Defining the mechanistic role of hSSB1 : a novel single-stranded DNA binding protein essential for DNA repair

Abstract

DNA is under constant attack from the external and internal environment. It is imperative to repair and maintain the vital genetic information of DNA. Otherwise it leads to an accumulation of mutations that alters the normal function of DNA and in turn causes a disorder in cellular metabolism. During repair, DNA unwinds into single-stranded DNA (ssDNA) and is even more vulnerable to damage. This is where human single-strand DNA binding protein 1 (hSSB1) binds and protects ssDNA. It is known that hSSB1 exists both as part of the MRN (MRE11, RAD50, NBS1) repair complex and the SOSS1 complex (made up of INTS3 and C9orf80). It is also known to initiate an appropriate repair mechanism by recruiting other proteins to the site of damage. There is an accumulating body of research on hSSB1 function in Double Strand Break Repair (DSBR). It promotes the ataxia telangiectasia mutated (ATM) kinase signalling cascade and also recruits the MRN complex to the site of double strand breaks (DSBs) in order to initiate DSBR via Homologous Recombination (HR). Recently, it was discovered that hSSB1 is capable of forming higher order oligomers and can function in the oxidative DNA damage response. The most common oxidative DNA damage is the 7,8-dihydro-8-oxoguanine (8-oxoG) adduct, which is the direct result of reactive oxygen species (ROS) produced during regular cellular respiration. If this damage goes unrepaired it may result in G:C to T:A transversions. These lesions are normally processed by the Base Excision Repair (BER) pathway, which involves human oxoguanine glycosylase (hOGG1) that cleaves the DNA backbone and removes the offending base. So far, it is understood that hSSB1 levels increase in response to oxidative damage; also, cells depleted of hSSB1 are hypersensitive to oxidative damage and are also unable to efficiently remove 8-oxoG adducts. The recruitment of hOGG1 to chromatin is dependent on hSSB1 and hSSB1 can promote hOGG1 cleavage of 8-oxoG. This thesis examines the mechanism of hSSB1 oligomerisation under oxidative conditions. hSSB1 forms dimers and tetramers and this oligomerisation is likely mediated by inter-domain disulfide bond formation. These oligomers can also be synthetically created through the use of a thiol reactive cross-linker. Oxidised hSSB1 binds to 8-oxoG damaged ssDNA with higher affinity than non-damaged ssDNA, likely indicating a direct role for oxidised hSSB1 in the recognition of 8-oxo-G lesions. Furthermore, hSSB1 and hOGG1 directly interact with a moderate binding affinity in the presence of 8-oxoG damaged ssDNA. Finally, a model of the tetramer is proposed using the recent crystal structure of monomeric hSSB1 as a template. The data presented here along with the proposed structural model allows hSSB1 to be placed in the oxidative DNA damage response pathway and gives crucial insight into the possible role of the oligomer in this process. As heightened levels of oxidative stress are associated with cancer (as well as aging and Alzheimer's disease), understanding the molecular mechanisms of how cells repair oxidative DNA damage will be essential in the development of novel therapeutic treatments.

Date of Award	2016
Original language	English