Autoensamblaje del BCP1-Triazol PDMS-b-PMMA-Triazol y BCP2-Triazol PDMS-b-PS-Triazol

The S. cerevisiae subtelomeric region is one of the best characterised. They comprise -2 5 kb and contain two elements: The Y ’ element and the conserved core X element.

The Y ’ elements are not found at all chromosome ends, however, up to four of them can be found in a single subtelomeric region. They are organised in tandem arrays and separated by short regions of telomeric repeats G2-3(TG) i_6.

The other major component o f the subtelomeric region is the Core X element. This element is found at all chromosome ends. It is 473 bp in length and is found proximal to the Y ’ elements. The Core X element has binding sites for the yeast origin recognition complex (ORC) and the Abfl transcription factor. Therefore, this element could have functional roles at telomeres, probably facilitating the silencing at chromosome ends (Pryde and Louis, 1999). Figure 1.14 illustrates the structure of yeast telomeric and subtelomeric regions.

X A ________ Core 1 X telomere X x _ > X Core

ZZZ

Figure 1.14 Examples o f some telomeric structures in S. cerevisiae. In budding yeast, subtelomeres contained X (a) or X and Y’ (b) elements. Protosilencers involved in the induction of the repression as well as anti-silencing regions are found in these elements. TG are the telomeric repeats TGj^.

1.5.3.3 Silencing at telomeres

Silenced chromatin at telomeres is formed when Raplp, with the help of yku80p and yku70p, recruits the SIR complex. This protein complex consists of an enzymatic component (Sir2p) and a structural component (Sir3p and Sir4p). Sir3p and Sir4p are recruited by Raplp to the telomeres. The interaction between Raplp-Sir3p and Raplp-Sir4p are independent of each other and the loss of these interactions can abolish transcriptional silencing. Telomere silencing can spread due to the binding affinity of Sir3p and Sir4p to the hypoacetylated form of H3 and H4 N-terminal tails at lysine residues, mainly at H4K16. This site is deacetylated by Sir2p creating high affinity binding sites for structural proteins Sir3p and Sir4p (Perrod and Gasser, 2003). Therefore, the activity of Sir2p is extremely important for the creation of silenced chromatin structure at telomeres (Chen and Widom, 2005).

Telomere silencing can be propagated from the end of the chromosome towards the centromere, a phenomenon called telomere position effect (TPE). The silencing or TPE at chromosome ends can be measured by the insertion of a reporter gene such as

URA3 at the subtelomeres (Sandell and Zakian, 1992). In the silenced chromatin

structure close to the telomere, the presence of RNA polymerase II, TFIIE and TFIIB at the promoters is reduced (Chen and Widom, 2005).

In truncated ends, as the distance from telomeric TG repeats increases, the TPE decreases. However, at native ends there are protosilencers in the subtelomere where the repression is greater. For example, the silencing is maximal at X-ACS region which is located at the Core X element. However, immediately internal to the

Chapter I. general introduction

telomeric sequence and in Y’ elements there is no repression (Pryde and Louis, 1999). Furthermore, the protein binding sites at subtelomeric region (mainly ORC but also Abflp and Raplp) are involved in the recruitment of the silencing complex.

The binding sites for Raplp are approximately every 35 bp in the whole telomere, however, if Raplp is removed TPE is alleviated (de Bruin et al., 2000). Chromatin in telomeres is more compacted when is not replicated facilitating the silencing (Sandell et al., 1994). Another factor that can impinge on transcriptional silencing is the temperature, at high temperature transcriptional silencing is enhanced and it is weakened by low temperature (Aylon and Kupiec, 2004).

Proteins involved in the SIR complex are very important for the telomeric silencing. For instance, there is an increase in silencing in SIR3 mutants when the

SIR4 gene dose is increased but there is loss of telomeric silencing when SIR4 and YKU80 genes are mutated (Benbow and Dubois, 2008). Mutations at natural

telomeres in SIR2 and SIR4 genes eliminate the silencing completely. Another protein involved in telomeric silencing is Hdflp, also called Yku70p. It has been reported that its deletion reduces the silencing at native telomeres (Pryde and Louis, 1999).Figure 1.15 illustrates some proteins involved in telomeric silencing

Telom eric DNA

Sir4p

histone

N -term inal tails

Figure 1.15 Silencing at telomeres. Raplp binds to the telomere and recruits Sir3p and Sir4p which can interact with each other. The telomere folds back and silencing is propagated throughout the subtelomeric region towards the centromere.

1.7 Aims of the project

The aim of my project was to study how chromatin structure and silencing at chromosome ends can affect NER efficiency. To eliminate any variation due to DNA sequence, yeast strains where the URA3 gene was moved to the subtelomeric region at different chromosome ends were employed. The URA3 gene was inserted where the

promoter is approximately 1.75 Kb centromere proximal to the TG1-3 telomeric

' __________________Chapter !, general introduction

at the subtelomere of the IIIR chromosome end and at the subtelomere of the XIL chromosomes end in a different strain.

In both strains URA3 was inserted at the same position in the subtelomere. However, the difference between these two locations is the silencing level. When

URA3 is inserted at the XIL chromosome end, the silencing is higher than at the IIIR

chromosome end (Pryde and Louis, 1999).

To investigate whether the disruption of silencing has a different effect in both strains, the SIR2 gene was deleted and the chromatin structure together with NER efficiency were studied.

All the studies were carried out by examining events in the coding region of

URA3. Figure 1.16 shows the different strains utilized in this thesis. The insertions of

the URA3 gene at the subtelomeres were performed previously (Pryde and Louis, 1999). A \ 116970 116167 URA 3 B) C) T G V Wild type IIIR CoreX STR ACS Abfl UR A3 T G ,,

I

Figure 1.16 Structure o f the chromosome ends with URA3 insertion. For this work three strains were employed. The wild type strain where URA3 gene is located at its natural location (chromosome V), a strain where URA3 is located at the IIIR chromosome end and a strain where URA3 was inserted at the XIL chromosome end.

STR - several subtelomeric repeats. In Chapter III (page 6 8) the strain where URA3 is

inserted in the IIIR was termend non-repressive end (NRE) and the strainwhere URA 3 is inserted in the XIL was termed the repressive end (RE).

Chapter II