Abstract
BackgroundOxidative damage to DNA, if not repaired, can be both miscoding and blocking. These genetic alterations can lead to mutations and/or cell death, which in turn cause cancer and aging. Oxidized DNA bases are substrates for two overlapping repair pathways: base excision (BER) and nucleotide incision repair (NIR). Hydantoin derivatives such as 5-hydroxyhydantoin (5OH-Hyd) and 5-methyl-5-hydroxyhydantoin (5OH-5Me-Hyd), major products of cytosine and thymine oxidative degradation pathways, respectively, have been detected in cancer cells and ancient DNA. Hydantoins are blocking lesions for DNA polymerases and excised by bacterial and yeast DNA glycosylases in the BER pathway. However little is known about repair of pyrimidine-derived hydantoins in human cells.Methodology/Principal FindingsHere, using both denaturing PAGE and MALDI-TOF MS analyses we report that the bacterial, yeast and human AP endonucleases can incise duplex DNA 5′ next to 5OH-Hyd and 5OH-5Me-Hyd thus initiating the NIR pathway. We have fully reconstituted the NIR pathway for these lesions in vitro using purified human proteins. Depletion of Nfo in E. coli and APE1 in HeLa cells abolishes the NIR activity in cell-free extracts. Importantly, a number of redundant DNA glycosylase activities can excise hydantoin residues, including human NTH1, NEIL1 and NEIL2 and the former protein being a major DNA glycosylase activity in HeLa cells extracts.Conclusions/SignificanceThis study demonstrates that both BER and NIR pathways can compete and/or back-up each other to remove hydantoin DNA lesions in vivo.
Highlights
Endogenous aerobic metabolism and variety of exogenous factors generate reactive oxygen species (ROS), which can damage macromolecules including lipids, proteins and nucleic acids
These results suggest that the Apn1 and Nfo-generated upper-band corresponds to the fragment containing a 59-dangling 5OH-5Me-Hyd nucleotide whereas the lower-band is similar to that observed with 5OH-HydNG substrate and may correspond to fragment containing a decomposed form of 5OH-5Me-Hyd
We investigated whether the AP endonucleases involved in the nucleotide incision repair (NIR) pathway recognize the pyrimidine-derived hydantoins in duplex DNA
Summary
Endogenous aerobic metabolism and variety of exogenous factors generate reactive oxygen species (ROS), which can damage macromolecules including lipids, proteins and nucleic acids. In the NIR pathway, an AP endonuclease makes an incision 59 next to a damaged base in a DNA glycosylase-independent manner, providing a proper 39OH group for DNA polymerization and a 59-dangling damaged nucleotide [3]. Oxidative damage to DNA, if not repaired, can be both miscoding and blocking These genetic alterations can lead to mutations and/or cell death, which in turn cause cancer and aging. Oxidized DNA bases are substrates for two overlapping repair pathways: base excision (BER) and nucleotide incision repair (NIR). Hydantoin derivatives such as 5hydroxyhydantoin (5OH-Hyd) and 5-methyl-5-hydroxyhydantoin (5OH-5Me-Hyd), major products of cytosine and thymine oxidative degradation pathways, respectively, have been detected in cancer cells and ancient DNA. Little is known about repair of pyrimidine-derived hydantoins in human cells
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