Linking replication stress with replication dynamics in Vicia faba root meristem cells

Abstract

DNA replication faces many challenges, both internally and externally, generally described as factors that induce replication stress. In this article, we describe how prolonged replication stress affects the dynamics of replication and fork activity in V. faba root meristem cells. V. faba seems to have a highly effective stress response system that includes a reaction to replication stress. We used 2.5 mM hydroxyurea (HU) for prolonged stress induction (32 hours) and measured changes in replication and fork activity after initial stress induction, prolonged exposure and after regeneration time in water. We also induced premature chromosome condensation (PCC) as a reference, to compare cells that express valid ATR/Chk1 S-phase checkpoint with cells that lack ATR functions. Our results included general changes in replication activity, obtained with 5-ethynyl-2'-deoxyuridine (EdU) labeling as well as an extended analysis of replication fork progression facilitated by double-labeling with EdU and 5-iodo-2’-deoxyuridine (IdU) which we found to be an appealing alternative to commonly used labeling with 5-chloro-2’-deoxyuridine (CldU) and IdU. A preliminary analysis of minichromo-some maintenance complex component 2 (MCM2), a subunit of minichromosome maintenance protein complex (MCM), were shown. We were able to pinpoint a mechanism that may contribute to the replication stress resistance of V. faba cells the most. We have also demonstrated that it is not only cells with ATR malfunctions where heterochromatin areas are extensively affected by replication stress.