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fbtwitterlinkedinvimeoflicker grey 14rssslideshare1
Fenwick, Georgina (2010)
Languages: English
Types: Doctoral thesis
Subjects: QD
The S.cerevisiae Ino80 chromatin remodelling complex is known to be involved in the DNA damage response at double strand breaks and has more recently also been shown to play a role at stalled replication forks. The many functions of this remodellor are likely to be mediated by different subunits of the complex. Interestingly, strains harbouring a deletion of the IES6 subunit are hypersensitive to hydroxyurea and fail to stabilise stalled replication forks, phenocopying strains lacking the catalytic subunit, INO80, indicating a role for Ies6 within the complex’s response to DNA damage. Although largely uncharacterised, Ies6 contains a YL1 domain, which is a putative DNA binding domain. In vitro DNA binding gel shift assays with recombinant Ies6 showed that this protein does possess DNA binding activity. Recombinant Ies6 bound both Holliday Junction and Y-fork DNA, as well as linear duplex DNA, displaying a small but reproducible preference for the two branch-structured DNAs. Recombinant Ies6 containing mutations in the protein’s C-terminal YL1 domain were generated and a quadruple mutant, ies6-T119A/K122A/S127A/T129A, exhibited significantly reduced DNA binding activity compared to the wild-type protein. The importance of the DNA binding activity was investigated in vivo, and, in contrast to the wild-type strain, the DNA binding mutant of IES6 failed to complement the deletion strain’s HU-hypersensitivity. Interestingly, overexpression of Top3 or Cdk1, but not Top2 or Clb2 also rescued the ies6 deletion strain’s HU-hypersensitivity to near wild-type levels. Further investigation revealed that Ies6 is also required for the maintenance of correct cellular ploidy, as in the absence of IES6, cellular ploidy is seen to increase prior to a drift towards unregulated ploidy and aneuploidy, which are hallmarks of cancer. Notably, the protein’s ability to bind DNA correlated with its ability to maintain cellular ploidy. We therefore propose that the Ino80 chromatin remodelling complex Ies6 subunit plays an important role in the maintenance of genomic stability.
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    • 2.1.1 Cloning strategy for the generation of plasmids containing ies6 point mutants by site-directed mutagenesis
    • 2.1.2 Holliday Junction, Y-fork and duplex DNA structures 3.1 Absence of Ies6 renders cells hypersensitive to hydroxyurea compared to absence of other Ies subunits
    • 3.2 The HU-hypersensitivity in the absence of Ies6 is comparable to the HU-hypersensitivity in the absence of Ino80
    • 3.3 Absence of Ies6 and absence of Arp8 lead to a similar HU-hypersensitivity phenotype
    • 3.4 In the absence of Ies6 replication forks collapse following acute exposure to HU 4.1 Purification of recombinant Ies6
    • 4.2 Preparation of in vitro DNA binding substrates to investigate the putative DNA binding activity of Ies6
    • 4.3 Recombinant Ies6 binds linear duplex DNA in vitro 4.4 Quantification of the recombinant Ies6 protein's DNA binding activity to linear duplex DNA in vitro
    • 4.5 Recombinant Ies6 is capable of binding Holliday Junction DNA in vitro 4.6 Quantification of the recombinant Ies6 protein's DNA binding activity to Holliday Junction DNA in vitro
    • 4.7 Recombinant Ies6 is able to bind Y-fork DNA in vitro xi
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  • Discovered through pilot similarity algorithms. Send us your feedback.

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