Analysis of the Ies6 subunit of the INO80 chromatin remodelling complex

Phelps, Sarah (2016) Analysis of the Ies6 subunit of the INO80 chromatin remodelling complex. Doctoral thesis (PhD), University of Sussex.

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Abstract

The INO80 complex is a large ATPase chromatin remodeller which contains 15 accessory subunits in S.cerevisiae. Its subunits include the highly conserved ATPases Ruvb1 and Ruvb2, the actin-related proteins Arp5, Arp8, Act1 and Arp4, Actin, and a number of IES (I̱noE̱ighty S̱pecific) subunits Ies1, Ies2, Ies3, Ies4, Ies5 and Ies6, in addition to subunits Nhp10 and Taf14. All 15 of the accessory subunits are assembled around a catalytic core component known as Ino80.
The INO80 complex has roles in transcription, DNA repair, replication, and chromosome segregation. These roles are in addition to its traditional nucleosome remodelling activities and the dispacement of H2A.Z from chromatin. Recent studies in S. cerevisiae have identified the subunit Ies6 as a critical component of the INO80 complex. Deletion of IES6, which encodes the small accessory subunit, clearly mimics the deletion f the gene encoding the catalytic subunit, INO80. Surprisingly, only one domain within Ies6 has been formally identified based on sequence analysis. This domain belongs to the L1_C class of domains. Such domains are commonly associated with DNA binding activity and transcription factors.
This stud has further characterised the Ies6 subunit both genetically and biochemically. Genetically, it has demonstrated that single point mutations at regions of proposed subunit-subunit interaction between the Arp5 or Rvb2 subunits, or within the YL1_C are not sufficient to disrupt Ies6 function. However, expression of a double point mutation, ies6(K114E/Y125A), in combination with rad50 deletion, caused a sensitivity to replication inhibition, but not chromosome segregation inhibition, indicating a potential separation of function in this utant due to the loss due of only one of the biological functions of Ies6.
Biochemically, we have confirmed that DBA binding capacity of Ies6 resides within the YL_C domain. In addition, although it has been demonstrated that the removal of H2A.Z acetylation exacerbates the increase in cellular ploidy observed in ies6 null cells, we found that overall levels of H2A.Z acetylation were not influenced by the loss of Ies6. This indicates that the role of H2A.Z acetylation in chromosome segregation may only affect ploidy status upon the loss of Ies6.
In addition, work on the R2TP complex (which contains the INO80 APases Ruvb1/Ruvb2, and subunits Tah1 and Phi1) has revealed the recruitment mechanism for the molecular chaperone, Hsp90, and the telomere length regulation protein, Tel2. Together, the R2TP complex, Hsp90 and Tel2 promote the stabilisation and maturation of multi-protein complexes. These include Phosphatidylinositol 3-kinase-related kinases (PIKKs, a family of kinases involved i Serine and Threonine phosphorylation), subunits of the INO80 complex and subunits of the SWR1 chromatin remodelling complex (a partner comlex to INO80 that incorporates H2A.Z into chromatin).

Item Type: Thesis (Doctoral)
Schools and Departments: School of Life Sciences > Biochemistry
Subjects: Q Science > QD Chemistry > QD0241 Organic chemistry > QD0415 Biochemistry
Q Science > QH Natural history > QH0301 Biology > QH0426 Genetics > QH0447 Genes. Alleles. Genome
Depositing User: Library Cataloguing
Date Deposited: 14 Jul 2016 10:42
Last Modified: 14 Jul 2016 10:42
URI: http://sro.sussex.ac.uk/id/eprint/61895

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