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4.1 Introduction

As discussed in the introduction to this thesis, the cohesin complex has a functional role in the repair of cellular DNA damage. A number of cohesin establishment factors have been shown to function in DNA damage repair pathway. It is believed that cohesin is recruited to sites of DNA damage to aid in the process of HR and stabilise the regions of DNA around the break. It is thought that cohesin establishment factors are actively recruit cohesin to these sites. ChlR1 is a cohesin establishment factor and the yeast homologue Chl1p is implicated in the cellular DNA damage repair response. Therefore, I hypothesise that ChlR1 functions in the recruitment and establishment of cohesin at sites of DNA damage.

Chl1p was initially discovered to function in the repair of DNA damage in S.cerevisiae. CHL1deleted cells were shown to be more sensitive to the DNA damage reagents methyl methanesulfonate (MMS), hydroxyurea (HU) and UV radiation [304, 305]. This led to the hypothesis that Chl1p is involved in the DNA damage sensing checkpoint or directly in the repair of DNA lesions. However CHL1 null yeast have been shown to have functional DNA damage checkpoint activation as Rad53 was phosphorylated when DNA damage was induced, although this yeast strain did show signs of DNA repair defects [304]. Similar effects were seen inCTF4nullS.cerevisiae[304]. Chl1p and Ctf4 were shown to associate with damaged chromatin during G2/M phase even though sister chromatid cohesion is

established in S phase [304]. NHEJ was shown not to be defective CHL1 and CTF4 deleted yeast but these cells had a defective HR pathway. Furthermore the frequency of unequal sister chromatid recombination in wild type cells increased with increasing doses of MMS. Conversely, this was not observed in the CHL1 and CTF4 deleted cells

suggesting that Chl1p and Ctf4 are involved in the recombination between sister chromatids [304].

An interaction between ChlR1 and TopBP1 has been shown through co-

immunoprecipitation experiments (Parish and Feeney, unpublished). TopBP1, as discussed in the introduction, is involved in the repair of double strand breaks that occur during DNA replication [70]. An interaction between ChlR1 and DNA-PKcs has also been isolated (Parish and Feeney, unpublished). This protein interaction was isolated using tandem affinity purification (TAP) and confirmed by co-immunoprecipitation experiments in hTERT-RPE1 cells. The interaction between ChlR1 and DNA-PKcs increased 6-fold in response to DNA damage caused by ionising radiation. DNA-PKcs as discussed in the introduction is involved in the NHEJ repair process and recruits components involved in the process through phosphorylation. Recently ChlR1 was shown to be phosphorylated during G1/S phase at a serine residue at amino acid 204 [306].

A number of other helicases related to ChlR1 have a functional role in the repair of DNA damage. The XPB and XPD helicases are members of the XPD family of helicases of which ChlR1 is closely related. This family of helicases contain iron sulphur cluster domains with XPD having a 5’-3’ directionality and XPB the opposite polarity [222]. These helicases are part of the transcriptional factor IIH complex (TFIIH) that functions in transcriptional initiation and nucleotide excision repair [222]. This process removes bulky adducts from DNA such as cisplatin lesions and photoproducts generated from UV light. The TFIIH complex opens the DNA around the promoter or damage site. It is believed that the helicase activity of XPB and XPD is important for this function. Mutations in either helicase results in the DNA repair syndrome xeroderma pigmentosum (XP) [307].

The FANCJ helicase that has been associated with the genetic disorder Fanconi anemia is also highly related to ChlR1. This helicase also has an iron sulphur cluster and is a member

of the DEAH family [308]. It was initially shown to have a role in DNA damage repair when it was shown to interact with BRCA1 [309]. FANCJ unwinds homologous recombination intermediates, which suggests it is involved in the HR pathway of double strand break repair [310]. Indeed FANCJ knockout cells have HR defect and are sensitive to ionising radiation [310]. Interestingly FANCJ has been shown to play a role in the response to replication stress. Depletion of FANCJ in Xenopus oocytes resulted in replication fork restart defects following treatment with camptothecin that causes DSB during DNA replication [311].

4.2 Hypotheses and Aims Hypothesis

ChlR1 is involved in DNA damage repair by recruiting cohesin to sites of DNA double strand breaks.

Aims

To confirm this hypothesis I aimed to

1) Confirm ChlR1 has a role in the repair of DNA damage using the DNA comet assay.

2) Confirm ChlR1 localises at sites of DNA double strand breaks by immunofluorescence.

3) Assay the effect of ChlR1 depletion on the recruitment of cohesin to DNA double strand breaks by ChIP assay.

4.3 The knockdown of ChlR1 Results in Inefficient Repair of DNA Damage

The evidence above suggests that ChlR1 may have a role in DNA damage repair. To confirm this hypothesis, cells were depleted of ChlR1 protein by RNA interference and the repair of DNA damage following ionising radiation assessed. The technique used to quantify the DNA damage in cells with ChlR1 knocked down was the comet assay. In this assay HeLa cells were transfected with either ChlR1 siRNA or scrambled siRNA as a non- specific control. Cells were then damaged with an appropriate dose of gamma radiation and allowed to repair for set time points before analysis. HeLa cells were used in this assay because I could efficiently knockdown ChlR1 in this cell line.

Initially, efficient knockdown of ChlR1 using RNA interference was optimised. A number of different siRNA oligonucleotides were tested, each of which targeted different regions of the ChlR1 mRNA. The first siRNA tested was ChlR1 212, which targets the ChlR1 open reading frame at nucleotide 212. The analysis of the knockdown is shown in figure 1. Efficient knockdown of ChlR1 protein using siRNA ChlR1 212 was achieved when the siRNA was transfected into cells at a concentration of 100nM.

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