Mechanisms modulating transcribed chromatin topology and suppressing replication fork instability

Résumé

ABSTRACT DNA replication is a risky process for the cell. Chromosomal DNA synthesis takes place at specialized structures (replication forks), which present intrinsic fragility and are preferred substrates of unscheduled recombination events. When replication fork progression is hampered, they can collapse priming DNA breaks and genomic rearrangements. Collision of replication forks with actively transcribed genes is a potential main determinant of replication fork stalling and a number of pathways have been involved in transcribed genomic regions protection. In budding yeast, DNA topoisomerase II (Top2) has been proposed to organize architectural domains to prevent DNA breaks following transcribed regions replication. Factors acting in the interface between transcription and mRNA export, like the THO/TREX complex, also suppress the accumulation of DNA:RNA hybrids forming at negatively supercoiled segments behind transcription bubble. DNA:RNA hybrids, when engaged by replication forks, prime aberrant recombination events and chromosomal abnormalities. Over 26 years ago, a topoisomerase-like catalytic active site was described in the Hpr1 THO component, raising the possibility that the complex modulates transcribed regions topology. To address this point, we mutagenized Hpr1 topoisomerase-like catalytic tyrosine to generate an hpr1-Y590A mutant allele. We found that hpr1-Y590A cells are sensitive to treatment with the replication inhibitor hydroxyurea, suggesting that Hpr1 might play a topological role in promoting replication fork stability. The hpr1-Y590A allele shows synthetic growth defects in combination with top2 mutations, and hpr1-Y590A top2-1 cells accumulate checkpoint activation signals following S-phase completion. This evidence suggests a degree of coordination between Hpr1 and Top2 in resolving topological stress and preventing chromosomal breaks at sites of replication transcription interference. Moreover, putative Hpr1 catalytic tyrosine mutation leads to the accumulation of negatively supercoiled topoisomers in Top1 compromised cells. We propose that the topoisomerase like catalytic tyrosine residue of Hpr1 contributes to modulate DNA topology at transcribed chromatin thus alleviating deleterious collisions between DNA replication and gene transcription machineries. Formation of secondary structures on the template DNA can also destabilize replication forks and prime chromosome rearrangements. Human minisatellite repeats, which show highly dynamic changes in repeat number, are prone to form G-quadruplexes, a four-stranded structure in which guanines within a single stranded DNA stretch pair and stack. Using budding yeast model system we have studied the replication of the human sub-telomeric minisatellites CEB25 and c-MYC. By 2D gel electrophoresis, we found that aberrant replication intermediates accumulate at CEB25 harboring regions in cells lacking Pif1 helicase. Likewise during the replication of c-MYC human minisatellite, we observed the accumulation of recombination intermediates in cells in which Pif1 activity is compromised. These defects observed are consistent with the proposed role of Pif1 in unwinding G-quadruplexes. We propose that these replication defects upon G-quadruplex stabilization in Pif1-ablated cells repeats reflect strand discontinuities in CEB25 minisatellite, while they determine fork breakage and unscheduled recombination repair attempts within the c-MYC minisatellite.