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Two components of the SMC5/6 complex have E3 ligase roles; NSMCE1 as an E3 ubiquitin ligase and NSMCE2 as an E3 SUMO ligase. These catalytic roles were initially proposed following sequence analysis of these subunits by (McDonald et al., 2003), where NSMCE1 featured a RING finger motif, suggesting its role as a possible E3 factor in ubiquitinylation and NSMCE2 featured a zinc finger similar to the DNA binding protein Miz1, indicating its role in SUMOylation. The ubiquitin ligase function of NSMCE1 was then confirmed in vitro, through the study of MAGE proteins such as MAGEG1 (Nse3 in yeast), where it was demonstrated that whilst NSMCE1 has its own weak ligase activity, NSMCE3 promotes and enhances this activity as part of the SMC5/6 complex (Doyle et al., 2010). The SUMOylation function of Nse2 was first confirmed in S. pombe, before this activity was then replicated in human cells (Andrews et al., 2005; Potts and Yu, 2005).

The roles of these ligase subunits have been studied intensely over the past 20 years, with their targets and functions still largely unidentified. However, quickly after the proteins discovery it was found that the Nse2/NSMCE2 SUMO ligase, in both yeast and human cells, had an important function in DNA damage repair and in the avoidance of DNA damage during normal mitotic cell division (Potts and Yu, 2005; Andrews et al., 2005). This led to the suggestion the NSMCE2 SUMO ligase may play a wide role in chromosome maintenance and RS recovery. Subsequently much of the research has focused upon this area.

In support of this suggestion, mutant Mms21/Nse2 S. cerevisiae cells were shown to spontaneously incur DNA damage as well as being very sensitive to RS, which resulted

in many cell cycle progression defects and chromosome breakage, possibly due to the non-functioning Nse2 SUMO ligase (Rai et al., 2011). Furthermore, several other yeast studies have also demonstrated a role for the Nse2 SUMO ligase in RS recovery, resolving recombination intermediates such as cruciform structures, produced following collapsed replication forks (Chavez et al., 2010; Branzei et al., 2006). As well as studies using yeast, there has also been support for the NSMCE2 DNA repair function in DT40 chicken cells, where the SUMO ligase ability was shown to be vital for DNA repair as well as recovery from RS following exogenous damage, with a strong suggestion for a role within HR (Kliszczak et al., 2012).

Primordial dwarfism, has now been determined to be the result of many DNA damage response/repair- associated genetic defects (Alcantara and O'Driscoll, 2014). Human patients with NSMCE2 mutations, have been described which present with primordial dwarfism, along with other defects such as gonadal failure and insulin resistance, indicating that the SUMO ligase function of NSMCE2 may be vital for DNA damage repair on a whole mammalian system level. The cells from these patients displayed increased numbers of micronuclei and nucleoplasmic bridges as well as reduced recovery from RS during DNA synthesis. These abnormalities could be reversed through the addition of wildtype NSMCE2 but not through the addition of ligase-dead NSMCE2, supporting the previous yeast studies that it is specifically the ligase function of NSMCE2 which carries out these RS recovery and repair roles. The same research group have also found that NSMCE2 knockdowns in Zebrafish produce the same dwarfism phenotype seen within the human patients. This again could be reversed through re-expression of wildtype NSMCE2 but not the ligase-dead form, further indicating that it may be the reduced tolerance to RS due to the removal of the SUMO ligase which causes the dwarfism (Payne et al., 2014).

Although there is now fairly strong evidence in support of a genome stability role for the NSMCE2 SUMO ligase, to date, only a handful of NSMCE2 SUMOylation targets have been identified. Initial studies in S. pombe revealed that Nse2 SUMOylates both itself, SMC6 and Nse3, whilst in vitro assays using human NSMCE2 have similarly confirmed NSMCE2 autoSUMOylation and modification of SMC6 (Andrews et al.,

2005) (Potts and Yu, 2005). It has been reported that SMC5 may also be SUMOylated, in both yeast and in human cells, but it is not yet clear whether this modification is solely NSMCE2 dependent (Bustard et al., 2012; Zhao and Blobel, 2005).

As well as components of the SMC5/6 complex itself, other reported targets of NSMCE2 SUMOylation include TRAX, the Sgs-1 -Top3-Rmi1 complex, KU70, Scc1, TRF1 and TRF2. These targets often support the case that the NSMCE2 SUMO ligase function lies within chromosome maintenance and RS repair. For example, the Sgs-1-Top3- Rmi1 (STR) complex is employed by the cell to remove and repair RS intermediates, and its ability to do so is provided by SUMOylation by NSMCE2. Upon SUMOylation, the STR complex accumulates at repair sites and is able to form inter-subunit interactions, promoting its activity, aiding RS tolerance (Bonner et al., 2016). The SMC5/6 complex has also been shown to SUMOylate Scc1, a human subunit of cohesin. The SUMOylation of Scc1 by NSMCE2 causes antagonisation of Wap1, allowing HR to be carried out within human cells, removing damage and providing protection from RS (Wu et al., 2012). As well as this, NSMCE2 is also known to SUMOylate the telomere binding proteins, TRF1 and TRF2. These proteins allow telomere elongation through HR to occur in a process named alternative lengthening of telomeres, often used by malignant cells (Potts and Yu, 2007).

As the range of NSMCE2 targets has only just beginning to be revealed, it is possible that nearly all of the genome stability and repair functions of the SMC5/6 may be explained through the NSMCE2 SUMO ligase ability. It is possible that the complex may act as a caretaker for the whole system, overseeing and directing the process to ensure genome stability throughout replication.