Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:2.7.7.7 (DNA polymerase)
 17,007 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

We present a new, rapid and simple method to study DNA Fragmentation Index (DFI) in sperm samples from boar under bright-field and fluorescence microscopy. Discrimination of sperm cells containing fragmented DNA relies on the extreme peripheral diffusion of their chromatin fragments, whereas those sperm nuclei without DNA fragmentation do not disperse or show very restricted spreading of DNA loops close to the flagellum. The basic methodology provided in the commercial kit Sperm-Sus-Halomax allows, in addition to a direct estimation of DFI in a sperm sample under bright field microscopy, a direct visualization of DNA breaks by incorporation of labelled nucleotides using the DNA polymerase I following the in situ nick translation assay (ISNT methodology not provided in the kit). An external control using DBD-FISH (DNA breakage detection-fluorescence in situ hybridization) on human and boar sperm samples was used in this experiment. The results obtained show (i) low levels of background DNA fragmentation (from 0.7 to 10%), (ii) no significant differences for DFI after the application of Sperm-Sus-Halomax and ISNT, with a tendency to be underestimated after using DBD-FISH and (iii) a characteristic chromatin organization in boar sperm nucleus, with a particular response to chromatin loop relaxation and preferential DNA labelling by ISNT at the proximal nuclear area, close to the flagellum. This methodology allows the routine assessment of boar sperm samples for DFI, as well as basic and clinical research on this relevant topic in any laboratory of semen analysis.
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PMID:A new method to analyze boar sperm DNA fragmentation under bright-field or fluorescence microscopy. 1599 25

DNA repair takes place in the context of chromatin. Previous studies showed that histones impair base excision repair (BER) of modified bases at both the excision and synthesis steps. We examined BER of uracil in a glucocorticoid response element (GRE) complexed with the glucocorticoid receptor DNA binding domain (GR-DBD). Five substrates were designed, each containing a unique C-->U substitution within the mouse mammary tumor virus promoter, one located within each GRE half-site and the others located outside the GRE. To examine distinct steps of BER, DNA cleavage by uracil-DNA glycosylase and Ape1 endonuclease was used to assess initiation, dCTP incorporation by DNA polymerase (pol) beta was used to measure repair synthesis, and DNA ligase I was used to seal the nick. For uracil sites within the GRE, there was a reduced rate of uracil-DNA glycosylase/Ape1 activity following GR-DBD binding. Cleavage in the right half-site, with higher GR-DBD binding affinity, was reduced approximately 5-fold, whereas cleavage in the left half-site was reduced approximately 3.8-fold. Conversely, uracil-directed cleavage outside the GRE was unaffected by GR-DBD binding. Surprisingly, there was no reduction in the rate of pol beta synthesis or DNA ligase activity on any of the fragments bound to GR-DBD. Indeed, we observed a small increase ( approximately 1.5-2.2-fold) in the rate of pol beta synthesis at uracil residues in both the GRE and one site six nucleotides downstream. These results highlight the potential for both positive and negative impacts of DNA-transcription factor binding on the rate of BER.
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PMID:Base excision repair in a glucocorticoid response element: effect of glucocorticoid receptor binding. 2062 60

At the moment, one of the actual trends in medical diagnostics is a development of methods for practical applications such as point-of-care testing, POCT or research tools, for example, whole genome amplification, WGA. All the techniques are based on using of specific DNA polymerases having strand displacement activity, high synthetic processivity, fidelity and, most significantly, tolerance to contaminants, appearing from analysed biological samples or collected under purification procedures. Here, we have designed a set of fusion enzymes based on catalytic domain of DNA polymerase I from Geobacillus sp. 777 with DNA-binding domain of DNA ligase Pyrococcus abyssi and Sto7d protein from Sulfolobus tokodaii, analogue of Sso7d. Designed chimeric DNA polymerases DBD-Gss, Sto-Gss and Gss-Sto exhibited the same level of thermal stability, thermal transferase activity and fidelity as native Gss; however, the processivity was increased up to 3-fold, leading to about 4-fold of DNA product in WGA which is much more exiting. The attachment of DNA-binding proteins enhanced the inhibitor tolerance of chimeric polymerases in loop-mediated isothermal amplification to several of the most common DNA sample contaminants-urea and whole blood, heparin, ethylenediaminetetraacetic acid, NaCl, ethanol. Therefore, chimeric Bst-like Gss-polymerase will be promising tool for both WGA and POCT due to increased processivity and inhibitor tolerance.
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PMID:Derivatives of Bst-like Gss-polymerase with improved processivity and inhibitor tolerance. 2893 94