Fanconis Anemia Proteins


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 Fanconis Anemia Proteins Background

Fanconi Anemia and its molecular pathway

Fanconi Anemia (FA) is a rare chromosome instability syndrome associated with increased cancer susceptibility that is inherited in an autosomal recessive pattern. FA was first described by Dr. Guido Fanconi in 1927 with his clinical observations on brothers who had various abnormal physical conditions, bone marrow failure and aplastic anemia. While the total number of FA patients is not documented worldwide, scientists estimate that the carrier frequency for Fanconi Anemia is somewhere between 1 in 600 and 1 in 100. Approximately 50% of FA-affected children have skeletal anomalies, and about 70% of them have hand and arm anomalies. Other birth defects also include skin discoloration, small head or eyes, and mental retardation or learning disabilities. People with Fanconi Anemia often develop leukemia and other cancers. In fact, FA patients have a much greater risk of developing acute myelogenous leukemia (AML) and cancers of the head and neck. Chromosomal instability, such as tri-radial or quadri-radial chromosomes, and increased numbers of chromosome breaks following exposure to DNA crosslinking agents such as mitomycin C (MMC), diepoxybutane (DEB), and cisplatin (CDDP), are common features observed in cells of patients with FA. Although the reason for this chromosome breakage has not been elucidated, the definitive diagnostic test for FA at the present time is the chromosome breakage test.

At least 13 complementation groups of FA have been identified using cell fusion experiments that correct sensitivity to DNA cross-linking agents: A, B, C, D1, D2, E, F, G, I, J, L, M, and N (Illustration 5). These genes account for almost all of the cases of Fanconi anemia. Mutations or deficiencies in complementation groups A, C, and G, are the most prevalent and account for 85% of all FA cases worldwide. The FA proteins (FANCs) are separated into three functionally distinct groups and thought to act cooperatively in a common pathway. The first group is the FA core complex consisting of FANC A, B, C, E, F, G, L, and M. The FA core complex acts as an E3 ligase to mono-ubiquitinate FANCD2 on Lysine 561 and FANCI (a paralogue of FANCD2) on Lysine 523 in response to genotoxin damage and also during S-phase. The second group is the FANCD2/FANCI complex (also termed 'ID complex'). Mono-ubiquitinated 'FANCD2-I' complex is recruited to chromatin and postulated to direct DNA repair, but the precise biochemical role(s) has not been characterized. FANCD1, FANCN, and FANCJ compose the third FA group; these proteins are not required for FANCD2-I mono-ubiquitination, but they are connected with the breast cancer susceptibility gene products of BRCA1, BRCA2, and to their partner proteins. FANCD1 is identical to BRCA2 and FANCN is identical to PALB2 (partner and localizer of BRCA2), a crucial regulator of the BRCA2 protein. Additionally, FANCJ is identical to BACH1 or BRIP1, a DNA helicase that interacts directly with BRCA1. FANCD2, FANCD1 (BRCA2), FANCN (PALB2) and BRCA1 co-localize in nuclear foci at the sites of DNA damage and BRCA1 itself is required for efficient formation of FANCD2 nuclear foci. Because of these interplays between FA and BRCA proteins, the FA pathway is also called the "FA-BRCA pathway" or "FA-BRCA network".

Summary of known FA genes proteins

Fig. 1 Summary of known FA genes/proteins.

Coordination of the FA pathway and other DNA repair mechanisms

Since FA-deficient cells are extremely sensitive to ICL-inducing DNA damage agents, the role of the FA pathway has been extensively studied in the context of ICL repair.

According to the most recent model for ICL repair studied in Xenopus egg extracts (Illustration. 6), (A) two DNA replication forks converge on the ICL, generating a structure in which the leading strands of each fork stall approximately 20 - 40 nucleotides from the ICL, (B) one leading strand is then extended to within 1 nucleotide of the ICL, a step which may require prior replisome remodeling, (C) subsequently the two sister chromatids are uncoupled via dual incisions on either side of the ICL, possibly by XPF and/or Mus81, (D) next, a trans-lesion DNA polymerase (possibly REV1) inserts a nucleotide across from the adducted base, (E) after which Polt, extends the nascent strand beyond the ICL, (F) finally, two fully repaired DNA duplexes are generated through the action of nucleotide excision repair (NER) on the top duplex and homologous recombination (HR) on the bottom duplex. Therefore, repair of ICL requires coordinated effects of different DNA repair pathways such as homologous recombination, trans-lesion synthesis, and nucleotide excision repair mechanisms. As it has been suggested that the FA pathway serves to coordinate these pathways, defective activities of these repair pathways in FA-deficient cells (or vice versa) have been demonstrated.

FA pathway in S-phase and DNA Replication

There is evidence suggesting that the FA pathway has a role during normal Sphase progression in mammalian cells. The FA core complex proteins, FANCA, C, and G are associated with chromatin in S phase and are then excluded from condensed chromosomes during mitosis. The recently identified FA core protein FANCM is also exclusively localized to chromatin during S-phase and undergoes cell cycle-dependent phosphorylation and dephosphorylation. Moreover, FANCD2 is mono-ubiquitinated only in S phase, leading to the association of mono-ubiquitinated FANCD2 with BRCA1 and RAD51 in replication foci. Using replication competent Xenopus extracts, Sobeck and colleagues recently showed that the xFA core complex and xFANCD2 proteins are recruited to chromatin in a manner that depends on replication initiation, both in S-phase and following MMC treatment. Interestingly, immunodepletion of xFANCA and xFANCD2 resulted in accumulation of replication-associated DSBs. Although several of the FA proteins associate with chromatin concomitant with DNA replication, the basis for the binding of FANC proteins to chromatin and the precise relationship of FANC proteins with replication licensing, initiation and elongation events in human cells are not understood. Moreover, although recent in vitro studies do indicate a role for xFANCs in repair of spontaneously occurring DSB during the elongation stage of DNA synthesis, it is unclear whether FANCs participate directly in DNA replication. Therefore, we investigated the consequences of FANCD2-deficiency on replication-related parameters in human cells, utilizing siRNA-mediated depletion of FA proteins in untransformed human cells.