Periodic Reporting for period 4 - REPSUMODDT (Mechanisms and regulators coordinating replication integrity and DNA damage tolerance.)
Reporting period: 2021-01-01 to 2021-12-31
This project aims to elucidate the origins of mutagenesis and chromosome structure instability generated in eukaryotic cells facing replication stress. In response to replication perturbations, cells need to activate specialized repair pathways known as DNA damage tolerance (DDT) in order to complete replication at sites of DNA damage. In this process, cells utilize either mutagenic specialized polymerases or copy the information from the undamaged sister strand in a recombination mediated process. The recombination mechanism is associated with the formation of recombination intermediates, which support error-free tolerance of DNA lesions, but they hold sister chromatids together. These structures need to be resolved before chromosome segregation and may influence mitotic chromosome structure. Understanding the mechanism and regulation of DDT and chromosome structural processes has crucial implications for modeling replication stress, which is prevalent in cancers. The project is important for the society, because the knowledge gained has high medical value.
The work in this project is structured towards three main aims. In the first aim, we are investigating the mechanism and regulation of recombination-mediated DNA damage tolerance and the principles that guide DDT pathway choice. In the second aim, we are examining the DNA dynamics at specific types of replication stress situations, including damaged, stalled and converging forks. In the third aim, we are investigating the principles that affect replication fork architecture and chromosome cohesion regulation and potential links between these processes.
The work in this project is structured towards three main aims. In the first aim, we are investigating the mechanism and regulation of recombination-mediated DNA damage tolerance and the principles that guide DDT pathway choice. In the second aim, we are examining the DNA dynamics at specific types of replication stress situations, including damaged, stalled and converging forks. In the third aim, we are investigating the principles that affect replication fork architecture and chromosome cohesion regulation and potential links between these processes.
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).
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).
The project is organized around three main aims. The progress beyond the state of the art and expected results is described below for each of the main Aims.
In Aim 1, major achievements were to: (1) identify local regulators of DDT by limiting the levels of the Srs2 anti-recombinase at sites of replication stress; (2) identify interplay between DDK and a SUMO protease, Ulp2, in facilitating early steps of replication (Psakhye et al, Mol Cell, 2019); (3) characterize roles of a conserved helicase, Chl1/DDX11, in facilitating recombination-mediated DDT (Abe et al, PNAS, 2018, and work in progress); (4) identify a Mms4-Mus81 regulatory mechanism operating in mitosis that limits the activity of the nuclease in the next cell cycle (Waizenegger et al, Nature Commun, 2020).
In Aim 2, we: (1) identified a role for Smc5/6 in promoting replication at natural pausing sites in budding yeast; (2) characterized the DNA intermediates arising at stalled replication forks and the functional interplay between Smc5/6 and STR in replication completion (Agashe et al, Nat Commun, 2021, accepted in principle for publication); (3) identified compensatory roles between Smc5/6 and MRX complexes at terminal forks (Villa et al, work in progress).
In Aim 3, we uncovered a role for Ctf4/AND-1 in protecting stalled replication forks from the action of nucleases (Abe et al, Nat Commun, 2018). Regarding cohesin, we identified a role for the cohesin regulator ESCO1/2 on interphase chromatin organization (Kawasumi et al, Genes Dev, 2017), uncovered synergy between DDX11 and CTF18 replication fork mediators in sister chromatid cohesion (manuscript in preparation) and identified a SUMO-regulated mechanism that regulates the turnover of cohesin and other SMC complexes on chromatin (Psakhye et al, in revision).
In Aim 1, major achievements were to: (1) identify local regulators of DDT by limiting the levels of the Srs2 anti-recombinase at sites of replication stress; (2) identify interplay between DDK and a SUMO protease, Ulp2, in facilitating early steps of replication (Psakhye et al, Mol Cell, 2019); (3) characterize roles of a conserved helicase, Chl1/DDX11, in facilitating recombination-mediated DDT (Abe et al, PNAS, 2018, and work in progress); (4) identify a Mms4-Mus81 regulatory mechanism operating in mitosis that limits the activity of the nuclease in the next cell cycle (Waizenegger et al, Nature Commun, 2020).
In Aim 2, we: (1) identified a role for Smc5/6 in promoting replication at natural pausing sites in budding yeast; (2) characterized the DNA intermediates arising at stalled replication forks and the functional interplay between Smc5/6 and STR in replication completion (Agashe et al, Nat Commun, 2021, accepted in principle for publication); (3) identified compensatory roles between Smc5/6 and MRX complexes at terminal forks (Villa et al, work in progress).
In Aim 3, we uncovered a role for Ctf4/AND-1 in protecting stalled replication forks from the action of nucleases (Abe et al, Nat Commun, 2018). Regarding cohesin, we identified a role for the cohesin regulator ESCO1/2 on interphase chromatin organization (Kawasumi et al, Genes Dev, 2017), uncovered synergy between DDX11 and CTF18 replication fork mediators in sister chromatid cohesion (manuscript in preparation) and identified a SUMO-regulated mechanism that regulates the turnover of cohesin and other SMC complexes on chromatin (Psakhye et al, in revision).