Preferential Repair of Oxidized Base Damage in the Transcribed Genes of Mammalian Cells

Abstract

Preferential repair of bulky DNA adducts from the transcribed genes via nucleotide excision repair is well characterized in mammalian cells. However, definitive evidence is lacking for similar repair of oxidized bases, the major endogenous DNA lesions. Here we show that the oxidized base-specific human DNA glycosylase NEIL2 associates with RNA polymerase II and the transcriptional regulator heterogeneous nuclear ribonucleoprotein-U (hnRNP-U), both in vitro and in cells. NEIL2 immunocomplexes from cell extracts preferentially repaired the mutagenic cytosine oxidation product 5-hydroxyuracil in the transcribed strand. In a reconstituted system, we also observed NEIL2-initiated transcription-dependent base excision repair of 5-hydroxyuracil in the transcribed strand, with hnRNP-U playing a critical role. Chromatin immunoprecipitation/reimmunoprecipitation studies showed association of NEIL2, RNA polymerase II, and hnRNP-U on transcribed but not on transcriptionally silent genes. Furthermore, NEIL2-depleted cells accumulated more DNA damage in active than in silent genes. These results strongly support the preferential role of NEIL2 in repairing oxidized bases in the transcribed genes of mammalian cells.

Preferential Repair of DNA Double-strand Break at the Active Gene in Vivo

“…Previous studies (17)(18)(19)(20)(21)(22)(23)(24)(35)(36)(37) have demonstrated transcription-coupled nucleotide/base excision repairs. However, it remained unknown whether transcription-coupled DSB repair exists.…”

“…In TC-NER, recognition of the lesion is tied to RNA polymerase stalling at the DNA damage site and involves Rad26p in yeast or Cockayne syndrome group B protein (CSB) in humans (25)(26)(27)(28)(29)(30)(31)(32)(33)(34). Like TC-NER, base excision repair is also coupled to transcription (35)(36)(37). However, it is yet to be determined whether DSB repair is coupled to transcription.…”

Enhancement of NEIL1 Protein-initiated Oxidized DNA Base Excision Repair by Heterogeneous Nuclear Ribonucleoprotein U (hnRNP-U) via Direct Interaction

“…Contrary to the common perception that the repair of oxidatively damaged bases in DNA via the BER pathway involves only a few conserved repair proteins, a more complex picture of BER is now emerging in that BER comprises several alternative subpathways that utilize distinct sets of repair enzymes and other proteins (16,19,21,35,46). We have been exploring the role of noncanonical proteins in oxidized base repair and have characterized the proteins present in immunoprecipitates of early BER enzymes including DGs.…”

“…Mammalian expression plasmids for C-terminally FLAG-tagged NEIL1 were previously described (33,35). Log-phase HEK293 cells (in 6-cm plates) were transfected with 1 g of NEIL1-FLAG or the empty vector plasmid (19). After 24 h, the cells were trypsinized and plated in fresh medium containing Zeocin (100 g/ml) to select clones carrying stably integrated plasmid DNA.…”

“…The immunoprecipitates (IPs) of both NEILs contain hnRNP-U. We previously characterized the involvement of NEIL2 in the preferential repair of transcribed genes, where NEIL2 interacts functionally with both RNA polymerase II and hnRNP-U (19). Unlike NEIL2, NEIL1 is up-regulated during the S-phase and interacts functionally with DNA replication proteins, including proliferating cell nuclear antigen (PCNA), replication protein A (RPA), FEN-1, and Werner helicase (WRN), etc.…”