Psy2/Pph3
Cells have evolved intricate and specialized responses to DNA damage, central to which are the DNA damage checkpoints that arrest cell cycle progression and facilitate the repair process. Activation of these damage checkpoints relies heavily on the activity of Ser/Thr kinases, such as Chk1 and Chk2 (S. cerevisiae Rad53), which are themselves activated by phosphorylation. Only more recently have we begun to understand how cells disengage the checkpoints to reenter the cell cycle. Activation of the checkpoint kinase Rad53 is a critical response to DNA damage that results in stabilization of stalled replication forks, inhibition of late-origin initiation, up-regulation of dNTP levels, and delayed entry to mitosis. Activation of Rad53 is well understood and involves phosphorylation by the protein kinases Mec1 and Tel1 as well as in trans autophosphorylation by Rad53 itself. However, deactivation of Rad53, which must occur to allow the cell to recover from checkpoint arrest, is not well understood. We have used genetic and biochemical methods to show that the type 2A-like protein phosphatase Pph3 forms a complex with Psy2 (Pph3–Psy2) that binds and dephosphorylates activated Rad53 during treatment with, and recovery from, methylmethane sulfonate- mediated DNA damage. In the absence of Pph3–Psy2, Rad53 dephosphorylation and the resumption of DNA synthesis are delayed during recovery from DNA damage. This delay in DNA synthesis reflects a failure to restart stalled replication forks, whereas, remarkably, genome replication is eventually completed by initiating late origins of replication despite the presence of hyperphosphorylated Rad53. These findings suggest that Rad53 regulates replication fork restart and initiation of late firing origins independently and that regulation of these processes is mediated by specific Rad53 phosphatases. We are currently further characterizing the different pathways that lead to different types of Rad53 deactivation, as well as their biological consequences. Future work will expand these studies to other kinases and phosphatases, as well as to other organisms.
Rnr4/Polδ
Under normal conditions the replicative polymerases, Polδ and Polε, synthesize DNA with remarkably high fidelity using deoxyribonucleotide phosphates provided by the enzyme ribonucleotide reductase (RNR). Rnr4 is one of the two, small, non-catalytic subunits of RNR in yeast, and we have found that it is induced by DNA damage and acts to stimulate dNTP production in a manner that facilitates translesion synthesis (TLS). However, the TLS facilitated by Rnr4 is not mediated by any of the conventional TLS polymerases (i.e. Polζ), but rather appears to be selectively mediated by Pol&delta and not by Polε. This pathway of induced mutation is distinct from the conventional pathways (i.e. post-replication repair (PRR) and conventional TLS polymerases such as Polζ). The potential importance of the Rnr4/Polδ pathway is illustrated by the fact that it not only contributes more to induced mutation by UV or the mutagen MMS than the PRR/TLS pathway, but also because it is essential for mutation induced by the mutagen EMS. EMS-induced mutation has long been enigmatic as it is not affected by deletion of the different components of the PRR/TLS system.
As an example of our long term interests, we have shown that low concentrations of HU, a potent inhibitor of RNR, potently inhibits DNA damage-induced mutation in S. cerevisiae (see below). Current efforts are focused on more fully characterizing the Rnr4/Polδ pathway and examining its potential contribution to induced mutation in human cells.
Esc4
When replication forks stall during DNA synthesis, cells respond by assembling multi-protein complexes to control the various pathways that stabilize the replication machinery, repair the replication fork, and facilitate the reinitiation of processive DNA synthesis. Increasing evidence suggests that cells have evolved scaffolding proteins to orchestrate and control the assembly of these repair complexes, typified in mammalian cells by several BRCT-motif containing proteins, such as Brca1, Xrcc1, and 53BP1. In S. cerevisiae, Esc4 contains six such BRCT domains and is required for the most efficient response to a variety of agents that damage DNA. We have shown that Esc4 interacts with a variety of proteins involved in the repair and processing of stalled or collapsed replication forks. However, the function of Esc4 does not appear to be restricted to a Rad55-dependent process, as we observed an increase in sensitivity to the DNA alkylating agent methane methylsulfonate (MMS) in a esc4Δ rad55Δ mutant, as well as double mutants of esc4Δ and other recombination genes, compared to the corresponding single mutants.
We also found that Esc4 forms multiple, S-phase specific nuclear foci in response to treatment with MMS or HU. Similar behavior is also observed in the absence of damage when either of the S-phase checkpoint proteins, Tof1 or Mrc1, is deleted. Thus, we propose that Esc4 associates with ssDNA of stalled forks and acts as a scaffolding protein recruit and/or modulate the function of other proteins required to reinitiate DNA synthesis.
Doa1
The cellular response to DNA damage requires not only direct repair of the damage, but also changes in the DNA replication machinery, chromatin, and transcription that facilitate survival. S. cerevisiae Doa1 helps to control the damage response by channeling ubiquitin from the proteosomal degradation pathway into pathways that mediate altered DNA replication and chromatin modification. DOA1 interacts with genes involved in PCNA ubiquitination as well as genes involved in histone H2B ubiquitination or deubiquitination.
In the absence of DOA1, damage-induced ubiquitination of PCNA does not occur. In addition, the level of ubiquitinated H2B is decreased under normal conditions and completely absent in the presence of DNA damage. In the case of PCNA, the defect associated with doa1Δ is alleviated by over-expression of ubiquitin, but in the case of H2B, it is not. The data suggest that Doa1 is the major source of ubiquitin for the DNA damage response and that Doa1 also plays an additional, essential and more specific role in the monoubiquitination of histone H2B.
