Muralidhar L. Hegde

Early steps in the DNA base excision/single-strand interruption repair pathway in mammalian cells

Base excision repair (BER) is an evolutionarily conserved process for maintaining genomic integrity by eliminating several dozen damaged (oxidized or alkylated) or inappropriate bases that are generated endogenously or induced by genotoxicants, predominantly, reactive oxygen species (ROS). BER involves 4-5 steps starting with base excision by a DNA glycosylase, followed by a common pathway usually involving an AP-endonuclease (APE) to generate 3′ OH terminus at the damage site, followed by repair synthesis with a DNA polymerase and nick sealing by a DNA ligase. This pathway is also responsible for repairing DNA single-strand breaks with blocked termini directly generated by ROS. Nearly all glycosylases, far fewer than their substrate lesions particularly for oxidized bases, have broad and overlapping substrate range, and could serve as back-up enzymes in vivo. In contrast, mammalian cells encode only one APE, APE1, unlike two APEs in lower organisms. In spite of overall similarity, BER with distinct subpathways in the mammals is more complex than in E. coli. The glycosylases form complexes with downstream proteins to carry out efficient repair via distinct subpathways one of which, responsible for repair of strand breaks with 3′ phosphate termini generated by the NEIL family glycosylases or by ROS, requires the phosphatase activity of polynucleotide kinase instead of APE1. Different complexes may utilize distinct DNA polymerases and ligases. Mammalian glycosylases have nonconserved extensions at one of the termini, dispensable for enzymatic activity but needed for interaction with other BER and non-BER proteins for complex formation and organelle targeting. The mammalian enzymes are sometimes covalently modified which may affect activity and complex formation. The focus of this review is on the early steps in mammalian BER for oxidized damage.

Interaction of the Human DNA Glycosylase NEIL1 with Proliferating Cell Nuclear Antigen THE POTENTIAL FOR REPLICATION-ASSOCIATED REPAIR OF OXIDIZED BASES IN MAMMALIAN GENOMES

NEIL1 and NEIL2 compose a family of DNA glycosylases that is distinct from that of the other two DNA glycosylases, OGG1 and NTH1, all of which are involved in repair of oxidized bases in mammalian genomes. That the NEIL proteins, unlike OGG1 and NTH1, are able to excise base lesions from single-stranded DNA regions suggests their preferential involvement in repair during replication and/or transcription. Previous studies showing S phase-specific activation of NEIL1, but not NEIL2, suggested NEIL1 involvement in the repair of replicating DNA. Here, we show that human NEIL1 stably interacts both in vivo and in vitro with proliferating cell nuclear antigen (PCNA), the sliding clamp for DNA replication. PCNA stimulates NEIL1 activity in excising the oxidized base 5-hydroxyuracil from single-stranded DNA sequences including fork structures. PCNA enhances NEIL1 loading on the substrate. In contrast, although present in the NEIL2 immunocomplex, PCNA does not stimulate NEIL2. NEIL1 interacts with PCNA via a domain that is located in a region near the C terminus, dispensable for base excision activity. The interacting sequence in NEIL1, which lacks the canonical PCNA-binding motif, includes a sequence conserved in DNA polymerase δ and implicated in its PCNA binding. Mammalian two-hybrid analysis confirmed PCNA interaction with NEIL1. The G127A mutation in PCNA reduces its stimulatory activity, suggesting that the interdomain connector loop, a common binding interface of PCNA, is involved in NEIL1 binding. These results strongly support in vivo function of NEIL1 in preferential repair of oxidized bases in DNA prior to replication.

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

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.

The Human Werner Syndrome Protein Stimulates Repair of Oxidative DNA Base Damage by the DNA Glycosylase NEIL1

The mammalian DNA glycosylase, NEIL1, specific for repair of oxidatively damaged bases in the genome via the base excision repair pathway, is activated by reactive oxygen species and prevents toxicity due to radiation. We show here that the Werner syndrome protein (WRN), a member of the RecQ family of DNA helicases, associates with NEIL1 in the early damage-sensing step of base excision repair. WRN stimulates NEIL1 in excision of oxidative lesions from bubble DNA substrates. The binary interaction between NEIL1 and WRN (KD = 60 nm) involves C-terminal residues 288-349 of NEIL1 and the RecQ C-terminal (RQC) region of WRN, and is independent of the helicase activity WRN. Exposure to oxidative stress enhances the NEIL-WRN association concomitant with their strong nuclear co-localization. WRN-depleted cells accumulate some prototypical oxidized bases (e.g. 8-oxoguanine, FapyG, and FapyA) indicating a physiological function of WRN in oxidative damage repair in mammalian genomes. Interestingly, WRN deficiency does not have an additive effect on in vivo damage accumulation in NEIL1 knockdown cells suggesting that WRN participates in the same repair pathway as NEIL1.

Physical and Functional Interaction between Human Oxidized Base-specific DNA Glycosylase NEIL1 and Flap Endonuclease 1

The S phase-specific activation of NEIL1 and not of the other DNA glycosylases responsible for repairing oxidatively damaged bases in mammalian genomes and the activation of NEIL1 by proliferating cell nuclear antigen (PCNA) suggested preferential action by NEIL1 in oxidized base repair during DNA replication. Here we show that NEIL1 interacts with flap endonuclease 1 (FEN-1), an essential component of the DNA replication. FEN-1 is present in the NEIL1 immunocomplex isolated from human cell extracts, and the two proteins colocalize in the nucleus. FEN-1 stimulates the activity of NEIL1 in vitro in excising 5-hydroxyuracil from duplex, bubble, forked, and single-stranded DNA substrates by up to 5-fold. The disordered region near the C terminus of NEIL1, which is dispensable for activity, is necessary and sufficient for high affinity binding to FEN-1 (KD ≅ 0.2 μm). The interacting interface of FEN-1 is localized in its disordered C-terminal region uniquely present in mammalian orthologs. Fine structure mapping identified several Lys and Arg residues in this region that form salt bridges with Asp and Glu residues in NEIL1. NEIL1 was previously shown to initiate single nucleotide excision repair, which does not require FEN-1 or PCNA. The present study shows that NEIL1 could also participate in strand displacement repair synthesis (long patch repair (LP-BER)) mediated by FEN-1 and stimulated by PCNA. Interaction between NEIL1 and FEN-1 is essential for efficient NEIL1-initiated LP-BER. These studies strongly implicate NEIL1 in a distinct subpathway of LP-BER in replicating genomes.

Activation of Ras Signaling Pathway by 8-Oxoguanine DNA Glycosylase Bound to Its Excision Product, 8-Oxoguanine

Background: 8-Oxo-7,8-dihydroguanine (8-oxoG) is an abundant DNA base lesion repaired by 8-oxoguanine glycosylase (OGG1) via the base excision repair pathway. Results: OGG1 binds to its repair product 8-oxoG and activates canonical Ras family GTPases, causing gene activation via MAPK signaling. Conclusion: OGG1 complexed with 8-oxoG has guanine nucleotide exchange factor activity. Significance: OGG1 modulates cellular signaling via its DNA repair-independent function. 8-Oxo-7,8-dihydroguanine (8-oxoG), arguably the most abundant base lesion induced in mammalian genomes by reactive oxygen species, is repaired via the base excision repair pathway that is initiated with the excision of 8-oxoG by OGG1. Here we show that OGG1 binds the 8-oxoG base with high affinity and that the complex then interacts with canonical Ras family GTPases to catalyze replacement of GDP with GTP, thus serving as a guanine nuclear exchange factor. OGG1-mediated activation of Ras leads to phosphorylation of the mitogen-activated kinases MEK1,2/ERK1,2 and increasing downstream gene expression. These studies document for the first time that in addition to its role in repairing oxidized purines, OGG1 has an independent guanine nuclear exchange factor activity when bound to 8-oxoG.

Prereplicative repair of oxidized bases in the human genome is mediated by NEIL1 DNA glycosylase together with replication proteins

Significance Repair of mutagenic oxidized bases in the genome is required before replication to prevent mutations. It is unknown how such base lesions, which do not block replication, are flagged for repair in the single-stranded replicating template. We demonstrate here that the repair-initiating, S-phase–activated Nei-like (NEIL) 1 DNA glycosylase binds to but does not excise the base lesion and cleave the template DNA strand, which would lead to a lethal double-strand break. Instead, NEIL1 blocks progression of the replication fork, which then regresses to allow lesion repair. In the absence of NEIL1, the related glycosylase NEIL2 serves as a backup enzyme. Base oxidation by endogenous and environmentally induced reactive oxygen species preferentially occurs in replicating single-stranded templates in mammalian genomes, warranting prereplicative repair of the mutagenic base lesions. It is not clear how such lesions (which, unlike bulky adducts, do not block replication) are recognized for repair. Furthermore, strand breaks caused by base excision from ssDNA by DNA glycosylases, including Nei-like (NEIL) 1, would generate double-strand breaks during replication, which are not experimentally observed. NEIL1, whose deficiency causes a mutator phenotype and is activated during the S phase, is present in the DNA replication complex isolated from human cells, with enhanced association with DNA in S-phase cells and colocalization with replication foci containing DNA replication proteins. Furthermore, NEIL1 binds to 5-hydroxyuracil, the oxidative deamination product of C, in replication protein A-coated ssDNA template and inhibits DNA synthesis by DNA polymerase δ. We postulate that, upon encountering an oxidized base during replication, NEIL1 initiates prereplicative repair by acting as a “cowcatcher” and preventing nascent chain growth. Regression of the stalled replication fork, possibly mediated by annealing helicases, then allows lesion repair in the reannealed duplex. This model is supported by our observations that NEIL1, whose deficiency slows nascent chain growth in oxidatively stressed cells, is stimulated by replication proteins in vitro. Furthermore, deficiency of the closely related NEIL2 alone does not affect chain elongation, but combined NEIL1/2 deficiency further inhibits DNA replication. These results support a mechanism of NEIL1-mediated prereplicative repair of oxidized bases in the replicating strand, with NEIL2 providing a backup function.

MOF Phosphorylation by ATM Regulates 53BP1-Mediated Double-Strand Break Repair Pathway Choice

Cell-cycle phase is a critical determinant of the choice between DNA damage repair by nonhomologous end-joining (NHEJ) or homologous recombination (HR). Here, we report that double-strand breaks (DSBs) induce ATM-dependent MOF (a histone H4 acetyl-transferase) phosphorylation (p-T392-MOF) and that phosphorylated MOF colocalizes with γ-H2AX, ATM, and 53BP1 foci. Mutation of the phosphorylation site (MOF-T392A) impedes DNA repair in S and G2 phase but not G1 phase cells. Expression of MOF-T392A also blocks the reduction in DSB-associated 53BP1 seen in wild-type S/G2 phase cells, resulting in enhanced 53BP1 and reduced BRCA1 association. Decreased BRCA1 levels at DSB sites correlates with defective repairosome formation, reduced HR repair, and decreased cell survival following irradiation. These data support a model whereby ATM-mediated MOF-T392 phosphorylation modulates 53BP1 function to facilitate the subsequent recruitment of HR repair proteins, uncovering a regulatory role for MOF in DSB repair pathway choice during S/G2 phase.

Role of Human DNA Glycosylase Nei-like 2 (NEIL2) and Single Strand Break Repair Protein Polynucleotide Kinase 3′-Phosphatase in Maintenance of Mitochondrial Genome

The repair of reactive oxygen species-induced base lesions and single strand breaks (SSBs) in the nuclear genome via the base excision (BER) and SSB repair (SSBR) pathways, respectively, is well characterize, and important for maintaining genomic integrity. However, the role of mitochondrial (mt) BER and SSBR proteins in mt genome maintenance is not completely clear. Here we show the presence of the oxidized base-specific DNA glycosylase Nei-like 2 (NEIL2) and the DNA end-processing enzyme polynucleotide kinase 3′-phosphatase (PNKP) in purified human mitochondrial extracts (MEs). Confocal microscopy revealed co-localization of PNKP and NEIL2 with the mitochondrion-specific protein cytochrome c oxidase subunit 2 (MT-CO2). Further, chromatin immunoprecipitation analysis showed association of NEIL2 and PNKP with the mitochondrial genes MT-CO2 and MT-CO3 (cytochrome c oxidase subunit 3); importantly, both enzymes also associated with the mitochondrion-specific DNA polymerase γ. In cell association of NEIL2 and PNKP with polymerase γ was further confirmed by proximity ligation assays. PNKP-depleted ME showed a significant decrease in both BER and SSBR activities, and PNKP was found to be the major 3′-phosphatase in human ME. Furthermore, individual depletion of NEIL2 and PNKP in human HEK293 cells caused increased levels of oxidized bases and SSBs in the mt genome, respectively. Taken together, these studies demonstrate the critical role of NEIL2 and PNKP in maintenance of the mammalian mitochondrial genome.

8-Oxoguanine DNA glycosylase-1 links DNA repair to cellular signaling via the activation of the small GTPase Rac1

8-Oxo-7,8-dihydroguanine (8-oxoG) is one of the most abundant DNA base lesions induced by reactive oxygen species (ROS). Accumulation of 8-oxoG in the mammalian genome is considered a marker of oxidative stress, to be causally linked to inflammation, and is thought to contribute to aging processes and various aging-related diseases. Unexpectedly, mice that lack 8-oxoguanine DNA glycosylase-1 (OGG1) activity and accumulate 8-oxoG in their genome have a normal phenotype and longevity; in fact, they show increased resistance to both inflammation and oxidative stress. OGG1 excises and generates free 8-oxoG base during DNA base-excision repair (BER) processes. In the present study, we report that in the presence of the 8-oxoG base, OGG1 physically interacts with guanine nucleotide-free and GDP-bound Rac1 protein. This interaction results in rapid GDP→GTP, but not GTP→GDP, exchange in vitro. Importantly, a rise in the intracellular 8-oxoG base levels increases the proportion of GTP-bound Rac1. In turn Rac1-GTP mediates an increase in ROS levels via nuclear membrane-associated NADPH oxidase type 4. These results show a novel mechanism by which OGG1 in complex with 8-oxoG is linked to redox signaling and cellular responses.