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The response of the dps-like gene to hydrogen peroxide is already well documented. The detoxifying effects of this gene product after oxidative stress are reported in many organisms, E. coli (Zhao, Ceci et al. 2002), bacteria (Andrews, Robinson et al. 2003), P. furiosus (Ramsay, Wiedenheft et al. 2006), H. salinarum (Reindel, Schmidt
et al. 2006) and S. solfataricus (Wiedenheft, Mosolf et al. 2005).
(Almiron, Link et al. 1992), hence the name DNA protection protein from starved cells (Dps).
S. solfataricus possesses a Dps-like protein encoded by sso2079, it is a 22 kDa protein, and twelve copies of the Dps protein assemble to form a hollow cage, with an inner diameter of ~5 nm and an outer diameter of ~10 nm, see Figure 4.12 (Wiedenheft, Mosolf et al. 2005).
Figure 4.12 3D Image reconstruction of the S. solfataricus Dps-like protein Image taken from Widenheft, Mosolf et al. 2005. Scale Bar 2.5 nm.
The structure of SsoDps is similar to the structure of ferritin proteins (iron storage proteins) and like the ferritin proteins SsoDps also sequesters iron within the interior of the protein cage. However, unlike the ferritins, which use oxygen as an oxidant to mineralise their stored iron (Arosio and Levi 2002), SsoDps uses hydrogen peroxide as an oxidant. In this way SsoDps is able to remove two of the components of the Fenton Reaction, see Figure 4.13, and so reduce the production of extremely damaging hydroxyl radicals (Gauss, Benas et al. 2006).
Figure 4.13 Fenton reaction
The Fenton reaction occurs between hydrogen peroxide and ferrous iron and results in the formation of extremely damaging hydroxyl radicals.
transcription assays performed by Dr Sonia Paytubi, which showed that expression from the dps promoter was strong in vitro (shown in Chapter 5, Figure 5.1). A closer look at the ssodps-like gene showed that it shares a divergent promoter with an nramp
gene. Natural-resistance-associated-macrophage proteins (Nramp) are a family of divalent metal transporters involved in balancing the levels of metals within the cell (Forbes and Gros 2001). Metal ions are necessary cofactors for the activity of enzymes such as superoxide dismutase, which protect against oxidative stress. According to the S. solfataricus P2 complete genome sequencing project website (http://www-archbac.u-psud.fr/projects/sulfolobus/) there is a transposon interrupting the nramp gene. This is likely to adversely effect the expression of this gene and so the function of the protein, which may make the cells more sensitive to hydrogen peroxide damage. To determine whether our lab strain of S. solfataricus P2 contained the transposon, PCR primers for both halves of the gene, and the transposon were designed, see Figure 4.14.
Figure 4.14 Diagram of the nramp gene and its transposon
The arrows represent primers. Primers were designed that would amplify the whole gene, including the transposon (sso2076F and Sso2078R). Primers for the transposon were also designed that could be used with the sso2076 and sso2078 primers to confirm the presence of the transposon.
PCR reactions were set up with the nramp and transposon specific primers; the results of the PCR can be seen in Figure 4.15.
Figure 4.15 Agarose gel showing PCR products of nramp/transposon PCR
The expected product size with primers in Set 1 (sso2076F & sso2078R) if the transposon is present was 2149 base pairs; there was a very faint band at around this size. The expected product size of primers of Set 2 (sso2077F & sso2077R) was 165 base pairs; there is a faint band round that size however there are two larger bands of around 1200 and 1500 base pairs that are significantly brighter. Set 3 (sso2076F & sso2077R) produced multiple bands. The expected product size was 809 base pairs but the brightest of the multiple bands was bigger, around 1200 base pairs. There were no amplification products from Set 4 (sso2077F & sso2078R) the expected product size was 1463 base pairs. In each set a and b are replicates while c is the negative (no DNA) control.
The predicted size of the PCR product from Set 1 (sso2076F and sso2076R), if the transposon is present is 2149 base pairs, and if the transposon is absent is 1045 base pairs. There was a faint band running just above 2000 base pair band indicated by the ladder, suggesting the transposon is present. The primers within the transposon, Set 2 (sso2077F and sso2077F), produced multiple bands. This may be due to the fact that the S. solfataricus genome possesses more than one transposon (Redder, She et al. 2001). PCR reactions with primer set 3 (sso2076F sso2077R) which amplifies the first half of the nramp gene and half of the transposon, showed multiple products none of which appeared to be the predicted size (809 bases). There was no amplification with primer set 4, which amplifies half of the transposon and the second half of the nramp
gene. The sso2077R primer appears to have more than one possible binding site in the genome, as PCR reactions with this primer resulted in multiple products. This could be because part of the transposon is inserted in other regions of the S. solfataricus
4.5 Discussion
After studying the genome wide response to UV damage using microarray technology and RT real time PCR a number of genes that maybe involved in repair were identified. To determine whether the induction of this set of genes wasstandard after all types of damage S. solfataricus was challenged with damaging agents that result in different types of damage, and the expression of the set of genes monitored by RT real time PCR.
The first damaging agent tested was Mitomycin C, which produces cross-links within the DNA, leading to stalling of replication. However, either the high temperature or acidic conditions of the Sulfolobus media appeared to inhibit its effect, or destroy the chemical altogether, as the effect on growth was minimal see Figure 4.1 - 4.3. The expression of sso0280 (tfb-3) and sso0771 (cdc6-2) did increase after the addition of MMC by 2-fold one hour after damage. Although this is a modest increase it still shows some effect of the MMC on these two genes, it is possible that there is a transient effect of the MMC before the Sulfolobus media destroys it.
Although there was little change in growth of the cells after Methyl methane sulfonate addition, there was a small induction of the sso0771 (cdc6-2), sso0280 (tfb-3) and
sso1459 (dpoII) genes. It has been suggested that sso0771 (cdc6-2) may be involved in control of replication and sso0280 (tfb-3) may be involved in controlling transcription (Gotz, Paytubi et al. 2007) if the prediction of these genes functions is correct, both would potentially be involved in the response to numerous kinds of damage.
The addition of Phleomycin had a noticeable effect on growth but strangely a minimal effect on gene expression. There was a small change in the expression of the sso0280 (tfb-3) gene, which increased around 1.8 fold by the 120 minute time point, but the ratio for the rest of the genes stayed around 1, indicating there was little change in expression in the control and treated samples.
Hydrogen peroxide had the most marked effect of any of the damaging agents and growth of the cells was severely affected by concentrations above 5 µM. Of the genes looked at, the expression of five increased more than 2-fold, sso0771 (cdc6-2), sso0446 (tfb-1), sso0959 (xpb-1), sso1459 (dpoII) and sso2079 (dps-like). The expression ratio of the first four genes was between 2 - 3.5, however the increase in expression of sso2079, the ssodps-like gene was huge. At 30 minutes after damage there was a 350-fold increase in expression (see Figure 4.11). Hydrogen peroxide induces oxidative stress, as does UV irradiation (although to a lesser extent), which may explain why many of the same proteins were up regulated after both kinds of damage. Oxidative stress results from an imbalance between ROS and antioxidants in the cells, leading to numerous types of damage, such as DNA breaks, damage to lipids and proteins, as ROS scavenges electrons wherever they find them (Valko, Rhodes et al. 2006). Because of the indiscriminate damage caused by oxidative stress, genes involved in many different pathways could be induced after addition of hydrogen peroxide.
The set of genes as a whole does not appear to be induced as standard for all types of damage. However the results shown here indicate that sso0280 (tfb-3) and sso0771 (cdc6-2) are induced by all of the damaging agents tested. When faced with DNA damage the cells DNA replication and cell division processes are slowed down or stopped to allow repair or take place, and to prevent the transmission of damage to daughter cells. S .solfataricus possess three Cdc6 proteins and it has been predicted that they have a function in control of replication initiation. The Cdc6-2 protein is able to bind the Cdc6-1 and Cdc6-3 binding sites, possibly blocking access of these proteins in order to pause replication (Robinson, Dionne et al. 2004). The increased expression of sso0771 (cdc6-2) in all the damage condition tested lends further weight to this idea.
The difference in the levels of the three S. solfataricus tfb genes could provide some insight as to how this organisms controls transcription after being exposed to damage.
levels of tfb3 increased. Increase in the levels of tfb3 was observed with all damaging agent suggesting this protein is involved in the cells response to numerous types of damage. The TFB3 protein is a truncated version of TFB1 and 2, missing the B-finger and DNA binding helix-turn-helix domain, leaving it unable to bind to promoter DNA and presumably unable to stimulate RNAP as the B-finger is predicted to perform this function. Surprisingly TFB3 has been shown to have a stimulatory effect in vitro
when added to assays containing the basal transcription proteins (Paytubi unpublished). The mechanism of this stimulation is not yet known.
sso1459 DpoII was also induced by methyl methane sulfonate and hydrogen peroxide, but the significance of this is unknown.
The response of the dps-like gene to hydrogen peroxide damage is well documented, but how the expression of this gene is controlled in S. solfataricus is not known. Transcription assays showed that transcription from the gene is strong in vitro (see Chapter 5, Figure 5.1) suggesting a repressor in vivo. Further investigation of the dps
promoter and identification of a possible transcriptional repressor are presented in Chapter 5.