Chromatin recruitment of DNA repair proteins: lessons from the fanconi anemia and double-strand break repair pathways

Abstract

In response to DNA damage, eukaryotic cells must rapidly load DNA repair proteins onto damaged chromatin. Chromatin recruitment often entails ubiquitination of a damage-specific DNA repair protein, interaction with a ubiquitin binding factor, assembly of a multisubunit DNA repair complex, and eventually a deubiquitination event once the DNA repair reaction has been completed. This review focuses on the recent discoveries in the Fanconi Anemia (FA) and DNA double-strand break (DSB) repair pathways, which underscore the importance of regulated chromatin loading in the DNA damage response. Interestingly, these two pathways share several features, suggesting a more general mechanism for DNA-repair regulation.

Figure 1. The Fanconi Anemia pathway regulates the loading of DNA repair complexes

1) The FANCD2 and FANCI proteins are phosphorylated on multiple amino acid residues in response to a DNA crosslink. 2) The phosphorylated FANCD2 and FANCI proteins are monoubiquitinated by the FA core complex, containing of the FANCA, FANCB, FANCC, FANCE, FANCF, FANCG, FANCL and FANCM protein subunits. FANCL is the E3 ligase subunit. 3) _[SC16]In the absence of genotoxic stress FANCD2 and FANCI are deubiquitinated by the USP1/UAF1 complex to prevent the accumulation of monoubiquitinated FANCD2 and FANCI. In response to DNA damage, the level of USP1/UAF1 complex decreases, resulting in the cellular increase in ubiquitinated FANCD2 and FANCI. 4) Monoubiquitinated FANCD2 and FANCI translocate to sites of damage on the chromatin, putatively with the help of a recruitment protein X. On chromatin the proteins assemble around the site of damage together with other FA proteins, including FANCD1(BRCA2), FANCN(PALB2) and FANCJ(BRIP1,BACH1). 5) The assembled FA DNA repair complex repairs the DNA damage in conjunction with other DNA repair proteins. 6) Once the repair process has been completed, FANCD2 and FANCI are deubiquitinated by the USP1/UAF1 complex, resulting in a disassembly of the FA DNA repair complex and dissociation from the chromatin.

Figure 2. Conserved elements of the Fanconi Anemia and DSB repair pathways

1) In response to either a DNA crosslink or double strand break, ATR or ATM phosphorylates FANCD2 or H2AX, respectively. 2) The phosphorylated proteins are now substrates for a ubiquitination event, carried out either by the FA core complex or the RNF8/UBC13 complex. 3) Monoubiquitinated FANCD2 protein can now be recruited to sites of damage on the chromatin by a recruitment factor, Protein X. Polyubiquitinated H2AX recruits RAP80 to sites of damage on the chromatin. 4) The properly localized FANCD2 protein associates with other DNA repair proteins and the DNA damage is repaired. In addition to RAP80, other DNA repair proteins are recruited to H2AX, and the DNA damage is repaired. 5) Once the DNA repair process has been completed, the recruited protein complex must be disassembled. This is achieved by deubiquitinating the FANCD2 protein by the USP1/UAF1 complex, and H2AX by an unknown deubiquitinating enzyme. 6) After the DNA repair process has been completed, FANCD2 is dephosphorylated by an unknown phosphatase, and H2AX is dephosphorylated by PP2A (Chowdhury et al., 2005). For simplicity, this model only shows a subset of the proteins known to participate in ICL and DSB repair. For instance, only FANCD2 is shown, although the paralog protein, FANCI, is believed to act in a similar fashion.