Key histone chaperones have distinct roles in replisome progression and genomic stability

Abstract

Replication-coupled (RC) nucleosome assembly is an essential process in eukaryotic cells in order to maintain chromatin structure during DNA replication. The deposition of newly synthesized H3/H4 histones during DNA replication is facilitated by specialized histone chaperones. Although the contribution of these histone chaperones to genomic stability has been thoroughly investigated, their effect on replisome progression is much less understood. By exploiting a time-lapse microscopy system for monitoring DNA replication in individual live cells, we examined how mutations in key histone chaperones including CAC1, RTT106, RTT109 and ASF1, affect replication fork progression. Our experiments revealed that mutations in CAC1 or RTT106 that directly deposit histones on the DNA, slowdown replication fork progression. In contrast, analysis of cells mutated in the intermediary ASF1 or RTT109 histone chaperones revealed that replisome progression is not affected. We found that mutations in histone chaperones including ASF1 and RTT109 lead to extended G2/M duration, elevated number of RPA foci and in some cases, increased spontaneous mutation rate. Our research suggests that histone chaperones have distinct roles in enabling high replisome progression and maintaining genome stability during cell cycle progression. Author SummaryHistone chaperones (HC) play key roles in maintaining the chromatin structure during DNA replication in eukaryotic cells. Despite extensive studies on HCs, little is known regarding their importance for replication fork progression during S-phase. Here, we utilized a live-cell imaging approach to measure the progression rates of single replication forks in individual yeast cells mutated in key histone chaperones. Using this approach, we show that mutations in CAC1 or RTT106 HCs that directly deposit histones on the DNA lead to slowdown of replication fork progression. In contrast, mutations in ASF1 or RTT109 HCs that transfers H3/H4 to CAC1 or RTT106, do not affect replisome progression but lead to post replication defects. Our results reveal distinct functions of HCs in replication fork progression and maintaining genome stability. IntroductionDNA replication in eukaryotic cells is a complex process that requires the accurate copying of the DNA itself and the formation of a precise chromatin structure [1,2]. The basic unit of chromatin is the nucleosome, composed of ~146 base pairs of DNA wrapped around an octamer of histones. A nucleosome is composed of a core of (H3-H4) 2 tetramer and two flanking H2A-H2B dimmers [3,4].During DNA replication, nucleosomes must be disassembled to allow replication fork progression and subsequently must be reassembled to establish the accurate chromatin state. Histone chaperones are essential for the process of DNA replication-coupled (RC) nucleosome deposition by facilitating correct histone assembly, post-translational modifications and localization during DNA replication [5][6][7][8].Newly synthesized histones are assembled into nucleosomes by several histone chap...