Surprising complexity of the Asf1 histone chaperone-Rad53 kinase interaction

Abstract

The histone chaperone Asf1 and the checkpoint kinase Rad53 are found in a complex in budding yeast cells in the absence of genotoxic stress. Our data suggest that this complex involves at least three interaction sites. One site involves the H3-binding surface of Asf11 with an as yet undefined surface of Rad53. A second site is formed by the Rad53-FHA1 domain binding to Asf1-T 270 phosphorylated by casein kinase II. The third site involves the C-terminal 21 amino acids of Rad53 bound to the conserved Asf1 N-terminal domain. The structure of this site showed that the Rad53 C-terminus binds Asf1 in a remarkably similar manner to peptides derived from the histone cochaperones HirA and CAF-I. We call this binding motif, ðR∕KÞRðI∕A∕VÞ × ðL∕PÞ, the AIP box for Asf1-Interacting Protein box. Furthermore, C-terminal Rad53-F 820 binds the same pocket of Asf1 as does histone H4-F 100 . Thus Rad53 competes with histones H3-H4 and cochaperones HirA/CAF-I for binding to Asf1. Rad53 is phosphorylated and activated upon genotoxic stress. The Asf1-Rad53 complex dissociated when cells were treated with hydroxyurea but not methyl-methane-sulfonate, suggesting a regulation of the complex as a function of the stress. We identified a rad53 mutation that destabilized the Asf1-Rad53 complex and increased the viability of rad9 and rad24 mutants in conditions of genotoxic stress, suggesting that complex stability impacts the DNA damage response.checkpoint | chromatin | DNA damage | NMR | X-ray structure

ANTI-SILENCING FUNCTION1 Proteins Are Involved in Ultraviolet-Induced DNA Damage Repair and Are Cell Cycle Regulated by E2F Transcription Factors in Arabidopsis

“…In yeast, Asf1 and the checkpoint kinase Rad53 are found in a complex in budding yeast cells in the absence of genotoxic stress (Jiao et al, 2012). Upon replication stress caused by hydroxyurea, the Asf1-Rad53 complex dissociates, suggesting that the complex is regulated by genotoxic stress conditions (Jiao et al, 2012). In addition, Asf1 is important for the transcriptional derepression of two DNA damage response genes during the S phase in response to hydroxyurea (Minard et al, 2011).…”

“…Moreover, we here demonstrate that major CPD removal mechanisms are unaffected in mutant plants, suggesting that ASF1 activities participate in CPD photorepair in Arabidopsis, because in the RNAi plants, chromatin is more accessible to damage accumulation, and not because ASF1 proteins regulate the expression of repair enzymes. In yeast, Asf1 and the checkpoint kinase Rad53 are found in a complex in budding yeast cells in the absence of genotoxic stress (Jiao et al, 2012). Upon replication stress caused by hydroxyurea, the Asf1-Rad53 complex dissociates, suggesting that the complex is regulated by genotoxic stress conditions (Jiao et al, 2012).…”

Replisome Function During Replicative Stress Is Modulated by Histone H3 Lysine 56 Acetylation Through Ctf4

“…The checkpoint kinase Mec1 and the Ddc1-Mec3-Rad17 sliding clamp regulate the interaction between Asf1 and Rad53 (Hu 2001;Sharp et al 2001;Burgess et al 2014). Since the asf1-V94R mutation also affects Asf1-Rad53 interaction (Jiao et al 2012;Dennehey et al 2013), we tested the viability of the Rad53-A806R-L808R (rad53-ALRR) mutant, which is strongly affected in its interaction with Asf1 (Jiao et al 2012) in the rrm3D background. We found that the rad53-ALRR rrm3D double mutant is viable ( Figure 1C, iii).…”

“…Asf1 participates in the assembly and disassembly of chromatin through its H3 histone chaperone activity, in transcriptional silencing via an interaction with Hir1 and in several aspects of the cellular response to genotoxic stress through an interaction with H3 or Rad53 (Hu 2001;Sharp et al 2001;Sutton et al 2001;Celic et al 2006;Recht et al 2006;Takahata et al 2009;Jiao et al 2012;Burgess et al 2014).…”

“…Given that HIR2 is required for the integrity of the HIR complex and, consequently, for histone deposition activity of Asf1/HIR (Green et al 2005;Silva et al 2012), these observations demonstrate that the replicationindependent chromatin assembly function of Asf1 is not required for rrm3D cell viability. We next evaluated the contribution of the single-residue substitution V94R, important for histone H3 interaction (Mousson et al 2005;Jiao et al 2012).…”