preterm infants after delivery and/or the efficacy and toxicity of therapeutic interventions affecting RBC measurements is the inability of blood Hct values to accurately reflect circulating RBC volume-owing to changes in plasma volume that influence Hct (i.e., a fall in plasma volume concentrates RBCs to increase Hct; a rise in plasma volume dilutes RBCs to decrease Hct). STUDY DESIGN AND METHODS: As part of a randomized, clinical trial testing the hypothesis that delayed clamping of the umbilical cord at delivery expands neonatal circulating RBC volume, blood Hct was compared to circulating RBC volume results measured directly with autologous, biotinylated RBCs or estimated mathematically with neonatal body weight and Hct values in neonates after immediate or delayed (60 sec) cord clamping. RESULTS: Circulating RBC volume measured directly with biotinylated RBCs significantly increased (p=0.04) in neonates after delayed (42.1 +/- 7.8 mL/kg) versus immediate (36.8 +/- 6.3 mL/kg) cord clamping-a difference not detected indirectly by measuring Hct or estimating circulating RBC volume mathematically. CONCLUSIONS: Because true hematologic effects of delayed versus immediate cord clamping may not be apparent or may be misinterpreted, when based on indirect measurements of Hct or calculations of circulating RBC volume, it is important to measure circulating RBC volume directly-as done with autologous, biotinylated RBCs-to document whether delayed cord clamping truly results in a transfer of significant quantities of RBCs from placenta to neonate. The clinical benefits and potential toxicities of increased RBC transfer to neonates require further studies.

Biotin↗
PubMed2000

Suprarenal aortic cross-clamping in elective abdominal aortic aneurysm surgery.

INTRODUCTION: this retrospective study was undertaken to evaluate whether suprarenal aortic cross-clamping increased the perioperative mortality and morbidity as compared to infrarenal clamping, in order to create the rationale for a more extensive application of this apparently more traumatic manoeuvre. MATERIALS AND METHODS: in a series of 734 elective aortic substitutions for abdominal aneurysm (AA), performed consecutively from January 1992 to June 1999, aortic cross-clamping was performed at a suprarenal level in 56 juxtarenal aneurysms, i.e. aneurysms extending to the lower edge of the renal arteries (8%, Group 1), and at an infrarenal level in 634 subrenal aneurysms (92%, Group 2). When analysing preoperative data, the diameter of aneurysms was larger in Group 1 than in Group 2 (p<0. 005). No significant differences were found between the two groups as regards age, sex, postinfarction cardiomyopathy, chronic obstructive pulmonary disease, chronic renal insufficiency and ASA classification of operative risks. RESULTS: the average time of renal exclusion in the juxtarenal aneurysms was 20 min (range 12-35 min). There is no difference between the two groups as regards the time of aortic clamping (mean 50 vs. 60 min) or the need for homologous blood transfusion (7% vs. 11% of patients). Perioperative (30 days) mortality did not differ: 3.6% vs. 1.9% (n.s.); nor did the incidence of acute myocardial infarction (3.6% vs. 2.3%). Renal function deteriorated in 8 (14%) vs. 0 (0%) (p<0.001) and 1 patient (2%) required permanent dialysis, as compared to 0% in Group 2. The incidence of ischaemic colitis was also significantly higher in Group 1 (7%) than in Group 2 (2%, p<0.01). CONCLUSION: this data shows that suprarenal clamping, which is necessary for the radical treatment of juxtarenal aortic aneurysms, can be performed with a low risk.

Aged↗
PubMed2001

Spinal cord metabolism during thoracic aortic cross-clamping in pigs with special reference to the effect of allopurinol.

OBJECTIVE: investigate the metabolic response of the spinal cord and the effect of allopurinol following cross clamping of the descending thoracic aorta in a porcine model. DESIGN: experimental animal study. MATERIALS: twelve domestic swine. Six pigs were pre-treated with allopurinol, while six pigs served as controls. METHODS: measurement of extracellular concentrations of glucose, pyruvate, lactate, glycerol and glutamate using microdialysis in the lumbar spinal cord. Measurement of lumbar spinal blood flow using laser Doppler technique. RESULTS: for all animals there was a significant decrease in concentrations of glucose and pyruvate together with a significant increase in the lactate-pyruvate ratio during aortic cross clamping. There was also a significant increase in glycerol concentrations 60 min after cross clamping, and a significant decrease in glutamate concentrations after 50 min. No differences in concentrations of glucose, pyruvate, lactate and glutamate or the glutamate-pyruvate ratio were observed between animals used as controls and those treated with allopurinol. The laser Doppler flux decreased to 40% of pre cross-clamp level, returning to normal values at declamping. CONCLUSION: the changes in energy-related metabolites reflect a considerable ischaemia in the spinal cord tissue but there was no convincing effect of allopurinol on the lumbar spinal cord metabolism during thoracic aortic cross clamping in this model.

Allopurinol↗
PubMed2005

Analysis of diastolic function in patients undergoing aortic aneurysm repair and impact on hemodynamic response to aortic cross-clamping.

OBJECTIVES: The purpose of this study was to analyze left ventricular diastolic function in patients undergoing aortic aneurysm repair and to investigate the effects of laparotomy and aortic cross-clamping on diastolic function. DESIGN: Prospective clinical study. SETTING: University hospital. PARTICIPANTS: Forty-five consecutive patients undergoing open aortic aneurysm repair. INTERVENTIONS: Left ventricular diastolic function and hemodynamic variables were evaluated using transesophageal Doppler echocardiography and a pulmonary artery catheter at baseline, after laparotomy, and at 1 and 10 minutes after cross-clamping. Diastolic function was determined by Doppler derivatives of mitral inflow (E/A ratio, deceleration time of early inflow) and pulmonary venous flow (S/D ratio). MEASUREMENTS AND MAIN RESULTS: Twenty of 39 patients revealed signs of diastolic dysfunction at baseline. Of these 20 patients, 14 displayed delayed relaxation and 6 displayed a pseudonormal filling pattern. Patients with pseudonormal filling exhibited a lower stroke volume (p = 0.02) and cardiac index (p < 0.01) in comparison to patients with normal diastolic function. Laparotomy was associated with an improvement of diastolic function in 9 of 20 patients with preexisting diastolic dysfunction. Only 3 patients suffered impairment of diastolic function after cross-clamping. The hemodynamic response to cross-clamping did not differ between patients with normal and abnormal diastolic function. CONCLUSIONS: About 50% of patients undergoing aortic aneurysm repair exhibit signs of diastolic dysfunction. The majority of these patients showed delayed relaxation. Patients with pseudonormal filling displayed a significantly lower cardiac index. Laparotomy resulted in an improvement in diastolic function in about half of patients with preexisting diastolic dysfunction. The effects of cross-clamping on diastolic function are minimal.

Aged↗
PubMed2004

[Use of the "c-clamp" in the emergency treatment of unstable pelvic fractures].

UNLABELLED: It was the objective of this investigation to describe the results after stabilisation of pelvic fractures with the pelvic clamp in polytraumatized patients with unstable pelvic ring fractures. PATIENTS: Between 1999 and 2001 11 polytraumatized patients with an average age of 38 years with unstable fractures of the pelvic ring were treated with the "pelvic clamp" (PC). METHOD: The patient's data were analysed retrospectively from the moment they were admitted to our department until 48 hours after the treatment with the pelvic clamp. The following data were observed: -- The time period until the pelvic clamp was placed. -- The mean blood pressure. -- The oxygenation level (PaO (2)/FiO (2) coefficient). -- The number of requested blood units. -- The time period until hemodynamic stabilisation took place. RESULTS: 8 patients survived their injuries. 5 of them were admitted primarily to our department (ISS 39.8, PTS 35), the other 3 were secondarily admitted to our department (ISS 48.3, PTS 39). 3 of 11 patients (27 %) died averaged within the first 45 minutes after admittance. They also were treated in other units before admittance to our department. 8 surviving patients showed -- a hemodynamic stabilisation 6 hours after the treatment with the PC. -- an increase of the mean blood pressure about 25 % 20 minutes after the treatment with the PC. -- a stabilisation of the oxygenation level 6 hours after the treatment with the PC. -- a decrease of the number of requested blood units 6 hours after treatment with the PC. CONCLUSION: Even if the number of patients who were treated is small, the study shows a positive trend in terms of stabilisation of the vital parameters after stabilisation of the pelvic fracture with the pelvic clamp.

Adult↗
PubMed2000

A 60 minute hyperglycemic clamp is sufficient to assess both phases of insulin secretion.

The hyperglycemic clamp is considered to be the gold standard for determining both first and second phase insulin secretion. In order to achieve a reasonable insulin plateau for the second phase, it has become common practice to clamp for 120 or even 180 minutes at 10 mM. It is unknown whether earlier insulin determinations would be sufficient to predict second phase insulin secretion. We therefore reviewed the hyperglycemic clamp data of 58 subjects with different degrees of glucose tolerance to assess whether one or more insulin concentrations determined at earlier time points of the clamp could predict second phase insulin secretion (insulin and C-peptide concentration at 120 minutes). The correlation coefficients between second-phase insulin secretion and plasma insulin or C-peptide at 60 min were 0.95 and 0.96, respectively (both p<0.00005). Averaging plasma insulin or C-peptide over 2 or more adjacent time points did not improve the correlation. In conclusion, a one-hour hyperglycemic clamp can provide the standard measurement of first phase insulin secretion plus a good approximation of second phase insulin secretion.

Adult↗
PubMed2000

Prevention of paraplegia in pigs by selective segmental artery perfusion during aortic cross-clamping.

PURPOSE: During thoracoabdominal aortic aneurysm repair, a prolonged interruption of the spinal cord blood supply can result in irreversible spinal cord damage. The aim of this study was to investigate whether selective segmental artery perfusion during aortic clamping could prevent paraplegia in pigs. METHODS: Specially designed segmental artery perfusion catheters, which could be attached to an extracorporeal bypass graft system, were used. In experiment I (n = 10), it was assessed whether selective segmental artery perfusion could reverse electrophysiologic evidence of spinal cord ischemia and maintain transcranial motor evoked potentials (tc-MEPs) during 60 minutes of aortic cross-clamping. The abdominal aorta, containing critical segmental arteries, was bypassed through use of an aortoaortic bypass graft system. After the disappearance of tc-MEPs, an aortotomy was followed by selective segmental artery perfusion. In experiment II (n = 10), the aim was to determine whether selective segmental artery perfusion could prevent paraplegia. In five animals (group A), aortic cross-clamping was followed by selective segmental artery perfusion; five control animals (group B) underwent segmental artery blockade only. Postoperative hind limb function and spinal cord histopathology were evaluated on the third postoperative day. RESULTS: In experiment I, tc-MEPs disappeared within 3.7 +/- 3.7 minutes after cross-clamping and returned in all animals in 8.5 +/- 5.3 minutes after selective perfusion. During the study period, tc-MEP amplitudes recovered to a median of 49% (range, 28%-113%) of baseline values. Total bypass graft flow was 880 +/- 294 mL/min, of which 184 +/- 54 mL/min was directed to the selective perfusion catheters. The flow in individual catheters was 52 +/- 13 mL/min. In experiment II, all perfused animals demonstrated normal hind limb function, whereas four of five control animals were paraplegic on day 3 (P =.04) In the perfused animals, histopathologic examination showed either no spinal cord damage or eosinophilic neurons only, whereas in paraplegic controls there was infarction in large areas of the cord (P <.0001). CONCLUSION: In pigs, selective segmental artery perfusion can provide sufficient spinal cord blood flow to prevent paraplegia resulting from 60 minutes of aortic clamping, as shown by clinical outcomes and histopathologic examination.

Animals↗
PubMed2003

Loading of the human 9-1-1 checkpoint complex onto DNA by the checkpoint clamp loader hRad17-replication factor C complex in vitro.

The human DNA damage sensors, Rad17-replication factor C (Rad17-RFC) and the Rad9-Rad1-Hus1 (9-1-1) checkpoint complex, are thought to be involved in the early steps of the DNA damage checkpoint response. Rad17-RFC and the 9-1-1 complex have been shown to be structurally similar to the replication factors, RFC clamp loader and proliferating cell nuclear antigen polymerase clamp, respectively. Here, we demonstrate functional similarities between the replication and checkpoint clamp loader/DNA clamp pairs. When all eight subunits of the two checkpoint complexes are coexpressed in insect cells, a stable Rad17-RFC/9-1-1 checkpoint supercomplex forms in vivo and is readily purified. The two individually purified checkpoint complexes also form a supercomplex in vitro, which depends on ATP and is mediated by interactions between Rad17 and Rad9. Rad17-RFC binds to nicked circular, gapped, and primed DNA and recruits the 9-1-1 complex in an ATP-dependent manner. Electron microscopic analyses of the reaction products indicate that the 9-1-1 ring is clamped around the DNA.

Adenosine Triphosphate↗
PubMed1994

Antitumor bisdioxopiperazines inhibit yeast DNA topoisomerase II by trapping the enzyme in the form of a closed protein clamp.

The mechanism of inhibition of eukaryotic DNA topoisomerase II [DNA topoisomerase (ATP-hydrolyzing), EC 5.99.1.3] by a member of the bisdioxopiperazine family of anticancer drugs, ICRF-193, was investigated by using purified yeast DNA topoisomerase II. In the absence of ATP, ICRF-193 has little effect on the binding of the enzyme to various forms of DNA. In the presence of ATP, the drug converts the enzyme to a form incapable of binding circular DNA. Incubation of a preformed circular DNA-enzyme complex with ICRF-193 and ATP converts the complex to a form stable in molar concentrations of salt. These results can be interpreted in terms of the ATP-modulated protein-clamp model of type II DNA topoisomerases [Roca, J. & Wang, J. C. (1992) Cell 71, 833-840]; ICRF-193 can bind to the closed-clamp form of the enzyme and prevents its conversion to the open-clamp form. This interpretation is further supported by the finding that whereas both ATP and the drug are needed to form the salt-stable circular DNA-enzyme complex, ATP is not needed for maintaining this complex; furthermore, a signature of the closed-clamp form of the enzyme, Staphylococcus aureus strain V8 endoproteinase cleavage site at Glu-680, is observed if the enzyme is incubated with both ATP and ICRF-193. Inhibition of interconversion between the open- and closed-clamp forms of type II DNA topoisomerases offers a new mechanism in the selection and design of therapeutics targeting this class of enzymes.

Adenosine Triphosphate↗
PubMed1996

Dynamics of loading the beta sliding clamp of DNA polymerase III onto DNA.

A "minimal" DNA primer-template system, consisting of an 80-mer template and 30-mer primer, supports processive DNA synthesis by DNA polymerase III core in the presence of the beta sliding clamp, gamma complex clamp loader, and single-stranded binding protein from Escherichia coli. This primer-template system was used to measure the loading of the beta sliding clamp by the gamma complex in an ATP-dependent reaction. Bound protein-DNA complexes were detected by monitoring fluorescence depolarization of DNA. Steady state and time-resolved anisotropies were measured, and stopped-flow pre-steady state fluorescence measurements allowed visualization of the loading reactions in real time. The rate of loading beta onto DNA was 12 s-1, demonstrating that clamp assembly is rapid on the time scale required for lagging strand Okazaki fragment synthesis. The association rate appears to be limited by an intramolecular step occurring prior to the clamp-loading reaction, possibly the opening of the toroidal beta dimer.

Adenosine Triphosphate↗
PubMed1998

Replication factor C disengages from proliferating cell nuclear antigen (PCNA) upon sliding clamp formation, and PCNA itself tethers DNA polymerase delta to DNA.

Replication factor C (RF-C) and proliferating cell nuclear antigen (PCNA) assemble a complex, called sliding clamp, onto DNA. The clamp in turn loads DNA polymerases (pol) delta and epsilon to form the corresponding holoenzymes, which play an essential role in replication of eukaryotic chromosomal DNA and in several DNA repair pathways. To determine the fate of RF-C after loading of PCNA onto DNA, we tagged the RF-C subunit p37 with a protein kinase A recognition motif, so that the recombinant five-subunit RF-C complex could be 32P-labeled and quantitatively detected in femtomolar amounts. Nonspecific binding of RF-C to DNA was minimized by replacing the p140 subunit with an N-terminally truncated p140 subunit lacking the previously identified nonspecific DNA binding domain. Neither of these modifications impaired the clamp loading activity of the recombinant RF-C. Using gel filtration techniques, we demonstrated that RF-C dissociated from the DNA after clamp loading or pol delta holoenzyme assembly, while PCNA or PCNA.pol delta complex remained bound to DNA. PCNA catalytically loaded onto the template-primer was sufficient by itself to tether pol delta and stimulate DNA replication. The readdition of RF-C to the isolated PCNA.DNA complex did not further stimulate pol delta DNA synthesis. We conclude that pol delta holoenzyme consists of PCNA and pol delta core and that RF-C serves only to load PCNA clamp.

Animals↗
PubMed1999

The carboxyl terminus of the bacteriophage T4 DNA polymerase contacts its sliding clamp at the subunit interface.

The location of the interaction of the COOH terminus of the bacteriophage T4 DNA polymerase with its trimeric, circular sliding clamp has been established. A peptide corresponding to the COOH terminus of the DNA polymerase was labeled with a fluorophore and fluorescence spectroscopy used to show that it forms a specific complex with the sliding clamp by virtue of its low K(D) value (7.1 +/- 1.0 microM). The same peptide was labeled with a photoaffinity probe and cross-linked to the sliding clamp. Mass spectrometry of tryptic digests determined the sole linkage point to be Ala-159 on the sliding clamp, an amino acid that lies on the subunit interface. These results demonstrate that the COOH terminus of the DNA polymerase is inserted into the subunit interface of its sliding clamp, thereby conferring processivity to the DNA polymerase.

Bacteriophage T4↗
PubMed2000

Dynamics of beta and proliferating cell nuclear antigen sliding clamps in traversing DNA secondary structure.

Chromosomal replicases of cellular organisms utilize a ring shaped protein that encircles DNA as a mobile tether for high processivity in DNA synthesis. These "sliding clamps" have sufficiently large linear diameters to encircle duplex DNA and are perhaps even large enough to slide over certain DNA secondary structural elements. This report examines the Escherichia coli beta and human proliferating cell nuclear antigen clamps for their ability to slide over various DNA secondary structures. The results show that these clamps are capable of traversing a 13-nucleotide ssDNA loop, a 4-base pair stem-loop, a 4-nucleotide 5' tail, and a 15-mer bubble within the duplex. However, upon increasing the size of these structures (20-nucleotide loop, 12-base pair stem-loop, 28-nucleotide 5' tail, and 20-nucleotide bubble) the sliding motion of the beta and proliferating cell nuclear antigen over these elements is halted. Studies of the E. coli replicase, DNA polymerase III holoenzyme, in chain elongation with the beta clamp demonstrate that upon encounter with an oligonucleotide annealed in its path, it traverses the duplex and resumes synthesis on the 3' terminus of the oligonucleotide. This sliding and resumption of synthesis occurs even when the oligonucleotide contains a secondary structure element, provided the beta clamp can traverse the structure. However, upon encounter with a downstream oligonucleotide containing a large internal secondary structure, the holoenzyme clears the obstacle by strand displacing the oligonucleotide from the template. Implications of these protein dynamics to DNA transactions are discussed.

Base Pairing↗