Profiling DNA damage response following mitotic perturbations

Abstract

Genome integrity relies on precise coordination between DNA replication and chromosome segregation. Whereas replication stress attracted much attention, the consequences of mitotic perturbations for genome integrity are less understood. Here, we knockdown 47 validated mitotic regulators to show that a broad spectrum of mitotic errors correlates with increased DNA breakage in daughter cells. Unexpectedly, we find that only a subset of these correlations are functionally linked. We identify the genuine mitosis-born DNA damage events and sub-classify them according to penetrance of the observed phenotypes. To demonstrate the potential of this resource, we show that DNA breakage after cytokinesis failure is preceded by replication stress, which mounts during consecutive cell cycles and coincides with decreased proliferation. Together, our results provide a resource to gauge the magnitude and dynamics of DNA breakage associated with mitotic aberrations and suggest that replication stress might limit propagation of cells with abnormal karyotypes.

Distinct cellular responses to replication stress leading to apoptosis or senescence

“…This histone modification supports chromatin remodeling and the recruitment of repair proteins . In addition to γH2AX, it has recently been shown that 53BP1 forms large nuclear structures around persisting DNA lesions induced by RS to protect these vulnerable regions until repair . Both γH2AX and 53BP1 were frequently bound to whole condensed chromosomes or only to a part of these chromosomes in cells exposed to replication inhibitor FLU and its combination with Chk1i.…”

Importance of a Conserved Lys/Arg Residue for Ligand/PDZ Domain Interactions as Examined by Protein Semisynthesis

“…In order to explore the importance of the conserved Lys/Arg residue forp eptide-ligand binding to PDZ domains, we employedt he prototypical PSD-95 PDZ2 domain along with four 5/6-tetramethylrhodamine (TAMRA)-labelled peptide ligands, representing both canonical and noncanonicall igands. The canonical ligands were GluN2B C-terminal peptide( TAMRA-YEKLSSIESDV-COOH;G luN2B-COOH) and CRIPT (TAMRA-DTKNY-KQTSV-COOH;C RIPT-COOH);t he noncanonical ligandsw ere a nNOS b-finger mimetic (TAMRA-cyclo(CTHLETTFTGDGTPKTIRV-TQpG);n NOS b-finger) [16,[31][32] and aC -terminally amidated GluN2B peptide (TAMRA-YEKLSSIESDV-CONH 2 ;G luN2B-CONH 2 ). We replacedL ys165 in PSD-95 PDZ2 with Argand four nonproteogenic amino acids:L ys variants homolysine (hLys) and orni- ChemBioChem 2016ChemBioChem , 17,1936ChemBioChem -1944 www.chembiochem.org thine (Orn), where the number of methylene groups to the primary amine is varied, and Arg variants citrulline (Cit) and acetyl-ornithine (AcOrn), where the degree of protonation, as well as hydrogen bonding are varied ( Figure 1C).…”

“…The ligand-binding site is as hallowp ocket between the b2s trand and a2h elix, where the ligand forms an extended antiparallel b strand ( Figure 1A). [14][15][16] The canonical peptide-binding mode involves ap eptideC -terminal carboxylate, which interactswith aconserved GLGF motif immediately before b2i nt he PDZ domain ( Figure 1B). The GLGF motif (carboxylate-bindings ite) forms ac radle of backbone amides that aligns ah ydrogen bond network to the peptide C-terminal carboxylate and hence stabilizes the interaction between the PDZd omain and the peptide.…”

“…The GLGF motif (carboxylate-bindings ite) forms ac radle of backbone amides that aligns ah ydrogen bond network to the peptide C-terminal carboxylate and hence stabilizes the interaction between the PDZd omain and the peptide. Additionally,a ne xtended backbone-backbone hydrogen bond network between the ligand and b2, [16,17] together with side-chain interactions primarily governed by the four last amino acids, are also fundamental fort he canonical binding of C-terminal peptides to PDZ domains. Thef ourC -terminal amino acids of the peptide ligand contains am otif (P À3 -P 0 ); residues P 0 and P À2 are particularly important for affinity and selectivity.…”

“…and form interactions similart ot he canonical binding mode: the P 0 site accommodates an aromatic side chain (Phe111)o f the nNOS b-finger,a nd ah ydrogen bond forms in the P À2 site between an a2H is residue and ah ydroxyl group (Thr109) in nNOS. [16,21,[23][24][25] Structural analysiso fP DZ domains has identified additional interactions between the side chain of ah ighly conserved basic residue( Arg/Lys) in the flexible b1-b2l oop (Lys165 and Arg318i nP SD-95 PDZ2 and PDZ3, respectively). These, together with the GLGF loop, stabilizet he carboxylate of C-terminal ligands through aw ater-mediated hydrogen bond ( Figure 1A).…”