DNA clamp function of the monoubiquitinated Fanconi anaemia ID complex

Abstract

The FANCI-FANCD2 (ID) complex, mutated in the Fanconi Anemia (FA) cancer predisposition syndrome, is required for the repair of interstrand crosslinks (ICL) and related lesions 1 . The FA pathway is activated when a replication fork stalls at an ICL 2 , triggering the mono-ubiquitination of the ID complex. ID mono-ubiquitination is essential for ICL repair by excision, translesion synthesis and homologous recombination, but its function was hitherto unknown 1 , 3 . Here, the 3.5 Å cryo-EM structure of mono-ubiquitinated ID (ID Ub ) bound to DNA reveals that it forms a closed ring that encircles the DNA. Compared to the cryo-EM structure of the non-ubiquitinated ID complex bound to ICL DNA, described here as well, mono-ubiquitination triggers a complete re-arrangement of the open, trough-like ID structure through the ubiquitin of one protomer binding to the other protomer in a reciprocal fashion. The structures, in conjunction with biochemical data, indicate the mono-ubiquitinated ID complex looses its preference for ICL and related branched DNA structures, becoming a sliding DNA clamp that can coordinate the subsequent repair reactions. Our findings also reveal how mono-ubiquitination in general can induce an alternate structure with a new function.

A PCNA-K164R mutation impinges on origin activation and mitotic DNA synthesis

“…Thus, FANCD2 could be recruited to chromatin by either unmodified PCNA, through its PCNAinteracting peptide (PIP) box (117), and/or by ubiquitinated PCNA through its coupling of ubiquitin conjugation to endoplasmic reticulum degradation (CUE) domain (118). Recent studies have demonstrated that unmodified FANCD2 does not bind to DNA (88,89). FANCD2 exists as a homodimer but in response to replication stress, FANCI is exchanged for one subunit of FANCD2, forming a FANCD2-FANCI (D2-I) heterodimer…”

“…that adopts an open conformation capable of binding DNA. Binding of DNA converts the D2-I complex into a closed conformation, and ubiquitination of FANCD2 by the FA core complex locks the complex on the DNA (88,89). Moreover, it is known that monoubiquitination of FANCD2 is carried out by the E3 ligase FANCL (119,120).…”

Structural insight into FANCI–FANCD2 monoubiquitination

“…The molecular details of FANCI–FANCD2:DNA interactions were resolved in the cryo-electron microscopy (EM) structure of FANCI–FANCD2 bound to interstrand cross-linked double-stranded DNA (dsDNA) (PDB: 6VAA , average resolution 3.8 Å) ( Figure 1 C) [ 25 ]. The structure of FANCI–FANCD2 revealed that dsDNA runs though the heterodimer [ 25 ], consistent with biochemical studies that indicate dsDNA is required for monoubiquitination to occur [ 17 , 31 ]. Interestingly, the monoubiquitination sites remain buried in the complex, which retains the same open trough-like shape as apo FANCI–FANCD2.…”

“…The previously observed preferential binding of FANCI to DNA [ 28 , 32 ] suggests that the tight binding of FANCI to DNA acts as an ‘entry point’ for FANCI–FANCD2 binding to stalled replication forks. Both FANCI and FANCD2 C-terminal domains (CTDs) are required for DNA binding within the ID2 complex [ 21 , 25 ]. This finding is consistent with previous low-resolution cryo-EM structure of FANCI–FANCD2 showing a C-terminal ‘Tower’ DNA-binding domain in FANCD2 [ 20 ].…”

“…Another recent structure study revealed that FANCD2 homodimers can exist, when the protein is purified in the absence of FANCI [ 21 ]. A cryo-EM structure of isolated FANCD2 (PDB: 6TN1 , average resolution 3.4 Å) revealed an overall closed trough-like structure that is not associated with DNA [ 21 , 25 ] ( Figures 2 A,B and 3 B). We hypothesize that because the ‘entry point’ of FANCI:DNA interaction is absent, the ubiquitination sites of FANCD2 remain buried in the FANCD2 NTD –FANCD2 NTD interface.…”