Uracil in duplex DNA is a substrate for the nucleotide incision repair pathway in human cells

Abstract

Spontaneous hydrolytic deamination of cytosine to uracil (U) in DNA is a constant source of genome instability in cells. This mutagenic process is greatly enhanced at high temperatures and in single-stranded DNA. If not repaired, these uracil residues give rise to C→T transitions, which are the most common spontaneous mutations occurring in living organisms and are frequently found in human tumors. In the majority of species, uracil residues are removed from DNA by specific uracil-DNA glycosylases in the base excision repair pathway. Alternatively, in certain archaeal organisms, uracil residues are eliminated by apurinic/apyrimidinic (AP) endonucleases in the nucleotide incision repair pathway. Here, we characterized the substrate specificity of the major human AP endonuclease 1, APE1, toward U in duplex DNA. APE1 cleaves oligonucleotide duplexes containing a single U·G base pair; this activity depends strongly on the sequence context and the base opposite to U. The apparent kinetic parameters of the reactions show that APE1 has high affinity for DNA containing U but cleaves the DNA duplex at an extremely low rate. MALDI-TOF MS analysis of the reaction products demonstrated that APE1-catalyzed cleavage of a U·G duplex generates the expected DNA fragments containing a 5′-terminal deoxyuridine monophosphate. The fact that U in duplex DNA is recognized and cleaved by APE1 in vitro suggests that this property of the exonuclease III family of AP endonucleases is remarkably conserved from Archaea to humans. We propose that nucleotide incision repair may act as a backup pathway to base excision repair to remove uracils arising from cytosine deamination.spontaneous DNA base deamination | alternative excision repair | evolution

Differential expression of APE1 and APE2 in germinal centers promotes error-prone repair and A:T mutations during somatic hypermutation

“…Therefore, in ung −/− ape2 Y/− GC B cells, which have SMUG1 and only very low levels of APE1, this pathway would be very inefficient at generating SSBs that can lead to mutations at A:T bp. Alternatively, or in addition, our data are consistent with the possibility that APE2 might incise the DNA backbone at dU bases, in addition to AP sites, consistent with a recent report (54). Although APEs are thought to act specifically at AP sites, this study showed that APE1 can incise DNA at dU in duplex DNA, albeit inefficiently.…”

Excision of Uracil from Transcribed DNA Negatively Affects Gene Expression

“…In contrast, the fate of U:G appears to be more complex because SMUG1 and TDG have been suggested to contribute to repair in addition to UNG1/2, at least in cell-free extracts (5,14). Moreover, alternative to a canonical BER, this lesion can undergo a direct incision by human APE1 (26) and may also be coprocessed by the mismatch repair if a nick or another uracil are present simultaneously in DNA (35). In our substrates containing a unique uracil, it was certainly not the mismatch repair activity that caused the inhibition of transcription (Fig.…”

“…This result confirms that UNG1/2 contributes to U:G excision but also indicates a significant input of another uracil-DNA glycosylase (or other glycosylases). Alternatively, the removal of uracil might take place by a recently described mechanism initiated directly by APE1 (26).…”

Removal of oxidatively generated DNA damage by overlapping repair pathways

“…It has been suggested that the NIR pathway can serve as a backup system for BER if the appropriate glycosylase is missing or inefficient. Once the AP endonuclease has produced the initial incision, the full regeneration and repair of the incised strand can occur as long as the other, subsequent repair factors are also available [18]…”