The role of hSSB1 in oxidative stress

Leong, Vincent Ching Shian (2013). The role of hSSB1 in oxidative stress PhD Thesis, School of Medicine, The University of Queensland.

       
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Author Leong, Vincent Ching Shian
Thesis Title The role of hSSB1 in oxidative stress
School, Centre or Institute School of Medicine
Institution The University of Queensland
Publication date 2013
Thesis type PhD Thesis
Supervisor Derek J Richard
Emma Bolderson
Total pages 135
Total colour pages 24
Total black and white pages 111
Language eng
Subjects 0604 Genetics
0601 Biochemistry and Cell Biology
Formatted abstract
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.
Keyword DNA repair
Oxidative stress
hSSB1
hOGG1
Base excision repair

 
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Created: Mon, 18 Nov 2013, 11:38:12 EST by Mr Ching Shian Leong on behalf of Scholarly Communication and Digitisation Service