br Materials and methods br

Materials and methods
Results
Discussion In this work, we have studied the contribution of different pathways to the slow but stable progression of replication forks through DNA damaged by MMS [15]. The in vivo analysis of DNA replication forks along a specific chromosome of S. cerevisiae has shown that nos inhibitor excision repair, homologous recombination and replicative DNA damage tolerance pathways are required to allow fork movement through alkylated DNA and cell viability. The absence of any of these pathways impedes the correct progression of replication forks under these DNA damaging conditions and causes cell death. Continuous removal of methylated bases by base excision repair during S phase appears as the primary requirement to eliminate the obstacles that interfere with the progression of replication forks in cells treated with MMS. In the absence of this activity, replication forks stall, which very strongly suggests that DNA lesions are responsible for the block. Homologous recombination and DNA damage tolerance pathways could be very likely involved in promoting fork restart once the lesions have been repaired by base excision repair and in the bypass of some replication blocking lesions originated by MMS that remained unrepaired. Recombination and DNA damage tolerance could also contribute to post-replication repair of some lesions behind the forks, such as small single-strand gaps or nicks [9], [12]. The long S phase in cells treated with MMS [33] is the result of checkpoint-dependent regulation of origin firing and checkpoint-independent slow fork movement [15]. Our results are consistent with the idea that fork slowing is due to the sum of stalling events rather than a different mode of replication or a global replication fork slowing down in the presence of DNA damage. We propose that replication forks moving through alkylated DNA collide stochastically with DNA lesions (methylated bases), which stop their progression. DNA replication forks are then stabilized by the S phase checkpoint and can subsequently overcome the obstacles that hinder their movement by the cooperation of repair, fork restart and damage tolerance activities. These need time to work, helping to explain the resultant slow but finally efficient fork movement. The S phase checkpoint, which stabilizes replication forks through damaged DNA, is active in the strains analyzed in this work. Therefore, the requirement for Mag1, Rad52 and Rad18 can be attributed to the lack of the relevant pathway and not to a deficient checkpoint response. These data also indicate that none of these pathways is necessary for checkpoint activation following DNA damage that is caused by MMS. This activation requires fork establishment [23] and the formation of a structure that would signal to initiate the checkpoint cascade, probably RPA-coated single-stranded DNA [50], for which the action of the pathways studied would not be required. The reduced viability of mag1 deficient cells exposed to MMS is reversed and stalled replication forks resume DNA synthesis if expression of the Mag1 protein is restored. In rad52 and rad18 mutants, removal of MMS allows the progression of enough replication forks to detect new DNA synthesis by flow cytometry, but does not restore viability. Moreover, unlike Mag1, there is no apparent contribution of newly synthesized Rad52 to replication fork progression or cell viability, and the contribution of new Rad18 is only modest. These results reflect that, although some forks can move, others could collapse irreversibly in cells lacking of Rad52 or Rad18, accounting for the irreversible loss of viability. In contrast, stalled forks maintain their integrity in the mag1 strain despite the absence of base excision repair. Other damaging agents affect replication differently to MMS. Thus, ionizing radiation slows down S phase progression due to transient blocks to origin firing, without affecting fork movement [51]. A different study has shown that neither recombination nor translesion synthesis, absent respectively in rad52Δ and rad18Δ mutants, seem to affect fork progression in the presence of irreparable UV-damage to DNA, although replication of this template is affected in these mutants, as the number of internal gaps is increased [31]. The different requirements for fork progression through DNA damaged by distinct agents makes sense, as the diverse nature of the lesions is likely to cause different kinds of blocks to DNA replication and a variety of structures at the damaged forks. For example, unlike irreparable UV damage, MMS does not originate a significant increase in ssDNA at replication forks [31].