Mechanisms of base selection by the E.coli mispaired uracil glycosylase

The repair of the multitude of single-base lesions formed daily in the cells of all living organisms is accomplished primarily by the base-excision repair (BER) pathway that initiates repair through a series of lesion-selective glycosylases. In this paper, single-turnover kinetics have been measured on a series of oligonucleotide substrates containing both uracil and purine analogs for the E. coli mispaired uracil glycosylase, MUG. The relative rates of glycosylase cleavage have been correlated with the free energy of helix formation, and with the size and electronic inductive properties of a series of uracil 5-substituents. Data is presented that MUG can exploit the reduced thermodynamic stability of mispairs to distinguish U:A from U:G pairs. Discrimination against the removal of thymine results primarily from the electron-donating property of the thymine 5-methyl substituent, while the size of the methyl group relative to a hydrogen atom is a secondary factor. A series of parameters have been obtained that allow prediction of relative MUG cleavage rates that correlate well with observed relative rates that vary over five orders of magnitude for the series of base analogs examined. We propose that these parameters may be common among DNA glycosylases, however, specific glycosylases may focus more or less on each of the parameters identified. The presence of a series of glycosylases which focus on different lesion properties, all coexisting within the same cell, would provide a robust and partially redundant repair system necessary for the maintenance of the genome

Postoperative Bowel Perforation due to Heterotopic Ossification (Myositis Ossificans Traumatica): A Case Report and Review of the Literature

Heterotopic ossification (HO) is the ectopic development of normal bone within soft tissue that can occur after traumatic injury. It is uncommon and may be missed or misdiagnosed, which can lead to complications. We report the case of an 84-year-old male with a previous history of a laparotomy who underwent resection of an intra-abdominal tumor through a midline incision. On postoperative day six, the patient was taken to the operating room, as succus was draining from the incision. Upon re-exploration, sharp bone-like material was found in the wound directly adjacent to an enterotomy. Pathology confirmed mature lamellar bone and the diagnosis of HO. This is the first report of postoperative intestinal perforation secondary to HO in a midline wound. We report this case to encourage accurate reporting of HO and its morbidity and complications for the benefit of appropriate surgical planning and epidemiologic tracking of outcomes

Characterization of Restriction-Modification Systems in KLEBSIELLA PNEUMONIAE

Two restriction-modification (R-M) systems KpnAI and KpnBl, found in Klebsiella pneumoniae strains M5a1 and GM236, respectively, have been studied and confirmed to he different from other R-M systems reported in K. pneumoniae. Mutant studies that the KpnAl and KpnBI systems may belong to either a type I or type III systems since approximately equal numbers of r-m+ and r-m- mutants were obtained. However, a DNA hybridization study using representative type I and type III probes from E. coli and S. typhimurium failed to show homologies to either KpnAI or KpnBI. The restriction endonuclease KpnBI was found to be temperature-sensitive with maximum restriction activity at 30°c and no restriction activity at 42°C. Further, the activity of endonuclease KpnBI was found to be reduced to almost zero level by growing the bacteria in the presence of 10% glycerol. Although the mechanism is not known, this is the first time such a phenomenon has been observed in any of the reported R-M systems. These studies also compared the efficiency of transformation in K. pneumoniae of three plasmid transformation methods; CaCl2 heat-shock; freezing and thawing in the presence of CaCl2; and electroporation. Electroporation was shown to be the most efficient method. Transformation efficiency in both the r+ kpnAL and r+ KpnBL strains was 20- to 100-fold less than the transformation efficiency of the r- strains, depending on plasmid size. Four different approaches have been used to clone the hsd genes of the KpnBI system. Two clones were obtained; these were named pKpnBl and pKpnB2. The pKpnBl and pKpnB2 clones were found to complement the restriction activity of a r-kpnBLm+ kpaBL K. pneumoniae mutant and were also found to complement both the restriction and modification activities of a r-kpnBLm+ kpaBL K. pneumoniae mutant. A quick subcloning method which involves making subclones from a plasmid clone in a single step was also developed. A preliminary analysis, based on complementation studies, of the gene structure suggested that the KpnBI system may consist of three structural genes, a characteristic of the type I R-M system

5-Halogenated pyrimidine lesions within a CpG sequence context mimic 5-methylcytosine by enhancing the binding of the methyl-CpG-binding domain of methyl-CpG-binding protein 2 (MeCP2)

Perturbations in cytosine methylation signals are observed in the majority of human tumors; however, it is as yet unknown how methylation patterns become altered. Epigenetic changes can result in the activation of transforming genes as well as in the silencing of tumor suppressor genes. We report that methyl-CpG-binding proteins (MBPs), specific for methyl-CpG dinucleotides, bind with high affinity to halogenated pyrimidine lesions, previously shown to result from peroxidase-mediated inflammatory processes. Emerging data suggest that the initial binding of MBPs to methyl-CpG sequences may be a seeding event that recruits chromatin-modifying enzymes and DNA methyltransferase, initiating a cascade of events that result in gene silencing. MBD4, a protein with both methyl-binding and glycosylase activity demonstrated repair activity against a series of 5-substituted pyrimidines, with the greatest efficiency against 5-chlorouracil, but undetectable activity against 5-chlorocytosine. The data presented here suggest that halogenated pyrimidine damage products can potentially accumulate and mimic endogenous methylation signals

Altered RECQ Helicase Expression in Sporadic Primary Colorectal Cancers

AbstractDeregulation of DNA repair enzymes occurs in cancers and may create a susceptibility to chemotherapy. Expression levels of DNA repair enzymes have been shown to predict the responsiveness of cancers to certain chemotherapeutic agents. The RECQ helicases repair damaged DNA including damage caused by topoisomerase I inhibitors, such as irinotecan. Altered expression levels of these enzymes in colorectal cancer (CRC) may influence the response of the cancers to irinotecan. Thus, we assessed RECQ helicase (WRN, BLM, RECQL, RECQL4, and RECQL5) expression in primary CRCs, matched normal colon, and CRC cell lines. We found that BLM and RECQL4 mRNA levels are significantly increased in CRC (P = .0011 and P < .0001, respectively), whereas RECQL and RECQL5 are significantly decreased (P = .0103 and P = .0029, respectively). RECQ helicase expression patterns varied between specific molecular subtypes of CRCs. The mRNA and protein expression of the majority of the RECQ helicases was closely correlated, suggesting that altered mRNA expression is the predominant mechanism for deregulated RECQ helicase expression. Immunohistochemistry localized the RECQ helicases to the nucleus. RECQ helicase expression is altered in CRC, suggesting that RECQ helicase expression has potential to identify CRCs that are susceptible to specific chemotherapeutic agents

Examination of the specificity of DNA methylation profiling techniques towards 5-methylcytosine and 5-hydroxymethylcytosine

DNA cytosine-5 methylation is a well-studied epigenetic pathway implicated in gene expression control and disease pathogenesis. Different technologies have been developed to examine the distribution of 5-methylcytosine (5mC) in specific sequences of the genome. Recently, substantial amounts of 5-hydroxymethylcytosine (5hmC), most likely derived from enzymatic oxidation of 5mC by TET1, have been detected in certain mammalian tissues. Here, we have examined the ability of several commonly used DNA methylation profiling methods to distinguish between 5mC and 5hmC. We show that techniques based on sodium bisulfite treatment of DNA are incapable of distinguishing between the two modified bases. In contrast, techniques based on immunoprecipitation with anti-5mC antibody (methylated DNA immunoprecipitation, MeDIP) or those based on proteins that bind to methylated CpG sequences (e.g. methylated-CpG island recovery assay, MIRA) do not detect 5hmC and are specific for 5mC unless both modified bases occur in the same DNA fragment. We also report that several methyl-CpG binding proteins including MBD1, MBD2 and MBD4 do not bind to sequences containing 5hmC. Selective mapping of 5hmC will require the development of unique tools for the detection of this modified base

Kinetics of deamination and Cu(II)/H2O2/Ascorbate-induced formation of 5-methylcytosine glycol at CpG sites in duplex DNA

Mutation in p53 tumor suppressor gene is a hallmark of human cancers. Six major mutational hotspots in p53 contain methylated CpG (mCpG) sites, and C →T transition is the most common mutation at these sites. It was hypothesized that the formation of 5-methylcytosine glycol induced by reactive oxygen species, its spontaneous deamination to thymine glycol and the miscoding property of the latter may account, in part, for the ubiquitous C →T mutation at CpG site. Here, we assessed the kinetics of deamination for two diastereomers of 5-methylcytosine glycol in duplex DNA. Our results revealed that the half-lives for the deamination of the (5S,6S) and (5R,6R) diastereomers of 5-methylcytosine glycol in duplex DNA at 37°C were 37.4 ± 1.6 and 27.4 ± 1.0 h, respectively. The deamination rates were only slightly lower than those for the two diastereomers in mononucleosides. Next, we assessed the formation of 5-methyl-2′-deoxycytidine glycol in the form of its deaminated product, namely, thymidine glycol (Tg), in methyl-CpG-bearing duplex DNA treated with Cu(II)/H2O2/ascorbate. LC-MS/MS quantification results showed that the yield of Tg is similar as that of 5-(hydroxymethyl)-2′-deoxycytidine. Together, our data support that the formation and deamination of 5-methylcytosine glycol may contribute significantly to the C →T transition mutation at mCpG dinucleotide site

Recognition of 5-Hydroxymethylcytosine by the Uhrf1 SRA Domain

Recent discovery of 5-hydroxymethylcytosine (5hmC) in genomic DNA raises the question how this sixth base is recognized by cellular proteins. In contrast to the methyl-CpG binding domain (MBD) of MeCP2, we found that the SRA domain of Uhrf1, an essential factor in DNA maintenance methylation, binds 5hmC and 5-methylcytosine containing substrates with similar affinity. Based on the co-crystal structure, we performed molecular dynamics simulations of the SRA:DNA complex with the flipped cytosine base carrying either of these epigenetic modifications. Our data indicate that the SRA binding pocket can accommodate 5hmC and stabilizes the flipped base by hydrogen bond formation with the hydroxyl group

Genomic mapping of 5-hydroxymethylcytosine in the human brain

Methylation at the 5-position of cytosine is a well-studied epigenetic pathway. In addition to 5-methylcytosine (5mC), substantial amounts of 5-hydroxymethylcytosine (5hmC) also referred to as the sixth DNA base have been detected in certain tissues, most notably the brain. However, the genomic distribution of this cytosine modification is unknown. Here, we have used an immunoprecipitation technique (5hmC-IP) to examine the occurrence of 5hmC in DNA from human brain frontal lobe tissue. The distribution of 5hmC was compared to that of 5mC. We show that 5hmC is more selectively targeted to genes than is 5mC. 5hmC is particularly enriched at promoters and in intragenic regions (gene bodies) but is largely absent from non-gene regions. 5hmC peaks at transcription start sites did not correlate with gene expression levels for promoters with intermediate or high CpG content. However, the presence of 5hmC in gene bodies was more positively correlated with gene expression levels than was the presence of 5mC. Promoters of testis-specific genes showed strong 5mC peaks in brain DNA but were almost completely devoid of 5hmC. Our data provide an overview of the genomic distribution of 5hmC in human brain and will set the stage for further functional characterization of this novel DNA modification