Measurement of deaminated cytosine adducts in DNA using a novel hybrid thymine DNA glycosylase

Abstract

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Characterization of a Novel Thermostable DNA Lyase Used To Prepare DNA for Next-Generation Sequencing

“…While a U:G mispair can be repaired by UDG, T:G mispairs are much more challenging to remove. , 5mC also deaminates at a similar rate to C but is much more poorly repaired by the human enzymes believed to be involved in their repair. , Consequently, T:G mispairs are believed to be one of the most common DNA damage adducts. Until recently, enzymes that remove T:G efficiently were not readily available . In addition, emerging methods, based upon NGS, are creating new opportunities to quantify and locate specific damage or enzymatic modifications in DNA isolated from biological sources using DNA repair enzymes .…”

“…The expression of His-tagged hyTDG-lyase was induced with IPTG (1 mM) at 250 rpm and 30 °C for 6 h. The cells were harvested by centrifugation at 4100 rpm for 5 min and stored −80 °C until use. The purification of the target protein was performed as previously described with slight modification . Briefly, the cell pellet was thawed and suspended in 4 mL of lysis buffer and sonicated on ice.…”

“…The value of this enzyme, in our hands, was its relatively good excision capability on a wide array of 5-substituted uracil analogues, including thymine, and the extremely high selectivity for uracil analogues mispaired with guanine . We attached a 29-amino acid peptide found in human TDG (hTDG) that increased the overall activity of hTDG to the catalytic domain of MIG to create a hybrid enzyme, hyTDG . MIG and hyTDG are monofunctional glycosylases that bind strongly to abasic sites, the product of the glycosylase activity.…”

Chemical and enzymatic modifications of 5-methylcytosine at the intersection of DNA damage, repair, and epigenetic reprogramming

“…To examine the substrate specificity of each glycosylase, 2.5 pmol of a FAM-labelled duplex containing a target pyrimidine pair opposite A or G was incubated with hUNG2 (0.07 mg, MW 40.0 kDa, 1.75 pmol), hTDG (1.5 mg, MW 48.4 kDa, 31 pmol), hSMUG1 (0.5 mg, 30.7 kDa, 16.3 pmol) in buffer containing 10 mM K 2 HPO 4 , 30mM NaCl, 40mM KCl, pH 7.9 or hMBD4 (0.5 mg, 66.9 kDa, 7.5 pmol) in buffer containing 20 mM tris-HCI, 1 mM DTT and 1mM EDTA at pH 8.0 and incubated for 1 h at 37°C. Recombinant hyTDG(Y163K) (0.5 mg, MW 29.7 kDa, 16.8 pmol) with no glycosylase activity, but efficient AP endonuclease activity [ 63 , 64 ], was added and incubated at 65°C for 1 h to cleave the phosphodiester backbone at the abasic site. An equal volume of formamide was added and samples were loaded onto a 20% polyacrylamide gel containing 6M urea and 1xTBE buffer and electrophoresed at 180 V for 40 min using a Bio-Rad Mini-PROTEAN Tetra Cell (Hercules, CA).…”

“…Following target bases excision, oligonucleotide cleavage can be accomplished with an AP lyase or NaOH. In this study, when analogs were paired with G, the phosphodiester backbone were cleaved with a Y163K mutant of the hyTDG we recently described [ 63 ], which has lost glycosylase activity but gained AP lyase activity [ 64 ]. The advantage of the AP lyase is that neutral conditions employed do not result in chemical damage to modified bases.…”