Error-Free DNA Damage Tolerance and Sister Chromatid Proximity during DNA Replication Rely on the Polα/Primase/Ctf4 Complex

摘要

class="head no_bottom_margin" id="sec1title">IntroductionFaithful DNA replication is crucial for genomic maintenance. When replication is perturbed, cells activate stress response networks that connect the detection of replication-blocking lesions with DNA damage tolerance (DDT) and repair pathways, chromatin modifications, cell-cycle control, and various other changes in cell physiology, often collectively referred to as the DNA damage response (DDR) (). Failures in these processes are implicated in the etiology of many developmental and neurological disorders and are thought to drive genome instability characteristic of cancer ().Genome duplication is carried out by the replisome machinery, initially assembled at replication origins (). Notably, replication initiation critically depends on the loading and activity of the Polymerase α (Polα)/Primase complex. This is the fundamental initiator of DNA replication in eukaryotic cells, as the replicative DNA polymerases can only elongate an existing RNA-DNA primer produced by this complex. The Primase produces short RNA fragments (about 7–12 nt long), which are subsequently subjected to limited extension by Polα. These RNA-DNA primers are then extended by the replicative polymerases Polε and Polδ ().Polα/Primase-mediated processes are not only relevant for origin-dependent replication initiation, but also for origin-independent initiation events, as is the case of lagging strand DNA synthesis, and possibly the restart of stalled forks downstream the blocking lesion under conditions of genotoxic stress (). The latter aspect is potentially crucial for efficient DDT and replication, especially in conditions in which fast replication is a requirement, such as at the early stages of development ().Two distinct modes of DDT, error-prone and error-free DDT, operate in all eukaryotic organisms (). Error-prone DDT is mediated by translesion synthesis (TLS) polymerases and largely accounts for mutagenesis. Error-free DDT uses a recombination-related mechanism known as template switching (TS), in which one newly synthesized strand serves as replication template for the other blocked nascent strand (). The choice between these DDT modes has profound consequences for genome stability, and to date, several factors have been implicated in DDT pathway choice: PCNA post-translational modifications with mono-ubiquitylation, poly-ubiquitylation, and SUMOylation (); genome architectural transitions coupled with early stages of replication (); and cell-cycle-specific changes in the abundance or regulation of key DDT factors ().Together with Polα/Primase, a number of structural proteins that tether the replicative minichromosome maintenance (MCM) helicase to the replicative polymerases are loaded at replication origins (). Ctf4 (AND-1 in mammalian cells) functions as such a replisome architectural factor, bridging the MCM helicase and two molecules of Polα/Primase (). It is of note, however, that while Polα and Primase are essential for cellular proliferation, Ctf4 is not. This indicates that even if uncoupled from the replicative helicase, Polα/Primase supports DNA synthesis. Besides its roles to maintain normal replisome architecture, Ctf4/AND-1 is also required for sister chromatid cohesion ().Increasing number of reports indicate “replication stress” at the basis of chromosomal instability, and as an important underlying factor of developmental anomalies (href="#bib18" rid="bib18 bib22" class=" bibr popnode">Halazonetis et al., 2008; Jackson and Bartek, 2009). However, the nature of the early chromosome lesions arising following such replication perturbations is largely unknown. Moreover, the connections between these replication dysfunctions and the observed chromatin structural alterations similarly triggered by mutations in cohesion factors remain elusive.Here we used budding yeast Saccharomyces cerevisiae cellular models of specific replication stress and sister chromatid cohesion defects to investigate a possible crosstalk between recombination-mediated DDT and chromatin structure/cohesion. Our results indicate that both replicative helicase-coupled re-priming and sister chromatid cohesion are important to facilitate error-free DDT by TS, but they do so via different mechanisms. The results shed light on how highly conserved replication-associated pathways crosstalk to each other and contribute to normal replication fork and chromatin structure, providing mechanistic insights into the molecular basis of human disorders caused by replication dysfunctions.