Dixon, Lily (2018) Investigating the role of NSMCE2 in cell cycle progression and genome maintenance. Masters thesis, UNSPECIFIED.
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Abstract
Preserving genome integrity is vital for protection against the development and progression of cancer. The SMC5/6 complex is an evolutionarily conserved protein complex thought to be involved in this process, specifically its SUMO ligase NSMCE2 subunit. Prior to this thesis, CRISPR-Cas-9 mutant NSMCE2 cell lines were produced; NSMCE2 knockout, NSMCE2 knockout with wildtype NSMCE2 re-expression and NSMCE2 knockout cells with ligase-dead NSMCE2 expression. The purpose of this research was to characterize these cell lines with the aim of suggesting possible roles for NSMCE2. Both the NSMCE2 knockout and NSMCE2 ligase-dead cell lines demonstrated proliferation defects and slowed S phase progression by FACS analysis, with the defect exacerbated in the ligase-dead cells. The remainder of this project investigated the cause of these defects, determining that it is not due to the activation of the replication checkpoint, although the ligase-dead cell lines did activate the S phase DNA damage checkpoint. Based on what is already known about NSMCE2 function, it is suggested that the most likely cause of the proliferation defects is un-sensed, unresolved replication stress characteristics. Whilst other proteins may compensate for the total loss of NSMCE2, the ligase-dead cell line is expected to be the result of a dominant-negative mutation, which may explain the more pronounced S phase defect. The NSMCE2 ligase-dead cell lines also demonstrate a >4N DNA content, so it is speculated that unresolved replication stress characteristics may also cause segregation difficulties in this cell line. However, immunofluorescence and decatenation inhibitor treatment has shown that NSMCE2 may instead or also be involved in activating the decatenation checkpoint to allow for efficient segregation. A bioSUMO method for isolation of specific SUMOylation targets has also been developed with the aim to analyse specific NSMCE2 modifications within the future.