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    • br Introduction Coping with DNA

    2019-08-14


    Introduction Coping with DNA damage is possible thanks to surveillance mechanisms (checkpoints), that detect the problem and promote its solution [1], [2], and to repair and tolerance pathways that remove the lesions or reduce the damage consequences [3], [4]. Failures in these processes have a high cost, as they are frequently linked to genome instability, a predominant characteristic of cancer cells [5], [6], [7]. Genome stability is especially at risk during chromosomal replication. DNA replication errors affect cell survival and are aggravated and many times originated by DNA damage. DNA synthesis can be compromised when replication forks encounter DNA lesions. Forks are then vulnerable and need to be stabilized, and lesions have to be repaired to complete DNA replication successfully [8], [9], [10], [11], [12], [13]. In BGJ 398 yeast, the S phase checkpoint, mediated by the conserved kinases Mec1 and Rad53 (human ATR and Chk2, respectively), stabilizes replication forks in response to DNA damage or replicative stress [14], [15]. It also protects forks at fragile sites [16], [17], regulates origin firing [18], [19], [20] and allows fork restart [21]. Moreover, the checkpoint helps to regulate the choice of the repair pathway [22]. The stabilization of stalled forks is the crucial downstream effector of the checkpoint in maintaining cell viability [23]. It is thought to occur, at least in conditions of dNTP depletion, by preserving the replisome at replication forks [24], [25], [26], [27] and through restraining recombination activities at forks [28], [29]. Fork stabilization prevents the accumulation of gapped, hemireplicated and four-branched molecules [30], [31], [32]. All these responses need previous checkpoint activation, which in turn requires the establishment of DNA replication forks [23]. A widely used agent in model studies on DNA damage is the alkylating compound methyl methanesulfonate (MMS). The MMS was used to define the S phase checkpoint [33] and to establish the parameters of this mechanism with respect to DNA replication [15]. Mec1 and Rad53 extend the S phase in the presence of MMS [33], and the increased duration of chromosome replication is due to the combination of a reduction in the rate of fork progression and inhibition of late origin firing [15]. Although the checkpoint is required for fork stability in cells exposed to MMS, the slow progression of forks under such conditions is checkpoint independent and has not been characterized yet. At present, the mechanisms that allow cells to replicate a DNA template that has been damaged by alkylation are poorly studied. MMS mainly methylates DNA on N7-deoxyguanine and N3-deoxyadenine. N3-methyladenine is toxic and inhibits DNA synthesis in vitro[34]. Understanding the processes that allow cells to cope with this kind of damage may help understand how replication fork progression succeeds in the presence of MMS. One of the major defences against DNA alkylation in the budding yeast Saccharomyces cerevisiae is the Mag1 3MeA DNA glycosylase, which initiates the base excision repair pathway by cleaving the glycosylic bond between 3MeA and the deoxyribose of the sugar phosphate backbone [35]. The other two main pathways implicated in the protection against MMS are recombination and DNA damage tolerance [36]. Indeed, mutants of RAD52, which is required for homologous recombination, and RAD18, a key component of the replicative DNA damage tolerance pathways, are highly sensitive to MMS [36]. Rad52 stimulates strand exchange and is involved in the repair of double-strand breaks and single-strand gaps [37]. Rad18 is a single-stranded DNA binding protein with E3 ubiquitin ligase activity. It forms a complex with the E2 ubiquitin-conjugating protein Rad6 that modifies PCNA [38], [39]. Post-replication repair genes belong to the RAD18/RAD6 epistasis group and contribute to damage bypass of lesions or to filling in of gaps formed in newly synthesized DNA. Previous studies have not addressed the importance of the base excision repair, homologous recombination, and DNA damage tolerance pathways for the progression of DNA replication forks in eukaryotic cells exposed to MMS. In this work, we have studied in vivo the contribution of these pathways to fork movement and fork stability after treatment of budding yeast cells with MMS. Using dense isotope transfer, the progression of DNA replication forks has been followed throughout a replicon on chromosome VI of S. cerevisiae in mag1, rad52 and rad18 mutants. Our results indicate that, in addition to a functional S phase checkpoint, base excision repair, homologous recombination and DNA damage tolerance activities are required for efficient replication fork progression and cell viability in the presence of alkylated DNA.