Methods for studying the recruitment of DDR proteins to DNA breaks. Scheme describing the multiple methods used to generate or mimic DNA breaks in vitro and in vivo and the techniques employed to monitor recruitment of DDR factors to such breaks.

Temporal regulation of DDR protein accumulation at DNA breaks. (A) Sequential recruitment of DDR factors to SSBs and DSBs generated by laser microirradiation. (B) Cell cycle regulation of DDR foci formation. (Solid line) Efficient focus formation; (dashed line) weak/undetectable foci.

Specialized binding modules for recognition of post-translational modifications (PTMs) at DNA breaks. The recruitment of DDR proteins to modified histones or other modified proteins at sites of DNA breaks is mediated by specific interactions between the post-translational modification and a dedicated binding module. BRCT and FHA domains, which are represented by red and green semicircles, bind phosphorylated serine or threonine residues; Tudor domains, chromodomains, and PDH finger domains bind methylated histones; bromodomains (Bromo) bind acetylated histones; and UBDs bind ubiquitylated proteins. The PAR-binding domain can take the form of a basic stretch of amino acids (Basic), a PAR-binding zinc finger (PBZ), or a macrodomain (Macro). Note that some of these modules are found as tandem domains and that not all post-translational modifications are damage-induced (asterisk [*] denotes constitutive modifications). The species of the proteins are indicated, unless only human proteins are listed. (H.s.) Homo sapiens; (S.c.) S. cerevisiae, (S.p.) S. pombe.

Protein dynamics to and from sites of DNA breaks. DNA damage checkpoint and repair factors and modulators of chromatin organization are recruited (green arrows) to DNA breaks (SSB and DSB), while transcription machineries are excluded from DDR foci (red arrows), and the dynamics of structural chromatin components operate in both directions (orange arrows).

Spatial organization of DDR protein accumulation at DNA DSBs. (A) DDR signal spreading. DDR proteins initially accumulate at DSB sites and then spread at distance via a positive feedback loop involving MDC1, which binds γH2AX, the MRN complex, and ATM kinase, which phosphorylates additional H2AX molecules further away from the break site. (B) Regional distribution of DDR proteins around DSBs. Factors involved in ATR signaling accumulate proximal to the break site on ssDNA generated by DNA end resection, while ATM signaling factors localize on flanking chromatin regions.

Binding platforms at DNA breaks. NBS1, MDC1, XRCC1, and XRCC4 act as binding platforms for the recruitment of other DDR factors to DNA breaks promoting DNA damage signaling and/or repair. Dotted lines indicate protein–protein interactions, while horizontal lines at the end of the dotted lines indicate interacting regions. Some interactions involve post-translational modifications. (P) Phosphorylation; (PAR) PARylation. The red and the green semicircles represent BRCT and FHA domains, respectively. (b) Basic region at the end of the first BRCT domain of XRCC1 that interacts with poly(ADP-ribosyl)ated PARPs.