In Aim 1, we are investigating the mechanism and regulation of recombination-mediated DDT in the context of replication. In previous work, we identified a SUMO-mediated mechanism involving the Slx5/8 SUMO-targeted ubiquitin ligase that locally enhances recombination at sites of replication stress. Here, we identified novel Slx5/8 substrates using a designed ligase trap and SILAC proteomics. The identified substrates include DDK, which is required for replication onset. We found that DDK must be protected by the Ulp2 protease involved in trimming SUMO chains against SUMO-chain buildup that causes its Slx5/8-mediated degradation (Psakhye et al, Mol Cell, 2019). Regarding recombination-mediated DDT, we uncovered roles of the Chl1 helicase in promoting postreplicative gap-filling via recombination (Reyes, Szakal et al, manuscript in preparation) and evidence for postreplicative DNA damage tolerance as a main pathway of bulky lesion repair also in vertebrate cells, in a manner dependent on the action of Chl1/DDX11 helicase (Abe et al., PNAS, 2018; Jegadesan and Branzei, manuscript in revision). The intermediates formed during DDT are largely processed by the Sgs1-Top3-Rmi1 complex, while persistent recombination structures are being resolved in G2/M, by the Mus81-Mms4 nuclease complex upon its activation by mitotic kinases. Here, we uncovered a mechanism by which the activated Mms4-Mus81 is downregulated in mitosis and in G1, via coordinated action between the Cul8 ubiquitin ligase, Esc2 and Slx5/8 SUMO-targeted ubiquitin ligase to limit error-prone resolution of replication-associated DNA structures (Waizenegger et al, Nat Commun, 2020).
In Aim 2, we are investigating the DNA dynamics at damaged, stalled and converging forks, with a particular focus on Smc5/6, involved in all these contexts. Here, we identified that Smc5/6 colocalizes with the Sgs1-Top3-Rmi1 (STR) complex at difficult to replicate genomic regions, known as natural pause sites, where the two complexes prevent accumulation of joint molecules to facilitate replication termination (Agashe et al, Nat Commun, 2021, accepted in principle for publication). Moreover, we uncovered compensatory roles of Smc5/6 and the Mre11-Rad50-Xrs2 complex at terminal forks (Villa et al, manuscript in preparation) and how DNA transitions at stalled fork are being modulated (Joseph, Reyes, Szakal, in progress).
In Aim 3, we are investigating potential relationship between replication fork architecture and sister chromatid cohesion. We uncovered that Ctf4/AND-1 is essential for proliferation in vertebrate cells by preventing fork resection (Abe et al, Nat Commun, 2018). We are testing whether Ctf4-mediated repriming may serve fork protection, while facilitating postreplicative recombination-mediated bypass (Dolce et al, ongoing). In regard to cohesin regulation, we reported roles for ESCO1/2 cohesin acetyltransferases in limiting the levels of cohesin on chromatin and influencing interphase chromatin structure (Kawasumi et al, Genes Dev, 2017), uncovered synergistic roles of Chl1/DDX11 and Ctf18 replication fork mediators in promoting sister chromatid cohesion (Kawasumi et al, in preparation), and identified a SUMO-regulated mechanism that regulates the turnover of cohesin on chromatin (Psakhye et al, in revision).
