Fanconi anemia proteins, DNA interstrand crosslink repair pathways, and cancer therapy

Abstract

DNA interstrand crosslinkers, a chemically diverse group of compounds which also induce alkylation of bases and DNA intrastrand crosslinks, are extensively utilized for cancer therapy. Understanding the cellular response to DNA damage induced by these agents is critical for more effective utilization of these compounds and for the identification of novel therapeutic targets. Importantly, the repair of DNA interstrand crosslinks (ICLs) involves many distinct DNA repair pathways, including nucleotide excision repair, translesion synthesis (TLS), and homologous recombination (HR). Additionally, proteins implicated in the pathophysiology of the multigenic disease Fanconi anemia (FA) have a role in the repair of ICLs that is not well understood. Cells from FA patients are hypersensitive to agents that induce ICLs, therefore FA proteins are potentially novel therapeutic targets. Here we will review current research directed at identifying FA genes and understanding the function of FA proteins in DNA damage responses. We will also examine interactions of FA proteins with other repair proteins and pathways, including signaling networks, which are potentially involved in ICL repair. Potential approaches to the modulation of FA protein function to enhance therapeutic outcome will be discussed. Also, mutation of many genes that encode proteins involved in ICL repair, including FA genes, increases susceptibility to cancer. A better understanding of these pathways is therefore critical for the design of individualized therapies tailored to the genetic profile of a particular malignancy. For this purpose, we will also review evidence for the association of mutation of FA genes with cancer in non-FA patients.

Fig. 1. Diagram of different types of DNA damage

(A) Modification of a single strand of DNA by chemicals, such as alkylating agents, induces a monoadduct. Such adducts can lead to crosslinks, either within a single DNA strand (DNA intrastrand crosslink) or between two paired DNA strands (DNA interstrand crosslink). (B) Other types of DNA damage, some of which can be induced in the process of repairing DNA interstrand crosslinks, include single-strand (SSBs) and double-strand DNA breaks (DSBs).

Fig. 2. Schematic outline of the repair of DNA interstrand crosslinks (ICLs)

A DNA interstrand crosslink blocks the progression of an advancing replication fork during S phase. This leads to signaling by the ATR checkpoint kinase, which recruits the machinery for DNA repair. Then the crosslink is unhooked, a process which involves incisions by endonucleases on both sides of the lesion on one DNA strand. This process requires the NER proteins ERCC1-XPF. This process also generates a DNA double-strand break which can initiate DNA repair by HR. To restore the integrity of the template strand for HR, the gap generated by unhooking the crosslink is filled in by the process of translesion synthesis (TLS) using bypass polymerases (indicated by a dotted gray line). The unhooked adduct is then removed by NER, or by spontaneous hydrolysis, and the gap is filled in by replicative DNA polymerases. End resection at the DNA double-strand break generates a 3′ single-strand overhang which can pair with the sister chromatid that has been restored by TLS. End resection may involve exonuclease activity of the MRE11-RAD50-NBS1 complex. Strand invasion involves RAD51 and BRCA2. Following branch migration, resolution of Holliday junction recombination intermediates permits the restart of DNA replication, a process which may involve the Mus81-Eme1 endonuclease.

Fig. 3. Diagram of interrelationships of Fanconi anemia (FA) proteins and of the regulation of FANCD2 monoubiquitination

At least eight of the FA proteins, FANC- A, B, C, E, F, G, L, and M, form a complex which is predominantly nuclear and which is required for the monoubiquitination of the FA proteins, FANCD2 and FANCI, either during S phase or in response to DNA damage. This biochemical pathway is termed the FA pathway. FAAP24 and FAAP100 are associated with the FA nuclear core complex but have not been identified as FA genes (indicated by lighter colored symbols for these proteins in the figure). The ATR checkpoint kinase and USP1/UAF1 have also not been identified as FA proteins, but are critical for the regulation of FANCD2 monoubiquitination. ATR is required for FANCD2 monoubiquitination in response to DNA damage by an unknown mechanism and USP1/UAF1 deubiquitinates FANCD2. Monoubiquitinated FANCD2 is targeted to chromatin, where it interacts with the FA protein BRCA2/FANCD1. Monoubiquitinated FANCD2 organizes BRCA2/FANCD1 foci in response to DNA damage. PALB2/FANCN is a recently identified FA protein which is a partner of BRCA2FANCD1 required for its stability and recruitment to chromatin. BRIP1/FANCJ, like BRCA2/FANCD1 and PALB2/FANCN, is not required for FANCD2 monoubiquitination and is therefore proposed to function downstream of, or in association with, monoubiquitinated FANCD2. The functional relationship of BRIP1/FANCJ to other FA proteins has not been determined.

Fig. 4. Diagram of the possible functions of Fanconi anemia (FA) proteins in promoting translesion synthesis (TLS) and homologous recombination (HR)

FA proteins could recruit or organize DNA damage response proteins, such as bypass polymerases (left) or BRCA2 (right) and thereby promote TLS or HR, respectively.