title: The C-terminal Domain (CTD) of Human DNA Glycosylase NEIL1 Is Required for Forming BERosome Repair Complex with DNA Replication Proteins at the Replicating Genome: DOMINANT NEGATIVE FUNCTION OF THE CTD.
abstract: The human DNA glycosylase NEIL1 was recently demonstrated to initiate prereplicative base excision repair (BER) of oxidized bases in the replicating genome, thus preventing mutagenic replication. A significant fraction of NEIL1 in cells is present in large cellular complexes containing DNA replication and other repair proteins, as shown by gel filtration. However, how the interaction of NEIL1 affects its recruitment to the replication site for prereplicative repair was not investigated. Here, we show that NEIL1 binarily interacts with the proliferating cell nuclear antigen clamp loader replication factor C, DNA polymerase δ, and DNA ligase I in the absence of DNA via its non-conserved C-terminal domain (CTD); replication factor C interaction results in ∼8-fold stimulation of NEIL1 activity. Disruption of NEIL1 interactions within the BERosome complex, as observed for a NEIL1 deletion mutant (N311) lacking the CTD, not only inhibits complete BER in vitro but also prevents its chromatin association and reduced recruitment at replication foci in S phase cells. This suggests that the interaction of NEIL1 with replication and other BER proteins is required for efficient repair of the replicating genome. Consistently, the CTD polypeptide acts as a dominant negative inhibitor during in vitro repair, and its ectopic expression sensitizes human cells to reactive oxygen species. We conclude that multiple interactions among BER proteins lead to large complexes, which are critical for efficient BER in mammalian cells, and the CTD interaction could be targeted for enhancing drug/radiation sensitivity of tumor cells.
authors.affiliation: Department of Radiation Oncology, Houston Methodist Research Institute, Houston, Texas 77030.
authors.forename: Pavana M
authors.initials: PM
authors.lastname: Hegde
authors.affiliation: Department of Radiation Oncology, Houston Methodist Research Institute, Houston, Texas 77030; Departments of Biochemistry and Molecular Biology, University of Texas Medical Branch, Galveston, Texas 77555.
authors.forename: Arijit
authors.initials: A
authors.lastname: Dutta
authors.affiliation: Department of Radiation Oncology, Houston Methodist Research Institute, Houston, Texas 77030.
authors.forename: Shiladitya
authors.initials: S
authors.lastname: Sengupta
authors.affiliation: Department of Radiation Oncology, Houston Methodist Research Institute, Houston, Texas 77030.
authors.forename: Joy
authors.initials: J
authors.lastname: Mitra
authors.affiliation: Department of Radiation Oncology, Houston Methodist Research Institute, Houston, Texas 77030.
authors.forename: Sanjay
authors.initials: S
authors.lastname: Adhikari
authors.affiliation: Department of Internal Medicine and University of New Mexico Cancer Center, University of New Mexico, Albuquerque, New Mexico 87131.
authors.forename: Alan E
authors.initials: AE
authors.lastname: Tomkinson
authors.affiliation: Department of Toxicology and Cancer Biology and Markey Cancer Center, University of Kentucky College of Medicine, Lexington, Kentucky 40536.
authors.forename: Guo-Min
authors.initials: GM
authors.lastname: Li
authors.affiliation: Departments of Microbiology and Immunology, University of Texas Medical Branch, Galveston, Texas 77555.
authors.forename: Istvan
authors.initials: I
authors.lastname: Boldogh
authors.affiliation: Departments of Internal Medicine, University of Texas Medical Branch, Galveston, Texas 77555.
authors.forename: Tapas K
authors.initials: TK
authors.lastname: Hazra
authors.affiliation: Department of Radiation Oncology, Houston Methodist Research Institute, Houston, Texas 77030; Departments of Biochemistry and Molecular Biology, University of Texas Medical Branch, Galveston, Texas 77555; Weill Medical College of Cornell University, New York, New York. Electronic address: smitra2@houstonmethodist.org.
authors.forename: Sankar
authors.initials: S
authors.lastname: Mitra
authors.affiliation: Department of Radiation Oncology, Houston Methodist Research Institute, Houston, Texas 77030; Weill Medical College of Cornell University, New York, New York; Houston Methodist Neurological Institute, Houston, Texas 77030. Electronic address: mlhegde@houstonmethodist.org.
authors.forename: Muralidhar L
authors.initials: ML
authors.lastname: Hegde
mesh_terms: D001483:Base Sequence; D004249:DNA Damage; D045647:DNA Glycosylases / Q000172:deficiency / Q000235:genetics*; D000072481:DNA Ligase ATP; D011088:DNA Ligases / Q000235:genetics / Q000378:metabolism; D004258:DNA Polymerase III / Q000235:genetics / Q000378:metabolism; D004260:DNA Repair; D004261:DNA Replication; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005786:Gene Expression Regulation; D015894:Genome, Human; D057809:HEK293 Cells; D006801:Humans; D008969:Molecular Sequence Data; D018384:Oxidative Stress; D017434:Protein Structure, Tertiary; D011839:Radiation, Ionizing; D017382:Reactive Oxygen Species / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D051818:Replication Protein C; D016196:S Phase / Q000235:genetics / Q000528:radiation effects; D015398:Signal Transduction
chemical_list: C000607892:LIG1 protein, human; D017382:Reactive Oxygen Species; D011994:Recombinant Proteins; D004258:DNA Polymerase III; D045647:DNA Glycosylases; C473634:NEIL1 protein, human; D051818:Replication Protein C; D011088:DNA Ligases; D000072481:DNA Ligase ATP
keywords: BERosome; DNA damage; DNA damage response; DNA polymerase; DNA replication; NEIL1 DNA glycosylase; base excision repair (BER); dominant negative inhibition; prereplicative repair