Abstract
Reactive oxygen species (ROS) oxidize cellular nucleotide pools and cause double strand breaks (DSBs). Non-homologous end-joining (NHEJ) attaches broken chromosomal ends together in mammalian cells. Ribonucleotide insertion by DNA polymerase (pol) μ prepares breaks for end-joining and this is required for successful NHEJ in vivo. We previously showed that pol μ lacks discrimination against oxidized dGTP (8-oxo-dGTP), that can lead to mutagenesis, cancer, aging and human disease. Here we reveal the structural basis for proficient oxidized ribonucleotide (8-oxo-rGTP) incorporation during DSB repair by pol μ. Time-lapse crystallography snapshots of structural intermediates during nucleotide insertion along with computational simulations reveal substrate, metal and side chain dynamics, that allow oxidized ribonucleotides to escape polymerase discrimination checkpoints. Abundant nucleotide pools, combined with inefficient sanitization and repair, implicate pol μ mediated oxidized ribonucleotide insertion as an emerging source of widespread persistent mutagenesis and genomic instability.
Highlights
Reactive oxygen species (ROS) oxidize cellular nucleotide pools and cause double strand breaks (DSBs)
Most DNA polymerases discriminate against ribonucleotide insertion in order to prevent the mutagenesis and genomic instability associated with increased genomic ribonucleotides
Genomic ribonucleotide incorporation can lead to replication blockage, mutagenesis and DSB formation
Summary
Reactive oxygen species (ROS) oxidize cellular nucleotide pools and cause double strand breaks (DSBs). Ribonucleotide insertion by DNA polymerase (pol) μ prepares breaks for end-joining and this is required for successful NHEJ in vivo. We previously showed that pol μ lacks discrimination against oxidized dGTP (8-oxo-dGTP), that can lead to mutagenesis, cancer, aging and human disease. Exposure to reactive oxygen species (ROS) can oxidize cellular macromolecules causing DNA damage, such as chromosomal single and double strand breaks (DSBs)[1,2]. While limited information is available on the role and impact of damaged ribonucleotides on DNA replication and repair, insertion of oxidized dGTP (8-oxo-dGTP) by polymerases is known to be mutagenic[22,23]. Elevated genomic 8-oxo-dG levels have been implicated in development of cancer, aging and human disease[30], as well as in bacterial antibiotic resistance[31,32,33]
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