Conclusions and future work

The characterisation of the putative helicases XPB1, XPB2 and Hel308 and the hypothetical protein Sso1468 from S. solfataricus are described in this thesis.

XPB1 and XPB2 were shown to bind preferentially to ssDNA rather than a dsDNA substrate. XPB1 binds DNA with an affinity consistent with other SF2 helicases (Voloshin and Camerini-Otero, 2007), in contrast, XPB2 exhibited a very low DNA binding affinity for DNA. Both XPB1 and XPB2 contain the seven motifs characteristic of SF1 and SF2 helicase, although neither protein was able to displace streptavidin from a biotinylated probe. Furthermore, XPB1 and XPB2 were unable to catalyse DNA strand separation consistent with recently published studies with human XPB (Coin et al., 2007).

It is known that human XPB binds the DNA downstream of the promoter, which positions it too far from the melted DNA to act as a conventional helicase and it is thought to act as an ATP-dependent conformational switch (Kim et al., 2000; Lin et al., 2005). It would be of great interest to pursue this observation further in the archaeal system and investigate the positioning of SsoXPB1 and/or SsoXPB2 at the melted promoter and the possibility that both SsoXPB1 and SsoXPB2 could act as a “ratchet wrench” (Kim et al., 2000) to promote formation of the stable nascent transcription bubble.

Most archaeal genomes possess homologues of repair genes, such as XPB (Grogan, 2000; She et al., 2001). The presence of two xpb genes seemed to be a feature of the crenarchaeal genomes inspected, a feature most likely the consequence of a duplication event. RT PCR and western blot analysis detected more xpb1 mRNA and protein respectively, compared to that of XPB2 in the S. solfataricus cell. This suggested that the two proteins could fulfill separate functions within the cell, although this was by no means conclusive. Closer inspection of the archaeal genomes identified a gene in close association with the xpb2 gene. Conservation of this pair implied that the two gene products might interact physically and functionally, similar to the interaction between human XPB and p52 (Coin et al., 2007). SsoXPB2 and SacBax1 were shown to interact with each other. In contrast to p52, however, SacBax1 was unable to stimulate the activity of SsoXPB2, a result of a possible functional barrier presented by the

investigate whether Bax1 functions to recruit XPB2 in addition to a role in modulating the catalytic activity of XPB2 in archaeal transcription and/or repair. More information is required to understand the importance of the XPB2-Bax1 interaction; it would be invaluable to obtain recombinant XPB2 and Bax1 from the same organism. This would require either the recloning of the S. solfataricus or S. acidocaldarius genes into a range of expression vectors or cloning, expressing and purifying XPB2 and Bax1 from a different species.

In order to gain more of an insight onto the interaction between SsoXPB2 and SacBax1, mass spectrometry can be used to identify the interacting surface. The specific technique is referred to as isotope-coded protein label (ICPL) and is based on differential isotope labelling of specific amino groups on isolated intact proteins with either light or heavy tags (Schmidt et al., 2005). Quantitative analysis of the light and heavy labelled peptides is achieved by direct comparison of the relative signal intensities in the MS- spectra (Schmidt et al., 2005). To date, the results have been inconclusive (Wilkinson, 2007). This work, however, is ongoing and is being carried out by Dr Sally Shirran and Dr Catherine Botting (BMS Mass Spectrometry and proteomics facility, St Andrews University).

In order to confirm a role for XPB1 and XPB2 in NER, it would be interesting to carry out knock out studies in S. solfataricus. By knocking out both xpb genes, xpb1 or

xpb2 and assessing colony survival after UV irradiation an involvement of XPB1 and XPB2 in NER could be identified. Knocking out one gene at a time would focus investigations on a plausible compensatory role of XPB1 for XPB2, or vice versa, which may provide a possible explanation for the presence of two xpb genes in the genome of S. solfataricus and other crenarchaea.

In stark contrast to the results presented for XPB1 and XPB2, the putative helicase Hel308 was able to unwind duplex DNA. Hel308 bound preferentially to ssDNA and was able to displace streptavidin from a biotinylated DNA probe. A number of arginine residues thought to be directly involved in DNA binding, based on the crystal structure, were separately mutated to alanine residues. In each case reduced DNA binding and, therefore, reduced catalytic activity was evident.

Unexpectedly, mutation of the RAR motif in domain 5 of Hel308 or the removal of this entire domain produced a more efficient helicase. Domain 5 was, therefore, identified as an autoinhibitory domain. This was an unexpected result since domain 5 consists of two helix-turn-helix motifs, such motifs are known to be involved in DNA binding (Luscombe et al., 2000). Further research must be undertaken to understand the mechanism by which domain 5 inhibits the activity of the intact protein, its importance, and how it is relieved in vivo. There are a number of possibilities including protein- protein interactions, conformational changes or proteolysis. Identification of interacting protein partners could provide insight into the activation mechanism.

Hel308 is thought to be involved in clearing the lagging strand template at a stalled replication fork, either by displacing the lagging DNA strand or by the displacement of SSB, in order for RadA to load onto the DNA and promote strand exchange (as proposed in figure 5.18). Another possibility is that Hel308 may act to inhibit strand exchange by disruption of the RadA nucleofilament. Further investigations are required to identify the exact role of Hel308 at this stage of replication restart. This work on Hel308 is ongoing and is being carried out by Annie McRobbie (PhD student, Professor Malcolm White group, BMS, St Andrews University).

It will be interesting to identify any inhibitory effects of the DNA binding proteins SSB and Alba and the strand exchange protein RadA on the catalytic activities of Hel308. Since the block imposed by domain 5 must somehow be relieved, the effect of SSB, Alba and RadA on the activity of the truncation mutant could provide some insight into the inhibition release mechanism.

Nothing is known so far about the way in which Hel308 loads onto the DNA. The crystal structure shows that there are no covalent links between domains 2 and 4. This suggests that the open ring structure is flexible to allow Hel308 to load onto DNA that does not have a 3’ end, such as a stalled replication fork. A loop connecting domains 2 and 3 has been proposed to act as the hinge for this movement (Richards et al., 2007).

In order to investigate this movement further, spin labelling studies will be carried out. By incorporating a cysteine residue and subsequently a methanethiosulfonate label into domain 2 and domain 4 the distance between the domains can be measured. A spin label is an organic molecule that possesses an unpaired electron (usually on a nitrogen

which is dictated by the local environment and has profound effects on the Electron Paramagnetic Resonance (EPR) spectrum. EPR is the technique used to study molecules with unpaired electrons. Any conformational change in the ring structure will result in a change in the local environment and to the distance between the two domains and subsequently between the two methanethiosulfonate labels (Gross et al., 1999; Steinhoff, 2002).

This technique will also be used to investigate the conformational changes to Hel308 during ATP hydrolysis and, therefore, spin labels have been incorporated into domains 1 and domains 2. These investigations are being carried out by Annie McRobbie (PhD student, Professor Malcolm White group, BMS, St Andrews University). The hypothetical S. solfataricus protein Sso1468 was thought to be an AAA ATPase, based on a BLASTP alignment search, with a role as a branch migration protein. Initially no ATP hydrolysis could be detected, however, a conformational change upon ATP (and not ATPγS) binding was identified. After an autophosphorylation event was ruled out, it was shown that Sso1468 exhibits a very low turnover of ATP. In contrast to many AAA ATPases, Sso1468 did not form a hexameric complex but was identified as a monomer in the conditions used. Some AAA ATPases do exist as monomers and exhibit low ATPase activity. It is thought that these enzymes undergo transient and concentration dependent oligomerisation under ATPase assay conditions (Babst et al., 1998; Karata et al., 1999). Alternatively Sso1468 may require protein interactions to stimulate its own activity or that of the interacting partner.

It would be interesting to identify proteins that interact with Sso1468. Mutagensis studies of Sso1468 could provide some insight into its function; for example, Sso1468 could possess an autoinhibitory domain similar to that described for Hel308. No branch migration activity was detected with Sso1468, the experiments, however, lacked positive controls. This is another area of interest that would need further investigation.

The research presented in this thesis covers a number of S. solfataricus proteins that function to maintain the genome. The study of XPB1, XPB2, Sso1468 and in particular Hel308 from the archaeal system provides some insights into the complicated environment of the eukaryotic cell.