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Report

Teaser, summary, work performed and final results

Periodic Reporting for period 2 - REPSUMODDT (Mechanisms and regulators coordinating replication integrity and DNA damage tolerance.)

Teaser

Summary

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, DNA damage response (DDR) and DNA damage tolerance (DDT) pathways become activated and are crucial for detection and tolerance of lesions, for facilitating replication completion, and for supporting chromosome structural integrity. While important functions of these processes have been outlined, much less is known about the choreography and interplay between DDT/DDR and chromosome structural establishment processes. In this project, we are using a palette of genetic, molecular, genomic and proteomic-based experimental strategies to investigate the mechanisms and outcomes associated with replicative and chromosome structural stress, which are frequent in cancers and human diseases. Understanding the mechanism and regulation of these processes has crucial implications for modeling replication stress and is therefore of high medical value.
The work in this project is structured towards three main aims. We are interested in defining the principles that regulate DDT pathway choice and replication fork architecture in response to genotoxic and topological stress; the DNA dynamics at damaged, stalled and converging replication forks; and the relationship between chromosome structure, in particular sister chromatid cohesion and DDT. We also plan to identify or characterize key regulators of these processes, with a focus on kinases (checkpoint, cell cycle, DDK) and SUMO/Ubiquitin-mediated transactions.

Work performed

In Aim 1, we are investigating the mechanism and regulation of recombination-mediated DDT in the context of replication. We identified a SUMO-mediated mechanism that locally enhances recombination by limiting the levels of the Srs2 anti-recombinase at sites of replication stress. This involves the Slx5/8 SUMO-targeted ubiquitin ligase, for which we identified novel substrates using a designed ligase trap, SILAC proteomics, and two-hybrid. One of the substrates is DDK, which interacts with the SUMO protease Ulp2, involved in trimming SUMO chains, and required for replication onset. We found that DDK must be protected by the Ulp2 protease against SUMO-chain buildup that causes its Slx5/8-mediated degradation (Psakhye et al, submitted). Regarding the replication-associated recombination mechanism, we investigated roles of other conserved factors, such as the Chl1 helicase in this process. We uncovered that Chl1 makes use of its helicase activity, in a postreplicative manner, to promote recombination downstream of Rad51-mediated filament formation, and expect to be able to define the interplay between key DNA helicases (Chl1, Srs2, Pif1, Sgs1) in this process. Importantly, we found 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 and the checkpoint clamp 9-1-1 (Abe et al., PNAS, 2018). Importantly, DDX11-mediated repair mechanisms is also required for replication through abasic sites (Abe et al., PNAS, 2018). This function promotes immunoglobulin gene diversity and may contribute to normal development, which is drastically impaired in the absence of DDX11 in mouse and leads to a developmental genetic disorder in humans. In the context of recombination regulation investigated at Aim 1, the Mus81-Mms4 nuclease complex is a key player in G2/M, upon its activation by the Cdk1/Plk1 kinases, but its premature activation leads to deleterious crossover (Szakal and Branzei, EMBO J, 2013). We have uncovered a mechanism by which the activated Mms4-Mus81 is downregulated in mitosis and in G1 to limit error-prone resolution of the emerging replication-associated recombination intermediates (Waizenegger et al, in preparation).

In Aim 2 of the proposal we identified a role for Smc5/6 in replication through natural pausing sites. We have continued the investigations along the proposed lines, identifying a role for the Sgs1-Top3-Rmi1 complex in this process and currently characterizing the interplay between Smc5/6 and Top3 in this process. Moreover, we are investigating the compensatory roles of Smc5/6 and the Mre11-Rad50-Xrs2 complex in cellular proliferation and at stalled replication forks.

In Aim 3, we found that Ctf4/AND-1, coupling Polalpha/Primase with the replicative helicase, is required for template switching in budding yeast (Fumasoni et al., 2015). We recently reported that AND-1 is essential for proliferation in vertebrate cells by preventing nuclease-mediated ssDNA gap accumulation at the replication fork junction, which later results in DSB formation and cellular lethality (Abe, Kawasumi et al., Nat Commun, 2018). Thus, repriming may indirectly serve fork protection, while facilitating postreplicative recombination-mediated bypass, a hypothesis we are currently probing in the lab. In Aim 3, we are also interested in the roles of cohesin-regulated pathways in chromosome structure and fork stability. In this regard, we recently reported a role for ESCO1/2 in limiting the levels of cohesin on chromatin and influencing interphase chromatin structure (Kawasumi, Abe et al, Genes Dev, 2017).

Final results

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.

Aim 1 plans to identify regulators of chromosome replication and DNA damage tolerance (DDT), with a focus on SUMO-regulated DNA transactions. In this context, major achievements beyond the state of the art were to: (1) identify local regulators of DDT by limiting the levels of the Srs2 anti-recombinase at sites of replication stress (preliminary results cited in the grant proposal); (2) identify interplay between DDK and a SUMO protease in facilitating early steps of replication (Psakhye et al, manuscript submitted); (3) characterize roles of a conserved helicase, Chl1/DDX11, in modulating DDT events and promoting replication through abasic sites (Abe et al, PNAS, 2018; and work in progress on Chl1); (4) identification of a Mms4-Mus81 regulatory mechanism operating in mitosis and G1 that limits the activity of the transiently activated Mus81-Mms4 nuclease complex in the next cell cycle, thus limiting error-prone events (Waizenegger et al, in preparation).

Aim 2 aims to identify DNA transactions and key factors mediating the response to different replication stress situations. Here, we identified: (1) a role for Smc5/6 in promoting replication at natural pausing sites in budding yeast (preliminary results cited in the grant proposal); (2) advanced the understanding of the DNA intermediates arising at stalled replication forks and the interplay between Smc5/6 and the STR, MRN complexes (work in progress).

Aim 3 plans at uncovering factors/mechanisms implicated in replication fork architecture and chromosome structure regulation and potential links between these processes. We uncovered a role for Ctf4/AND-1 in protecting stalled replication forks from the action of nucleases (Abe, Kawasumi et al, Nat Commun, 2018, and work in progress). This function prevents the formation of double strand breaks in G2, likely by facilitating replication-associated recombination. This action may be mediated by the recruitment of Pol alpha to stalled replication forks, a hypothesis currently under investigation. Regarding the sister chromatid cohesion part, we identified a role for the cohesin regulator ESCO1/2 on interphase chromatin organization (Kawasumi, Abe et al, Genes Dev, 2017). How this function affects replication fork speed and other replication parameters is currently under investigation.