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At least 775 records · Page 43Linked to original sources
PubMed2004

Late-stage progressive corneal astigmatism after penetrating keratoplasty for keratoconus.

PURPOSE: Progressive corneal astigmatism occurring at least 10 years after penetrating keratoplasty for keratoconus is a late-phase complication of surgery. This report characterizes this condition in a series of patients from three corneal referral centers in the United States. METHODS: Charts were retrospectively reviewed which met the following criteria: penetrating keratoplasty performed for keratoconus at least 10 years ago, keratometry or simulated keratometry from topography as well as manifest refraction recorded at least 6 months after the last suture removal ("baseline"), and an increase in corneal astigmatism of at least three D over baseline recorded at least 5 years later. Patients who had any other corneal or intraocular surgery performed were excluded. RESULTS: Data from 15 patients (11 males and 4 females) who had penetrating keratoplasties performed by 8 different surgeons are included in this descriptive series. Postoperative follow-up averaged 17.27 years (range 11-24 years). The average donor button size was 7.83 mm (range 7.25-8.5 mm). Baseline corneal astigmatism was obtained an average of 5.2 years after penetrating keratoplasty (range 1.5-16 years) and was on average 3.57 +/- 1.8 D (10 with-the-rule [WTR], 3 against-the-rule [ATR], 2 oblique). Corneal astigmatism significantly increased to an average of 11.23 +/- 3.56 D (range 8.00-19.37 D, P < 0.0001) and most astigmatism was regular and WTR (9 WTR, 3 ATR, 3 oblique) 15.3 years (range 10-22 years) after surgery. Inferior steepening on topography was often noted, even those with oblique and ATR axes. CONCLUSIONS: High, late-stage, regular astigmatism after penetrating keratoplasty for keratoconus is described in a series of patients occurring at least 10 years after surgery. Possible mechanisms of this progressive astigmatism are recurrence of keratoconus in the graft, progressive corneal thinning of the host cornea, or progressive misalignment of the graft-host interface over time.

Astigmatism↗
PubMed1996

The acid tolerance response of Salmonella typhimurium provides protection against organic acids.

Salmonella typhimurium encounters a variety of acid stress situations during pathogenesis and in the natural environment. These include the extreme low pH encountered in the stomach and a less acidic intestinal environment containing large amounts of organic weak acids (volatile fatty acids). The acid tolerance response (ATR) is a complex defence system that can minimize the lethal effects of extreme low pH (pH3). The data presented illustrate that the ATR can also defend against weak acids such as butyric, acetic or propionic acids. Although an acid shock of pH 4.4 induced the ATR, growth in subinhibitory concentrations of weak acids did not. Various mutations shown to affect tolerance to extreme acid conditions (pH 3) were tested for their effects on tolerance to weak acids. An rpoS mutant lacking the alternative sigma factor sigma s failed to protect cells against weak acids as well as extreme acid pH. The fur (ferric uptake regulator) and atp (Mg(2+)-dependent ATPase) mutants defective in extreme acid tolerance showed no defects in their tolerance to weak acids. Curiously, the atbR mutant that exhibits increased tolerance to extreme acid pH proved sensitive to weak acids. Several insertions that rendered cells sensitive to organic acids were isolated, all of which proved to be linked to the rpoS locus.

Acids↗
PubMed2004

Chk1 activation requires Rad9 S/TQ-site phosphorylation to promote association with C-terminal BRCT domains of Rad4TOPBP1.

To gain insight into the function and organization of proteins assembled on the DNA in response to genotoxic insult we investigated the phosphorylation of the Schizosaccharomyces pombe PCNA-like checkpoint protein Rad9. C-terminal T412/S423 phosphorylation of Rad9 by Rad3(ATR) occurs in S phase without replication stress. Rad3(ATR) and Tel1(ATM) phosphorylate these same residues, plus additional ones, in response to DNA damage. In S phase and after damage, only Rad9 phosphorylated on T412/S423, but not unphosphorylated Rad9, associates with a two-BRCT-domain region of the essential Rad4(TOPBP1) protein. Rad9-Rad4(TOPBP1) interaction is required to activate the Chk1 damage checkpoint but not the Cds1 replication checkpoint. When the Rad9-T412/S423 are phosphorylated, Rad4(TOPBP1) coprecipitates with Rad3(ATR), suggesting that phosphorylation coordinates formation of an active checkpoint complex.

Binding Sites↗
PubMed2005

Functional MRI predicts memory performance after right mesiotemporal epilepsy surgery.

PURPOSE: Anterior temporal lobe resection (ATR) is a treatment option in drug-resistant epilepsy. An important risk of ATR is loss of memory because mesiotemporal structures contribute substantially to memory function. We investigated whether memory-activated functional MRI (fMRI) can predict postoperative memory loss after anterior temporal lobectomy in right-sided medial temporal lobe epilepsy (MTLE). METHODS: We included 16 patients (10 women) aged 16-54 years. The mean age at epilepsy onset was 12.5 years (range, 1-26 years). The patients' mean Wechsler IQ score was 95.2 (range, 62-125). The activation condition of fMRI consisted of retrieval from long-term memory induced by self-paced performance of an imaginative walk. All but one patient had left-sided speech dominance according to speech-activated fMRI. Outside the scanner, we evaluated the pre- and postoperative visual memory retention by using Rey Visual Design Learning Test. RESULTS: We found a correlation between the preoperative asymmetry index of memory-fMRI and the change between pre- and postsurgical measures of memory retention. Reduced activation of the mesiotemporal region ipsilateral to the epileptogenic region correlated with a favorable memory outcome after right-sided ATR. CONCLUSIONS: In light of the postoperative results, the theoretical implication of our study is that fMRI based on a simple introspective retrieval task measures memory functions. The main clinical implication of our study is that memory-fMRI might replace the invasive Wada test in MTLE by using a simple fMRI paradigm. Predictive power, however, will be studied in larger patient samples. Other studies are required for left-sided MTLE and neocortical epilepsies to assess the clinical usefulness of memory-fMRI.

Adolescent↗
PubMed2004

Polymorphism of renin-angiotensin system genes in IgA nephropathy.

BACKGROUND AND AIMS: Individuals are prone to disease because of certain disease-susceptible genes. The angiotensin I-converting enzyme (ACE) gene insertion/deletion (I/D), the angiotensinogen (AGT) gene, M235T, and the angiotensin II type 1 receptor (ATR) gene, A1166C, polymorphisms have been associated with IgA nephropathy (IgAN) and its progression. Several studies on Caucasians and Japanese patients have reported contradictory results. We determined these polymorphisms in 118 Chinese patients with IgAN and 94 healthy Chinese subjects to assess their clinical impact. METHODS: Genotyping was performed with DNA isolated from peripheral leucocytes, polymerase chain reaction amplification of the polymorphic sequence, restriction enzymes digestion, and separation and identification of DNA fragments. Clinical data at renal biopsy and final status on renal function were determined from patients' records. RESULTS: Comparing all IgAN patients with controls, AGT and ATR genotype distributions were similar, whereas there was a significant increase in the ACE DD genotype (P < 0.05). When comparing patients with end-stage renal failure (IgAN-ESRF) and those without (IgAN-nonESRF), there was no difference among the three gene polymorphisms. In contrast, there were significant differences in higher male prevalence (P < 0.05), increased serum creatinine at presentation (P < 0.05), more sclerosis (P < 0.01) and higher tubulointerstitial lesion score (P < 0.001) in the IgAN-ESRF group. CONCLUSION: Among the ACE, AGT and ATR gene polymorphisms, only the DD genotype may predispose the individual to IgAN in the Chinese population. None are significant for prognosticating ESRF.

Adult↗
PubMed1991

Active fixation leads--long-term threshold reduction using a drug-infused ceramic collar.

Previous investigators, including our group, have reported the threshold reduction benefits of steroid-releasing leads. To date, all published literature has been for the passive fixation versions. The application of steroids should also enhance the performance of active fixation leads. We have developed and tested an atrial and a ventricular Accufix lead with a dexamethasone acetate-releasing, porous ceramic collar (DA DEC). A long-term sheep study has shown a significant reduction in thresholds (THR) when compared to standard Accufix leads without the collar (ACC) for atrial (ATR) and ventricular (VENT) versions (bipolar THR [0.5 msec] at 24 weeks: VENT DA DEC = 0.51 +/- 0.07, VENT ACC = 1.49 +/- 1.03; ATR DA DEC = 1.31 +/- 1.14, ATR ACC = 2.99 +/- 1.31). All other parameters tested, including pacing and sensing impedance as well as polarization overpotential, were similar for the two groups. The Accufix DEC leads therefore have excellent potential for low energy stimulation.

Aluminum Oxide↗
PubMed1996

Acid adaptation of Listeria monocytogenes can enhance survival in acidic foods and during milk fermentation.

We have previously shown that tolerance to severe acid stress (pH 3.5) can be induced in Listeria monocytogenes following a 1-h adaptation to mild acid (pH 5.5), a phenomenon termed the acid tolerance response (ATR) (B. O'Driscoll, C. G. M. Gahan, and C. Hill, Appl. Environ. Microbiol. 62:1693-1698, 1966). In an attempt to determine the industrial significance of the ATR, we have examined the survival of adapted and nonadapted cells in a variety of acidic foods. Acid adaptation enhanced the survival of L. monocytogenes in acidified dairy products, including cottage cheese, yogurt, and whole-fat cheddar cheese. Acid-adapted L. monocytogenes cultures also demonstrated increased survival during active milk fermentation by a lactic acid culture. Similarly, acid-adapted cells showed greatly improved survival in low-pH foods (orange juice and salad dressing) containing acids other than lactic acid. However, in foods with a marginally higher pH, such as mozzarella cheese, a commercial cottage cheese, or low-fat cheddar cheese, acid adaptation did not appear to enhance survival. We have previously isolated mutants of L. monocytogenes that are constitutively acid tolerant in the absence of an induction step (O'Driscoll et al., Appl. Environ. Microbiol. 62:1693-1698, 1996). In the present study, one such mutant, ATM56, demonstrated an increased ability to survive in low-pH foods and during milk fermentation when compared with the wild-type strain. Significant numbers of ATM56 could be recovered even after 70 days in both whole-fat and low-fat cheddar cheese. Collectively, the data suggest that ATR mechanisms, whether constitutive or induced, can greatly influence the survival of L. monocytogenes in low-pH food environments.

Adaptation, Physiological↗
PubMed1997

Intracellular pH is a major factor in the induction of tolerance to acid and other stresses in Lactococcus lactis.

This study demonstrates that exposure of log-phase Lactococcus lactis subsp. cremoris 712 cells to mildly acid conditions induces resistance to normally lethal intensities of environmental stresses such as acid, heat, NaCl, H2O2, and ethanol. The intracellular pH (pHi) played a major role in the induction of this multistress resistance response. The pHi was dependent on the extracellular pH (pHo) and on the specific acid used to reduce the pHo. When resuspended in fresh medium, cells were able to maintain a pH gradient even at pHo values that resulted in cell death. Induction of an acid tolerance response (ATR) coincided with an increase in the ability of cells to resist change to an unfavorable pHi; nevertheless, a more favorable pHi was not the sole reason for the increased survival at acid pHo. Cells with an induced ATR survived exposure to a lethal pHo much better than did uninduced cells with a pHi identical to that of the induced cells. Survival following lethal acid shock was dependent on the pHi during induction of the ATR, and the highest survival was observed following induction at a pHi of 5.9, which was the lowest pHi at which growth occurred. Increased acid tolerance and the ability to maintain a higher pHi during lethal acid stress were not acquired if protein synthesis was inhibited by chloramphenicol during adaptation.

Acetic Acid↗
PubMed1999

Relationship between acid tolerance, cytoplasmic pH, and ATP and H+-ATPase levels in chemostat cultures of Lactococcus lactis.

The acid tolerance response (ATR) of chemostat cultures of Lactococcus lactis subsp. cremoris NCDO 712 was dependent on the dilution rate and on the extracellular pH (pHo). A decrease in either the dilution rate or the pHo led to a decrease in the cytoplasmic pH (pHi) of the cells, and similar levels of acid tolerance were observed at any specific pHi irrespective of whether the pHi resulted from manipulation of the growth rate, manipulation of the pHo, or both. Acid tolerance was also induced by sudden additions of acid to chemostat cultures growing at a pHo of 7.0, and this induction was completely inhibited by chloramphenicol. The end products of glucose fermentation depended on the growth rate and the environmental pHo of the cultures, but neither the spectrum of end products nor the total rate of acid production correlated with a specific pHi. The rate of ATP formation was not correlated with pHi, but a good correlation between the cellular level of H+-ATPase and pHi was observed. Moreover, an inverse correlation between the cytoplasmic levels of ATP and pHi was established. Each pHi below 6. 6 was characterized by unique levels of ATR, H+-ATPase, and ATP. High levels of H+-ATPase also coincided with high levels of acid tolerance of cells in batch cultures induced with sublethal levels of acid. We concluded that H+-ATPase is one of the ATR proteins induced by acid pHi through growth at an acid pHo or a slow growth rate.

Adenosine Triphosphate↗
PubMed2003

Salmonella enterica serovar Typhimurium and Listeria monocytogenes acid tolerance response induced by organic acids at 20 degrees C: optimization and modeling.

An acid tolerance response (ATR) has been demonstrated in Listeria monocytogenes and Salmonella enterica serovar Typhimurium in response to low pH poised (i.e., adapted) with acetic or lactic acids at 20 degrees C and modeled by using dynamic differential equations. The ATR was not immediate or prolonged, and optimization occurred after exposure of L. monocytogenes for 3 h at pH 5.5 poised with acetic acid and for 2 h at pH 5.5 poised with lactic acid and after exposure of S. enterica serovar Typhimurium for 2 h at pH 5.5 poised with acetic acid and for 3 h at pH 5.5 poised with lactic acid. An objective mechanistic analysis of the acid inactivation data yielded estimates of the duration of the shoulder (t(s)), the log-linear decline (k(max)), and the magnitude of a critical component (C). The magnitude of k(max) gave the best agreement with estimates of conditions for optimum ATR induction made from the raw data.

Acetic Acid↗
PubMed2004

Model system for growing and quantifying Streptococcus pneumoniae biofilms in situ and in real time.

Streptococcus pneumoniae forms biofilms, but little is known about its extracellular polymeric substances (EPS) or the kinetics of biofilm formation. A system was developed to enable the simultaneous measurement of cells and the EPS of biofilm-associated S. pneumoniae in situ over time. A biofilm reactor containing germanium coupons was interfaced to an attenuated total reflectance (ATR) germanium cell of a Fourier transform infrared (FTIR) laser spectrometer. Biofilm-associated cells were recovered from the coupons and quantified by total and viable cell count methods. ATR-FTIR spectroscopy of biofilms formed on the germanium internal reflection element (IRE) of the ATR cell provided a continuous spectrum of biofilm protein and polysaccharide (a measure of the EPS). Staining of the biofilms on the IRE surface with specific fluorescent probes provided confirmatory evidence for the biofilm structure and the presence of biofilm polysaccharides. Biofilm protein and polysaccharides were detected within hours after inoculation and continued to increase for the next 141 h. The polysaccharide band increased at a substantially higher rate than did the protein band, demonstrating increasing coverage of the IRE surface with biofilm polysaccharides. The biofilm total cell counts on germanium coupons stabilized after 21 h, at approximately 10(5) cells per cm(2), while viable counts decreased as the biofilm aged. This system is unique in its ability to detect and quantify biofilm-associated cells and EPS of S. pneumoniae over time by using multiple, corroborative techniques. This approach could prove useful for the study of biofilm processes of this or other microorganisms of clinical or industrial relevance.

Bacteriological Techniques↗
PubMed1991

Inducible pH homeostasis and the acid tolerance response of Salmonella typhimurium.

The acid tolerance response (ATR) is an adaptive system triggered at external pH (pHo) values of 5.5 to 6.0 that will protect cells from more severe acid stress (J. Foster and H. Hall, J. Bacteriol. 172:771-778, 1990). Correlations between the internal pH (pHi) of adapted versus unadapted cells at pHo of 3.3 indicate that the ATR system produces an inducible pH-homeostatic function. This function serves to maintain the pHi above 5 to 5.5. Below this range, cells rapidly lose viability. Development of this pH homeostasis mechanism was sensitive to protein synthesis inhibitors and operated only to augment the pHi at pHo values below 4. In contrast, classical constitutive pH homeostasis was insensitive to protein synthesis inhibitors and was efficient only at pHo values above 4. Physiological studies indicated an important role for the Mg(2+)-dependent proton-translocating ATPase in affording ATR-associated survival during exposure to severe acid challenges. Along with being acid intolerant, cells deficient in this ATPase did not exhibit inducible pH homeostasis. We speculate that adaptive acid tolerance is important to Salmonella species in surviving acid encounters in both the environment and the infected host.

Acids↗
PubMed1993

The acid tolerance response of Salmonella typhimurium involves transient synthesis of key acid shock proteins.

Although Salmonella typhimurium prefers neutral-pH environments, it can adapt to survive conditions of severe low-pH stress (pH 3.3). The process, termed the acid tolerance response (ATR), includes two distinct stages. The first stage, called pre-acid shock, is induced at pH 5.8 and involves the production of an inducible pH homeostasis system functional at external pH values below 4.0. The second stage occurs following an acid shock shift to pH 4.5 or below and is called the post-acid shock stage. During this stage of the ATR, 43 acid shock proteins (ASPs) are synthesized. The present data reveal that several ASPs important for pH 3.3 acid tolerance are only transiently produced. Their disappearance after 30 to 40 min of pH 4.4 acid shock coincides with an inability to survive subsequent pH 3.3 acid challenge. Clearly, an essential feature of inducible acid tolerance is an ability to synthesize these key ASPs. The pre-acid shock stage, with its inducible pH homeostasis system, offers the cell an enhanced ability to synthesize ASPs following rapid shifts to conditions below pH 4.0, an external pH that normally prevents ASP synthesis. The data also address possible signals for ASP synthesis. The inducing signal for 22 ASPs appears to be internal acidification, while external pH serves to induce 13 others. Of the 14 transient ASPs, 10 are induced in response to changes in internal pH. Mutations in the fur (ferric uptake regulator) locus that produce an Atr- acid-sensitive phenotype also eliminate induction of six transiently induced ASPs.

Acids↗
PubMed1994

Acid-sensitive mutants of Salmonella typhimurium identified through a dinitrophenol lethal screening strategy.

Salmonella typhimurium exhibits a low-pH-inducible acid tolerance response (ATR) that can protect the adapted cell from severe acid challenge (pH 3.3). It is a two-stage system, with some proteins induced at pH 5.8 (pre-acid shock) and others induced below pH 4.5 (acid shock). The genetics of acid resistance was investigated through the use of a new screening medium. The medium contained 200 microM dinitrophenol (DNP) and was adjusted to pH 4.7 to 4.8. The medium will lower the internal pH of cells to a lethal level. However, cells capable of mounting an ATR will survive longer on this medium than acid-intolerant cells. Using this DNP lethal screening strategy, we isolated several acid-sensitive insertion mutants. Some mutants were defective in the pre-acid shock ATR stage but exhibited a normal or nearly normal post-acid shock-induced acid tolerance (atrB and atrC). Others could not induce acid tolerance by using either pre- or post-acid shock strategies (atrD, atrF, and atrG). The atrB locus was found to be part of a regulon under the control of a trans-acting regulator, atbR. An insertion in atbR caused constitutive acid tolerance because of overexpression of the regulon. Mutations in atrD and atrF affected iron metabolism and, in a manner analogous to ferric uptake regulator (fur) mutations, diminished acid resistance. The atrF mutation mapped within the ent cluster, probably in a fep uptake locus. The atrD locus mapped near metC and may represent an insertion into the S. typhimurium homolog of the Escherichia coli exbB or exbD locus. The mutation in atrC caused extreme UV light sensitivity and proved to occur within the polA (DNA polymerase I) locus. The results support the concept of overlapping acid protection systems in S. typhimurium.

2,4-Dinitrophenol↗
PubMed1996

The role of fur in the acid tolerance response of Salmonella typhimurium is physiologically and genetically separable from its role in iron acquisition.

The response of Salmonella typhimurium to low pH includes a low-pH protection system called the acid tolerance response (ATR). The iron-regulatory protein Fur has been implicated in the ATR since fur mutants are acid sensitive and cause altered expression of several acid shock proteins (J. W. Foster, J. Bacteriol. 173:6896-6902, 1991). We have determined that the acid-sensitive phenotype of fur mutations is indeed due to a defect in Fur that can be complemented by a fur(+)-containing plasmid. However, changes in cellular iron status alone did not trigger the ATR. Cells clearly required exposure to low pH in order to induce acid tolerance. The role of Fur in acid tolerance was found to extend beyond regulating iron acquisition. A mutation in fur converting histidine 90 to an arginine (H90R) eliminated Fur-mediated iron regulation of enterochelin production and deregulated an iroA-lacZ fusion but had no effect on acid tolerance. The H90R iron-blind Fur protein also mediated acid shock induction of several Fur-dependent acid shock proteins and acid control of the hyd locus. In addition, a Fur superrepressor that constitutively repressed iron-regulated genes mediated normal Fur-dependent acid tolerance and pH-controlled gene expression. The results indicate the acid-sensing and iron-sensing mechanisms of Fur are separable by mutation and reinforce the concept of Fur as a major global regulator in the cell.

Acids↗
PubMed2000

Defects in D-alanyl-lipoteichoic acid synthesis in Streptococcus mutans results in acid sensitivity.

In the cariogenic organism, Streptococcus mutans, low pH induces an acid tolerance response (ATR). To identify acid-regulated proteins comprising the ATR, transposon mutagenesis with the thermosensitive plasmid pGh9:ISS1 was used to produce clones that were able to grow at neutral pH, but not in medium at pH 5.0. Sequence analysis of one mutant (IS1A) indicated that transposition had created a 6.3-kb deletion, one end of which was in dltB of the dlt operon encoding four proteins (DltA-DltD) involved in the synthesis of D-alanyl-lipoteichoic acid. Inactivation of the dltC gene, encoding the D-alanyl carrier protein (Dcp), resulted in the generation of the acid-sensitive mutant, BH97LC. Compared to the wild-type strain, LT11, the mutant exhibited a threefold-longer doubling time and a 33% lower growth yield. In addition, it was unable to initiate growth below pH 6.5 and unadapted cells were unable to survive a 3-h exposure in medium buffered at pH 3.5, while a pH of 3.0 was required to kill the wild type in the same time period. Also, induction of the ATR in BH97LC, as measured by the number of survivors at a pH killing unadapted cells, was 3 to 4 orders of magnitude lower than that exhibited by the wild type. While the LTA of both strains contained a similar average number of glycerolphosphate residues, permeabilized cells of BH97LC did not incorporate D-[(14)C]alanine into this amphiphile. This defect was correlated with the deficiency of Dcp. Chemical analysis of the LTA purified from the mutant confirmed the absence of D-alanine-esters. Electron micrographs showed that BH97LC is characterized by unequal polar caps and is devoid of a fibrous extracellular matrix present on the surface of the wild-type cells. Proton permeability assays revealed that the mutant was more permeable to protons than the wild type. This observation suggests a mechanism for the loss of the characteristic acid tolerance response in S. mutans.

Acids↗
PubMed2004

Adaptive acid tolerance response of Streptococcus sobrinus.

Streptococcus mutans and Streptococcus sobrinus are the bacteria most commonly associated with human dental caries. A major virulence attribute of these and other cariogenic bacteria is acid tolerance. The acid tolerance mechanisms of S. mutans have begun to be investigated in detail, including the adaptive acid tolerance response (ATR), but this is not the case for S. sobrinus. An analysis of the ATR of two S. sobrinus strains was conducted with cells grown to steady state in continuous chemostat cultures. Compared with cells grown at neutral pH, S. sobrinus cells grown at pH 5.0 showed an increased resistance to acid killing and were able to drive down the pH through glycolysis to lower values. Unlike what is found for S. mutans, the enhanced acid tolerance and glycolytic capacities of acid-adapted S. sobrinus were not due to increased F-ATPase activities. Interestingly though, S. sobrinus cells grown at pH 5.0 had twofold more glucose phosphoenolpyruvate:sugar phosphotransferase system (PTS) activity than cells grown at pH 7.0. In contrast, glucose PTS activity was actually higher in S. mutans grown at pH 7.0 than in cells grown at pH 5.0. Silver staining of two-dimensional gels of whole-cell lysates of S. sobrinus 6715 revealed that at least 9 proteins were up-regulated and 22 proteins were down-regulated in pH 5.0-grown cells compared with cells grown at pH 7.0. Our results demonstrate that S. sobrinus is capable of mounting an ATR but that there are critical differences between the mechanisms of acid adaptation used by S. sobrinus and S. mutans.

Adaptation, Physiological↗
PubMed2006

DNA methyltransferase 1 knockdown activates a replication stress checkpoint.

DNA methyltransferase 1 (DNMT1) is an important component of the epigenetic machinery and is responsible for copying DNA methylation patterns during cell division. Coordination of DNA methylation and DNA replication is critical for maintaining epigenetic programming. Knockdown of DNMT1 leads to inhibition of DNA replication, but the mechanism has been unclear. Here we show that depletion of DNMT1 with either antisense or small interfering RNA (siRNA) specific to DNMT1 activates a cascade of genotoxic stress checkpoint proteins, resulting in phosphorylation of checkpoint kinases 1 and 2 (Chk1 and -2), gammaH2AX focus formation, and cell division control protein 25a (CDC25a) degradation, in an ataxia telangiectasia mutated-Rad3-related (ATR)-dependent manner. siRNA knockdown of ATR blocks the response to DNMT1 depletion; DNA synthesis continues in the absence of DNMT1, resulting in global hypomethylation. Similarly, the response to DNMT1 knockdown is significantly attenuated in human mutant ATR fibroblast cells from a Seckel syndrome patient. This response is sensitive to DNMT1 depletion, independent of the catalytic domain of DNMT1, as indicated by abolition of the response with ectopic expression of either DNMT1 or DNMT1 with the catalytic domain deleted. There is no response to short-term treatment with 5-aza-deoxycytidine (5-aza-CdR), which causes demethylation by trapping DNMT1 in 5-aza-CdR-containing DNA but does not cause disappearance of DNMT1 from the nucleus. Our data are consistent with the hypothesis that removal of DNMT1 from replication forks is the trigger for this response.

Animals↗