Cell Cycle Regulation of the Murine 8-Oxoguanine DNA Glycosylase (mOGG1): mOGG1 Associates with Microtubules During Interphase and Mitosis By: Kimberly Conlon, D. Zharkov and M. Berrios As Originally Published in DNA Repair, 2004 Tel: (631) 553-5284 Email Dr. Conlon

Abstract

8-Oxoguanine DNA glycosylase (OGG1) is a major DNA repair enzyme in mammalian cells. OGG1 participates in the repair of 8-oxoG, the most abundant known DNA lesion induced by endogenous reactive oxygen species in aerobic organisms. In this study, antibodies directed against purified recombinant human OGG1 (hOGG1) or murine (mOGG1) protein were chemically conjugated to either the photosensitizer Rose Bengal or the fluorescent dye Texas red. These dye-protein conjugates, in combination with binding assays, were used to identify associations between mOGG1 and the cytoskeleton of NIH3T3 fibroblasts. Results from these binding studies showed that mOGG1 associates with the cytoskeleton by specifically binding to the centriole and microtubules radiating from the centrosome at interphase and the spindle assembly at mitosis. Similar results were obtained with hOGG1. Together results reported in this study suggest that OGG1 is a microtubuleassociated protein itself or that OGG1 utilizes yet to be identified motor proteins to ride on microtubules as tracks facilitating the movement and redistribution of cytoplasmic OGG1 pools during interphase and mitosis and in response to oxidative DNA damage.

1. Introduction

Endogenous reactive oxygen species (ROS) are byproducts from aerobic metabolism. ROS formation in vivo increases when cells are exposed to environmental pollutants [1,2], certain drugs [3], nutrient deprivation [4], oxidizing agents or ionizing radiation [5-7] and during some pathological processes such as inflamation or ischemia-reperfusion [8]. Reaction between ROS and DNA leads to modifications of several types, including single or double strand breaks, as well as modifications of bases and sugars. Damage to genomic DNA often results in mutations that may lead to neoplasia, cell death and degenerative diseases [9-12]. Although cellular anti-oxidant defenses (e.g., catalase, peroxidase, superoxide dismutase) can effectively combat the effects of ROS, those that escape these defenses can diffuse into the nucleus to react with DNA [13].

ROS generate several base modifications in DNA including 7,8-dihydro-8-oxoguanine (8-oxoG). 8-OxoG is a relatively stable oxidized form of guanine frequently produced by ROS when interacting with either free nucleotides orDNA [14].At replication, 8-oxoG will often be mispaired with adenine, giving rise to G:C to T:A transversions, a common somatic mutation associated with human cancers. 8-OxoG has also been shown to be miscoding in vitro and mutagenic in vivo [15]. As a result, 8-oxoG has been used as a marker for oxidative DNA damage [13,16].

8-OxoG-DNA repair activities have been reported in mammals including mice [17,18], human leukocytes and HeLa cells [19,20]. The murine OGG1 (mOGG1) gene was localized to chromosome 6 [21], and the gene product was identified as a DNA 8-oxoG glycosylase [18]. The human OGG1 gene has been localized to the short arm of chromosome 3 (3p25/26) in a region that is commonly deleted in lung cancers [22]. Recently, liver cells from homozygous ogg-/- mice have shown a 10-fold increase in G:C to T:A transversion frequencies in their DNA [23,24].

Human OGG1 gene generates several isoforms by alternative splicing; all isoforms have been localized to mitochondria except for one containing a single NLS that has been localized to the nucleus [25-28]. Similarly, antibodies directed against hOGG1 or hMYH, the human homolog for MutY, another DNA glycosylase directed against oxidative DNA damage, localized these enzymes to mitochondria and nuclei of human cells, and their distribution was regulated by alternative splicing of each transcript [29,30]. Furthermore, using transiently expressed epitope-tagged human OGG1, hMYH and the human homolog of yet another repair enzyme edonuclease III (hNTH1), Takao and collaborators showed that these enzymes were localized mostly to the nucleus and mitochondria in transfected Cos7 cells [26]. Recently, using a stable transfectant cell line expressing human OGG1 fused at the C-terminus to GFP, it was shown that human OGG1 was preferentially associated with a nuclear matrix or karyoskeleton-enriched fraction and chromatin during interphase and becomes associated with mitotic chromosomes during mitosis [31]. It is now accepted that there are distinct nuclear and cytoplasmic pools of OGG1, with the former composed of a single isoform, OGG1-1a, and the latter, of several isoforms lacking the nuclear localization signal [26,27]. Activity, expression and subcellular distribution of OGG1 is modulated by a number of circumstances causing oxidative DNA damage [1-8].

We previously reported that cytoplasmic pools ofmOGG1 redistribute to the nucleus and nuclear periphery in response to nutrient deprivation and oxidative DNA damage [4]. The relatively rapid redistribution of mOGG1 observed in cells exposed to oxidative stress suggested that the movement of intracellular mOGG1 pools may be mediated, in part, by active transport rather than just diffusion through the cytosol. This recent observation together with a previous report showing the association of OGG1 with the nuclear matrix or karyoskeleton [31] suggests that relationships similar to those observed in the nucleus may also exist between OGG1 and elements of the cytoskeleton of eukaryotic cells. The cytoskeleton is a filamentous network spanning the cytoplasm and composed of three major cytoskeletal polymers: actin filaments, intermediate filaments and microtubules. These filaments interact with each other and with many different associated proteins to mediate the trafficking of macromolecules and small organelles through the cytoplasm [32-36]. The apparent universality of this bidirectional trafficking is substantiated by the wide range of macromolecules transported by cytoskeletal filaments, including ribosomal components, other ribonucleoproteins, cytosolic proteins, etc. For example, cells use the cytoskeleton to regulate the trafficking of mRNAs and cofactors from transcription and processing sites in the nucleus to translation and degradation sites in the cytosol [37,38].

Microtubules are hollow cylindrical structures approximately 25 nm in diameter and can reach 25 μm in length. Each is constructed of 13 protofilaments formed by tubulin molecules, relatively stable heterodimers of alpha and beta tubulin subunits. Besides serving other functions, microtubules, in conjunction with microtubule-associated proteins (MAPs) and molecular motor proteins, provide paths along which macromolecules (i.e. mRNAs and proteins) are transported to or from the nucleus or elsewhere in the cytosol [39,40]. Association with microtubules facilitates not only the distribution of specific macromolecules to a targeted cell region but also prevents these macromolecules from being diluted in the cytosol [38]. Microtubules also form the centrioles at the core of centrosomes (see, e.g., [41]). In addition to participating in the organization of microtubules and the mitotic spindle assembly, centrosomes have been shown to play a critical role in cell cycle progression and cellular responses to DNA replication defects and DNA damage [42,43].

Although most studies have been focused on elucidating the expression, enzyme mechanism and substrate specificity of the mammalian OGG1 protein, the role that cytoskeletal components may play in regulating the distribution of OGG1 pools within cells has not yet been evaluated. This study used several strategies to circumvent limitations associated with the use of actively growing tissue culture cells not over expressing OGG1 including the combination of binding assays, chemical cross-linking and in situ site-directed photochemistry to establish whether OGG1 associates with elements of the cytoskeleton as an effective pathway to regulate its redistribution in normally growing cells as well as in response to oxidative stress and DNA damage. Results from this study showed that OGG1 specifically binds to microtubules in actively growing cells. OGG1 remained largely bound to a network of microtubules and the mitotic spindle assembly during interphase and mitosis respectively suggesting that OGG1, and other DNA repair proteins, may bind to microtubules and/or use MAPs or molecular motors riding on microtubules to relocate repair enzyme pools in cells growing under normal and oxidative stress conditions and during the cell cycle.

2. Materials and methods

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