Principales Indicadores de la Postura Fiscal

5.1 Introduction

Cells have to maintain genome integrity. Therefore, the efficient repair of DNA damage from extracellular and intracellular agents is vital. One of the repair mechanisms is NER; this is a highly conserved repair mechanism that removes bulky DNA lesions including UV induced CPDs, (6-4) PPs and other chemical adducts (de Laat et al., 1999; Prakash and Prakash, 2000). Eukaryotic NER requires approximately 30 proteins to remove damage from naked DNA. As mentioned in Chapter I (page 16) the mechanism is divided into two subpathways; TC-NER, repairs the TS o f transcriptionally active genes and GG-NER repairs the NTS of transcriptionally active genes and both strands of transcriptionally inactive genes. The TC-NER subpathway tends to repair damage faster than GG-NER as restoring the ability to transcribe appears to be a priority. This faster repair is probably due to the recognition o f the damage undertaken by the stalled RNA polymerase II at the DNA lesion and other proteins that are shared between transcription and TC-NER (Coin et al., 2008; Li et al., 2006; Prakash and Prakash, 2000).

The core NER mechanism operation on naked DNA was first determined (Aboussekhra et al., 1995; Guzder et al., 1995) and afterwards, the NER efficiency was studied in a chromatin environment (reviewed by Teng et al., 2008; Waters and Smerdon, 2005). In vitro studies, using reconstituted nucleosome as templates showed that nucleosomes exert an inhibitory effect on NER since the packaging of DNA into nucleosomes in living cells and chromatin provides a completely different template from naked DNA. Thus, the overall repair of DNA damage by NER is less efficient in nucleosomes than in naked DNA (Wang et al., 1991).

Interactions between nucleosomes and NER have been long documented, and they reflect the influence of the static and dynamic aspects of nucleosomes on the process (Ferreiro et al., 2004; Livingstone-Zatchej et al., 2003). The chromatin organisation within the location of a particular DNA damage significantly affects the efficiency o f NER. For instance, high resolution mapping of CPD repair by NER revealed a faster repair o f lesions in linker DNA and towards the 5' end of positioned

nucleosomes and a slower repair in the centre or in the “internal protected region“of the nucleosomes. However, this modulation was only found in the NTS of active genes and in both strands of inactive genes in several S. cerevisiae loci (Ferreiro et al., 2004; Li and Smerdon, 2002; Tijsterman et a l, 1999; Wellinger and Thoma, 1997). In other words, repair modulation was found in the strands where the location of the nucleosomes and the chromatin is more stable or compact. Some examples where repair efficiency is regulated by the chromatin are at the URA3 gene (Tijsterman et al., 1999; Wellinger and Thoma, 1997), the GAL1-10 promoter (Allinen, 2002) and the

MET16 (Ferreiro et al., 2004).

On the contrary, for the TS of active genes the repair rate is more homogeneous since the chromatin is more relaxed possibly leading to an easier access o f the NER machinery. This also suggests that chromatin structure is a factor which can modulate the repair efficiency. Another evidence of the chromatin structure influence in NER is the work described by Ferreiro et al. (2004) where they examined the repair of

MET16 to clarify the relationship between chromatin, transcription and repair. In the M ET 16 gene, the Cbflp and the grown media can modulate the chromatin structure,

transcription and repair. For example, the transcription of MET16 is repressed when a

cbflA strain is grown in minimal medium. In this case, the MNase digestion pattern of

chromatin reveals a more compact region, mainly around the TATA box. However, in the wild type strain, the chromatin is more MNase sensitive at the TATA box. This modulation o f the chromatin structure influenced transcription which is induced in minimal medium (derepressing conditions) in the CBF1 wild type cells, but not in the mutant strain. When DNA repair rate was studied at MET16, a general influence of C bflp on the CPD repair rate was found; repair was less efficient in the cbflA compared to the wild type strain. Moreover, under the derepressing and repressing conditions, with both the wild type and mutant strains, the repair rate pattern for

M ET 16 is not homogeneous showing the nucleosome positions in the chromatin at the

NTS. It is worthy to note that in the cbflA strain, M ET 16 under derepressing and repressing conditions is lowly transcribed. On the contrary, in the wild type strain, under derepressing condition MET16 is highly transcribed and under repressing conditions is lowly transcribed. Repair trends may thus vary for genes with different status o f transcription and different chromatin structure.

Chromatin remodellers have been implicated in the modulation o f the chromatin structure and they are linked to permit transcription and repair processes. Evidence of

this is the Radl6p, a protein involved in the damage recognition during yeast GG- NER. This protein can modify the chromatin to allow damage recognition for the repair machinery. Furthermore, it has been proposed that Radl6p might be a chromatin remodeller from the SWI/SNIF family due to the fact that it shares homology with Snf2p, the catalytic subunits of SWI/SNF, and alters the chromatin environment leading to the recognition of the lesion (Bang et al., 1992; Bi et al., 2004; Teng et a l, 2008).

Other examples o f chromatin remodellers which are involved in modulating the chromatin to allow repair by the NER are the subunits Snf5p and Snf6p of the yeast chromatin remodelling complex SWI/SNF. These subunits can be co-purified with Rad4p and Rad23p, which are factors involved in early damage recognition in NER (Gong et al., 2006). A SWI/SNF remodeller complex has been reported to stimulate NER both in vivo and in reconstituted nucleosomes (Gong et al., 2006; Hara and Sancar, 2002), possibly by facilitating access of the Rad4p and Rad23p which triggers the recognition step of NER to the DNA lesion. Swi2p, another component of SWI/SNF complex, is involved in remodelling the chromatin structure after UV irradiation facilitating the DNA accessibility in the chromatin at the MFA2 promoter region (Yu et al., 2005).

Results shown in Chapter III and chapter IV strongly suggest that the chromatin environment is related to the subtelomeric URA3 expression. Thus, at the NRE the DNA is more accessible to MNase and the gene is expressed. On the contrary, at the RE, the DNA is less accessible to MNase and thus the gene is not expressed.

When the SIR2 gene was deleted, the chromatin structure and the expression of the URA3 gene were affected. The MNase sensitivity and the expression of the gene become similar at both, the RE and NRE. The influence of the SIR2 deletion was more dramatic at the RE than at the NRE. These results suggest that SIR2 negatively affects gene transcription and chromatin organisation. In addition, the NER rate may also be affected as a result of these chromatin organisations. In this chapter the repair efficiency o f CPD removal at the sequence level of the URA3 gene is described. Once again, the strains for this study were RE, NRE, NREs/r2zl and REsir2A.

This system hopefully would enable a better understanding of the correlations between chromatin structure and NER since the same sequence can be studied in different chromatin environments.

Chapter I Analysis o f DNA repair efficiency in the RE ami NRE

5.2. Results

5.2.1 Quality of DNA

Yeast cells were UV treated and allowed to recover in complete medium for 0, 1, 2, 3 or 4 repair hours. DNA from each sample was then extracted and the quality of the extracted DNA was analysed in agarose gels. Figure 5.1 shows typical gels to determine the quality of the extracted DNA for the NRE and RE strains. Figure 5.2 shows the typical quality of the extracted DNA for the NREsj>2zJ and REsir2A strains.

Sharp bright bands of DNA are observed in all of the gels (figure 5.1 and figure 5.2) indicating high quality DNA for all of the samples. Therefore, the analysis of CPD repair rate was undertaken with the DNA of samples such as shown below.

10K b

u

Recovering hours after UV

0 1 2 3 4

B U

Recovering hours after UV

0 1 2 3 4

Figure 5.1 Quality o f the extracted DNA from the NRE and RE strains. (U) Represent the sample taken before the UV and (0, 1, 2, 3, 4) represents the samples taken 0, 1, 2, 3, 4, hours after UV (A) shows the DNA from the NRE strains (B) shows the DNA from the RE strains.

hapier I' Analysis o f DNA repair efficiency m the RE and NRE

Recovering hours after UV

U 0 1 2 3 4

Recovering hours after UV

U 0 1 2 3 4

Figure 5.2 Quality o f extracted DNA from NREsir2A and REsir2A strains. (U) Represent the sample taken before the UV and (0, 1, 2, 3, 4) represents the samples taken 0, 1, 2, 3, 4, hours after UV (A) shows the DNA from the NREsir2A strains (B) shows the DNA from the REsir2A strains.

Next, good quality intact DNA was digested with the MseI restriction endonuclease and the digestion was analysed via another agarose gel. As an example, figure 5.3 illustrates digested DNA from the wild type NRE strain with the samples collected before and after UV.

Recovering hours after

Figure 5.3 Genomic DNA digested with Mse I restriction enzyme. A smear indicates a good digestion of the DNA. (U) Represent the sample taken before the UV and (0, 1, 2, 3, 4) represents the samples taken 0, 1, 2, 3, 4, hours after UV.

5.2.2 The NER efficiency in the NRE and RE strains

The NER of CPDs in the URA3 gene at the NRE or the RE was investigated at the sequence level. After the digestion with Mse I, the DNA was treated with a crude extract of M. luteus containing an endonuclease V, an enzyme which cuts where the CPDs are produced in the DNA. The fragments were labeled and ran on a polyacrylamide gel (described by Teng et al., 1997). Figure 5.4 shows DNA sequencing gels from the subtelomeric URA3 Mse I restriction fragment for the TS and NTS at both strains. Each band represents a specific position in the DNA where CPDs occur. The intensity of the bands indicates the extent of damage at particular positions

within the sequence. The non-radiated sample (U) provided a unique top strong band indicating the full-length fragment. UV-irradiated samples were taken either immediately after 150 J/m2 of UV (0 repair hour) or at various times after UV (1, 2, 3, 4 repair hours) to determine repair efficiency. These samples resulted in multiple fragments on the sequencing gel. Full size fragments indicate molecules where no damage was induced. The size of these fragments is smaller than the intact fragment and it represent the location of the DNA damage. Positions are determined by the Sanger sequence represented in the gel. The decrease in intensity of a particular band with increasing repair time indicates that the damage is repaired. The gels were quantified as described in Chapter II (page 54). CPD repair rates at the Mse I fragment are summarized in figure 5.5. This shows the average time (in hours) needed to repair 50% o f the initial amount of the lesion (at 0 hours) (T50%) o f three independent experiments at each CPD site.

When the differences in repair of this sequence at the RE and NRE are compared, it is clear that NER is much faster when URA3 is at the NRE for both the NTS (P<0.0001, Mann Whitney test) and the TS (PO.OOOl, Mann Whitney test). The differences in the repair between the TS and the NTS o f URA3 at the RE with the average Tso% for the entire fragment are smaller than at the NRE being 7.77 hrs and 8.18 hrs respectively. However, the repair is faster for the TS compared to NTS when

URA3 is at the NRE, with average T5o% of 2.85 hrs and 4.03 hrs respectively. This

faster repair o f CPDs in the TS as opposed to the NTS suggests that a TCNER subpathway is involved in the TS repair and the GGNER is involved in the NTS repair.

To conclude, the repair results obtained here correlate well with the MNase sensitivity pattern previously described. When URA3 is at the RE the chromatin is less accessible to MNase, and the repair of CPDs is slower. For instance, in the TS, the repair at the RE is markedly slower than at the NRE (P<0.0001, Mann Whitney test). One o f the maximum differences for individual repair o f CPDs between both strains (NRE and RE) in the TS was obtained around +561bp, +554/TTTCTCT/+562 (3-fold difference) and one of the minimum differences were found around +478bp, +477/TC/+480 (2-fold difference). At both repair locations, the repair data correlates with the MNase sensitivity. In the RE strain both repair points are a MNase protected region yet are non MNase protected at the NRE.

Figure 5.5 illustrates the differences in DNA repair efficiency of the URA3 sequence between the NRE and RE at the TS and NTS.

In the NTS repair was also slower at the RE than at the NRE (PO.OOOl, figure 5.5). One of the maximum differences in repair efficiency o f individual CPDs in the NTS between these SIR2 wild type strains was obtained around +654 bp, +655/CC/+658 (3.47-fold difference) and the minimal differences were around +407 bp, +395/TTT/+399 (1.14-fold difference). Here, once again both individual repair rates correlate with MNase sensitivity where chromatin was protected from MNase at the RE and sensitive at the NRE (figure 5.5).

Chapter i. Analysis oj i)A f repair efficiency in die k I: and ' A7 .