The role of hSSB1 in oxidative stress

氧化应激 DNA损伤 DNA修复 生物 同源重组 DNA DNA复制 端粒 氧化磷酸化 复制蛋白A 细胞生物学 基底切除修复术 基因 遗传学 压力(语言学) 基因组不稳定性 化学 抗氧化剂 活性氧 谷胱甘肽 DNA结合蛋白 生物化学 转录因子
作者
Vincent Leong
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摘要

Single-stranded DNA binding proteins (SSB) are essential to maintain the integrity of the genome in all organisms. Replication protein A (RPA) is a nuclear member of the SSB family and is found in all eukaryotes and required for a variety of DNA metabolic processes such as DNA replication, recombination, damage repair signaling, and telomere maintenance. However, Richard et al. (2008) have recently identified that the human genome encodes two additional mammalian homologs of SSB, hSSB1 and hSSB2. Sequence alignments have revealed that hSSB1and hSSB2 are more closely related to the simple archaeal and bacterial SSBs. Similar to RPA, hSSB1 is critical for the repair of DNA double-strand breaks (DSBs) through the homology-directed repair (HDR) pathway, forming repair foci independent of the cell-cycle phase, and influencing ATM-mediated checkpoint activation. Reactive oxygen metabolites generated as a consequence of aerobic life have been viewed as sources of the cellular oxidative stress and DNA damage, and has been implicated in several human pathophysiologic conditions such as chronic diseases and aging. Reactive oxygen species (ROS) interacts with DNA to form dozens of different types of DNA damage lesions, however the predominant form of oxidative damage is 7,8-dihydro-8-oxo-dGuanine (8-oxodG). The immediate response is the initiation of the 8-oxo-dG repair pathway that involves a series of enzymatic and excision processes. The functional interplay between human SSB and base excision repair pathway is complex and incompletely understood. In my thesis, I have tested the hypothesis that hSSB1 protects cells from oxidative stress by functioning as part of a base excision repair pathway. I have found that hSSB1 is more chromatin associated in cells grown at atmospheric oxygen conditions as compared to 8% oxygen. hSSB1 is also recruited to chromatin in U-2 OS cells after oxidative damage. This finding is further strengthened by showing that chromatin-bound hSSB1 was significantly lower in neonatal foreskin fibroblasts (NFFs) as compared to U-2 OS cells. NFFs are known suffer less oxidative damage than the transformed U2OS cancer cells. My investigation has further demonstrated that hSSB1-depleted cells show an increased sensitivity to H2O2 and are unable to repair H2O2-induced 8-oxo-dG. These data suggest that hSSB1 safeguards cells from cellular stress and play a role in the repair of oxidised base lesions that arise as a result of oxidative DNA damage. To elucidate the mechanism of action I used multiple sequence alignment tools, these identified three cysteines in hSSB1 that are conserved throughout the metazoans. I identified that the cysteine 41 (Cys 41) is critical for hSSB1 activity in vitro. Ablation of this cysteine disulphide bridge by mutagenesis prevents the tetramerization formation of hSSB1 under oxidative condition. These data suggest that oxidative stress induces a conformational change in hSSB1, leading to stabilisation of hSSB1 tetramers. Intriguingly using Biacore studies, I have demonstrated that oxidised hSSB1 has a 8-fold higher affinity for single-stranded DNA containing 8-oxo-dG moiety as compared to non-oxidised hSSB1. This puts forward a mechanism by which hSSB1 tetramerizes following oxidative stress, then rapidly binds to the damage site, potentially recruiting in repair factors such as hOGG1. Interestingly, I have demonstrated cells lacking hSSB1 are unable to activate p53 (Ser15) or ATM (Ser1981) following oxidative stress. Taken together, these data provide strong evidence that hSSB1 not only functions in the repair of double-strand DNA breaks, but also in the repair of 8-oxo-dG damaged DNA.

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