Abstract
Base excision repair (BER) is a key genome maintenance pathway. The NEIL1 DNA glycosylase recognizes oxidized bases, and likely removes damage in advance of the replication fork. The rs5745906 SNP of the NEIL1 gene is a rare human germline variant that encodes the NEIL1 G83D protein, which is devoid of DNA glycosylase activity. Here we show that expression of G83D NEIL1 in MCF10A immortalized but non-transformed mammary epithelial cells leads to replication fork stress. Upon treatment with hydrogen peroxide, we observe increased levels of stalled replication forks in cells expressing G83D NEIL1 versus cells expressing the wild-type (WT) protein. Double-strand breaks (DSBs) arise in G83D-expressing cells during the S and G2/M phases of the cell cycle. Interestingly, these breaks result in genomic instability in the form of high levels of chromosomal aberrations and micronuclei. Cells expressing G83D also grow in an anchorage independent manner, suggesting that the genomic instability results in a carcinogenic phenotype. Our results are consistent with the idea that an inability to remove oxidative damage in an efficient manner at the replication fork leads to genomic instability and mutagenesis. We suggest that individuals who harbor the G83D NEIL1 variant face an increased risk for human cancer.
Highlights
Base excision repair is critically important for maintaining genomic stability because it repairs at least 20,000-50,000 lesions per cell per day that arise from the inherent instability of DNA and the presence of reactive oxygen and nitrogen species (RONs)
G83D is deficient for DNA glycosylase activity but is able to bind to DNA with a thymine glycol (Tg):A base pair in vitro and exhibits increased retention time at psoralen crosslinks [14, 18]
Our results show that cells expressing G83D have significantly increased levels of stalled replication forks when compared to cells expressing WT NEIL1 in the presence of oxidative DNA damage induced by H2O2 (Figure 2B and 2C)
Summary
Base excision repair is critically important for maintaining genomic stability because it repairs at least 20,000-50,000 lesions per cell per day that arise from the inherent instability of DNA and the presence of reactive oxygen and nitrogen species (RONs) (for a review see [1]). Apurinic/apyriminidinic endonuclease I (APE I) recognizes and incises the abasic site generated by a monofunctional DNA glycosylase, leaving a single nucleotide gap with a 3’OH and a 5’-deoxyribose phosphate (dRP) group. This gap is filled in by DNA polymerase beta (pol ß), which removes the 5’dRP, and DNA ligase 3α (LIG3α) in complex with X-Ray Cross-Complimenting 1 (XRCC1) seals the nick (for a review see [3]). Pol ß binds to this substrate and fills in the resulting single nucleotide gap In both cases, the XRCC1/ Ligase IIIɑ or XRCC1/Ligase I complex catalyzes ligation of the resulting ends. NEIL3 exhibits weak lyase activity in the form of β-elimination, the base excision reaction is significantly more efficient than its lyase activity, suggesting that it functions predominantly as a monfunctional enzyme [5, 6]
Sign-in/Register to view highlights & summary 
Published Version (
Free)
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call