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

Abstract

The DNA of all living organisms is persistently damaged by endogenous reactions including deamination and oxidation. Such damage, if not repaired correctly, can result in mutations that drive tumor development. In addition to chemical damage, recent studies have established that DNA bases can be enzymatically modified, generating many of the same modified bases. Irrespective of the mechanism of formation, modified bases can alter DNA-protein interactions and therefore modulate epigenetic control of gene transcription. The simultaneous presence of both chemically and enzymatically modified bases in DNA suggests a potential intersection, or collision, between DNA repair and epigenetic reprogramming. In this paper, we have prepared defined sequence oligonucleotides containing the complete set of oxidized and deaminated bases that could arise from 5-methylcytosine. We have probed these substrates with human glycosylases implicated in DNA repair and epigenetic reprogramming. New observations reported here include: SMUG1 excises 5-carboxyuracil (5caU) when paired with A or G. Both TDG and MBD4 cleave 5-formyluracil and 5caU when mispaired with G. Further, TDG not only removes 5-formylcytosine and 5-carboxycytosine when paired with G, but also when mispaired with A. Surprisingly, 5caU is one of the best substrates for human TDG, SMUG1 and MBD4, and a much better substrate than T. The data presented here introduces some unexpected findings that pose new questions on the interactions between endogenous DNA damage, repair, and epigenetic reprogramming pathways.

Rapid excision of oxidized adenine by human thymine DNA glycosylase

“…Evidently, mechanisms that preclude excision of thymine from A·T pairs are relaxed for excision of oxoA paired with adenine. Notably, a recent study finds that TDG excision of fC and caC is similarly efficient when these bases are paired with adenine or guanine ( 54 ).…”

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

“…Similarly, 5mC deamination results in a T:G mispair, which also causes the same artifact in 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 .…”

“…The T:G mismatch is persistent in human DNA because members of the uracil-DNA glycosylase superfamily that remove T from a T:G mispair have much weaker activity for T:G than for U:G, the product of cytosine deamination. 38,39 The measurement of a T:G mispair presents some unique challenges. Traditional methods for the measurement of DNA adducts require the enzymatic or acid hydrolysis of DNA prior to the separation of the DNA nucleosides or bases and analysis by mass spectrometry-based methods.…”

“…In higher organisms, the replacement of C with 5mC is important in the epigenetic control of gene expression and also for chromatin structure. − Enzymatic DNA methylation usually occurs in the CpG dinucleotide, and C to T transition mutations at CpG dinucleotides represent the most frequent single-base change found in human tumors. − The deamination of 5mC to T generates a T:G mismatch and, upon DNA replication, results in a C to T mutation. The T:G mismatch is persistent in human DNA because members of the uracil-DNA glycosylase superfamily that remove T from a T:G mispair have much weaker activity for T:G than for U:G, the product of cytosine deamination. , …”

“…After DNA replication, this results in a C to T mutation. The T:G mismatch is persistent in human DNA because members of the uracil-DNA glycosylase superfamily presumed to remove T from a T:G mispair have much weaker activity for T:G than for U:G, the product of cytosine deamination. , Despite its prominent role in mutagenesis, methods to measure T:G mismatches have only recently been developed …”