The Fanconi anemia pathway and DNA interstrand cross-link repair

Fanconi anemia (FA) is an autosomal or X-linked recessive disorder characterized by chromosomal instability, bone marrow failure, cancer susceptibility, and a profound sensitivity to agents that produce DNA interstrand cross-link (ICL). To date, 15 genes have been identified that, when mutated, result in FA or an FA-like syndrome. It is believed that cellular resistance to DNA interstrand cross-linking agents requires all 15 FA or FA-like proteins. Here, we review our current understanding of how these FA proteins participate in ICL repair and discuss the molecular mechanisms that regulate the FA pathway to maintain genome stability.     

Tip60 is required for DNA interstrand cross-link repair in the Fanconi anemia pathway

The disease Fanconi anemia is a genome instability syndrome characterized by cellular sensitivity to DNA interstrand cross-linking agents, manifest by decreased cellular survival and chromosomal aberrations after such treatment. There are at least 13 proteins acting in the pathway, with the FANCD2 protein apparently functioning as a late term effecter in the maintenance of genome stability. We find that the chromatin remodeling protein, Tip60, interacts directly with the FANCD2 protein in a yeast two-hybrid system. This interaction has been confirmed by co-immunoprecipitation and co-localization using both endogenous and epitope-tagged FANCD2 and Tip60 from human cells. The observation of decreased cellular survival after exposure to mitomycin C in normal fibroblasts depleted for Tip60 indicates a direct function in interstrand cross-link repair. The coincident function of Tip60 and FANCD2 in one pathway is supported by the finding that depletion of Tip60 in Fanconi anemia cells does not increase sensitivity to DNA cross-links. However, depletion of Tip60 did not reduce monoubiquitination of FANCD2 or its localization to nuclear foci following DNA damage. The observations indicate that Fanconi anemia proteins act in concert with chromatin remodeling functions to maintain genome stability after DNA cross-link damage.     

Repair kinetics of genomic interstrand DNA cross-links: evidence for DNA double-strand break-dependent activation of the Fanconi anemia/BRCA pathway

The detailed mechanisms of DNA interstrand cross-link (ICL) repair and the involvement of the Fanconi anemia (FA)/BRCA pathway in this process are not known. Present models suggest that recognition and repair of ICL in human cells occur primarily during the S phase. Here we provide evidence for a refined model in which ICLs are recognized and are rapidly incised by ERCC1/XPF independent of DNA replication. However, the incised ICLs are then processed further and DNA double-strand breaks (DSB) form exclusively in the S phase. FA cells are fully proficient in the sensing and incision of ICL as well as in the subsequent formation of DSB, suggesting a role of the FA/BRCA pathway downstream in ICL repair. In fact, activation of FANCD2 occurs slowly after ICL treatment and correlates with the appearance of DSB in the S phase. In contrast, activation is rapid after ionizing radiation, indicating that the FA/BRCA pathway is specifically activated upon DSB formation. Furthermore, the formation of FANCD2 foci is restricted to a subpopulation of cells, which can be labeled by bromodeoxyuridine incorporation. We therefore conclude that the FA/BRCA pathway, while being dispensable for the early events in ICL repair, is activated in S-phase cells after DSB have formed.     

SCAI promotes error‐free repair of DNA interstrand crosslinks via the Fanconi anemia pathway

DNA interstrand crosslinks (ICLs) are cytotoxic lesions that threaten genome integrity. The Fanconi anemia (FA) pathway orchestrates ICL repair during DNA replication, with ubiquitylated FANCI‐FANCD2 (ID2) marking the activation step that triggers incisions on DNA to unhook the ICL. Restoration of intact DNA requires the coordinated actions of polymerase ζ (Polζ)‐mediated translesion synthesis (TLS) and homologous recombination (HR). While the proteins mediating FA pathway activation have been well characterized, the effectors regulating repair pathway choice to promote error‐free ICL resolution remain poorly defined. Here, we uncover an indispensable role of SCAI in ensuring error‐free ICL repair upon activation of the FA pathway. We show that SCAI forms a complex with Polζ and localizes to ICLs during DNA replication. SCAI‐deficient cells are exquisitely sensitive to ICL‐inducing drugs and display major hallmarks of FA gene inactivation. In the absence of SCAI, HR‐mediated ICL repair is defective, and breaks are instead re‐ligated by polymerase θ‐dependent microhomology‐mediated end‐joining, generating deletions spanning the ICL site and radial chromosomes. Our work establishes SCAI as an integral FA pathway component, acting at the interface between TLS and HR to promote error‐free ICL repair.     

Fanconi anemia and DNA replication repair

There has been a recent profusion of reviews on Fanconi anemia (FA), which will give readers a comprehensive outline of the field R.D. Kennedy, A.D. D'Andrea, The Fanconi anemia/BRCA pathway: new faces in the crowd, Genes Dev. 19 (2005) 2925-2940; L.J. Niedernhofer, A.S. Lalai, J.H. Hoeijmakers, Fanconi anemia (cross)linked to DNA repair, Cell 123 (2005) 1191-1198; H. Joenje, K.J. Patel, The emerging genetic and molecular basis of Fanconi anaemia, Nat. Rev. Genet. 2 (2001) 446-457. Here, we will focus on key areas that place the FA proteins in the context of DNA repair during replication. In addition, where possible we will put forward propositions that in our opinion need addressing, and where possible provide models that can be tested.     

DNA repair defects channel interstrand DNA cross-links into alternate recombinational and error-prone repair pathways

The repair of psoralen interstrand cross-links in the yeast Saccharomyces cerevisiae involves the DNA repair groups nucleotide excision repair (NER), homologous recombination (HR), and post-replication repair (PRR). In repair-proficient yeast cells cross-links induce double-strand breaks, in an NER-dependent process; the double-strand breaks are then repaired by HR. An alternate error-prone repair pathway generates mutations at cross-link sites. We have characterized the repair of plasmid molecules carrying a single psoralen cross-link, psoralen monoadduct, or double-strand break in yeast cells with deficiencies in NER, HR, or PRR genes, measuring the repair efficiencies and the levels of gene conversions, crossing over, and mutations. Strains with deficiencies in the NER genes RAD1, RAD3, RAD4, and RAD10 had low levels of cross-link-induced recombination but higher mutation frequencies than repair-proficient cells. Deletion of the HR genes RAD51, RAD52, RAD54, RAD55, and RAD57 also decreased induced recombination and increased mutation frequencies above those of NER-deficient yeast. Strains lacking the PRR genes RAD5, RAD6, and RAD18 did not have any cross-link-induced mutations but showed increased levels of recombination; rad5 and rad6 cells also had altered patterns of cross-link-induced gene conversion in comparison with repair-proficient yeast. Our observations suggest that psoralen cross-links can be repaired by three pathways: an error-free recombinational pathway requiring NER and HR and two PRR-dependent error-prone pathways, one NER-dependent and one NER-independent.     

Double-strand breaks induce homologous recombinational repair of interstrand cross-links via cooperation of MSH2, ERCC1-XPF, REV3, and the Fanconi anemia pathway

DNA interstrand cross-linking agents have been widely used in chemotherapeutic treatment of cancer. The majority of interstrand cross-links (ICLs) in mammalian cells are removed via a complex process that involves the formation of double-strand breaks at replication forks, incision of the ICL, and subsequent error-free repair by homologous recombination. How double-strand breaks effect the removal of ICLs and the downstream homologous recombination process is not clear. Here, we describe a plasmid-based recombination assay in which one copy of the CFP gene is inactivated by a site-specific psoralen ICL and can be repaired by gene conversion with a mutated homologous donor sequence. We found that the homology-dependent recombination (HDR) is inhibited by the ICL. However, when we introduced a double-strand break adjacent to the site of the ICL, the removal of the ICL was enhanced and the substrate was funneled into a HDR repair pathway. This process was not dependent on the nucleotide excision repair pathway, but did require the ERCC1-XPF endonuclease and REV3. In addition, both the Fanconi anemia pathway and the mismatch repair protein MSH2 were required for the recombinational repair processing of the ICL. These results suggest that the juxtaposition of an ICL and a DSB stimulates repair of ICLs through a process requiring components of mismatch repair, ERCC1-XPF, REV3, Fanconi anemia proteins, and homologous recombination repair factors.     

DNA repair: exploiting the Fanconi anemia pathway as a potential therapeutic target

DNA repair is an active cellular process to respond to constant DNA damage caused by metabolic processes and environmental factors. Since the outcome of DNA damage is generally adverse and long term effects may contribute to oncogenesis, cells have developed a variety of DNA repair mechanisms, which operate depending on the type of DNA damage inflicted. At least 15 Fanconi anemia (FA) proteins interact in a common pathway involved in homologous recombination. Inherited homozygous mutations in any of these FA genes cause a rare disease, Fanconi anemia, characterized by congenital abnormalities, progressive bone-marrow failure and cancer susceptibility. Heterozygous germline FA mutations predispose to various types of cancer. In addition, somatic FA mutations have been identified in diverse cancer types. Evidence exists that cells deficient in the FA pathway become dependent on alternative pathways for survival. Additional inhibition of such alternative pathways is thus expected to result in cell death, creating a relationship of synthetic lethality. Identifying these relationships can reveal yet unknown mechanisms of DNA repair and new targets for therapy.     

Repair of DNA interstrand cross-links

DNA interstrand cross-links (ICLs) are very toxic to dividing cells, because they induce mutations, chromosomal rearrangements and cell death. Inducers of ICLs are important drugs in cancer treatment. We discuss the main properties of several classes of ICL agents and the types of damage they induce. The current insights in ICL repair in bacteria, yeast and mammalian cells are reviewed. An intriguing aspect of ICLs is that a number of multi-step DNA repair pathways including nucleotide excision repair, homologous recombination and post-replication/translesion repair all impinge on their repair. Furthermore, the breast cancer-associated proteins Brca1 and Brca2, the Fanconi anemia-associated FANC proteins, and cell cycle checkpoint proteins are involved in regulating the cellular response to ICLs. We depict several models that describe possible pathways for the repair or replicational bypass of ICLs.     

DNA interstrand cross-links induce futile repair synthesis in mammalian cell extracts

DNA interstrand cross-links are induced by many carcinogens and anticancer drugs. It was previously shown that mammalian DNA excision repair nuclease makes dual incisions 5' to the cross-linked base of a psoralen cross-link, generating a gap of 22 to 28 nucleotides adjacent to the cross-link. We wished to find the fates of the gap and the cross-link in this complex structure under conditions conducive to repair synthesis, using cell extracts from wild-type and cross-linker-sensitive mutant cell lines. We found that the extracts from both types of strains filled in the gap but were severely defective in ligating the resulting nick and incapable of removing the cross-link. The net result was a futile damage-induced DNA synthesis which converted a gap into a nick without removing the damage. In addition, in this study, we showed that the structure-specific endonuclease, the XPF-ERCC1 heterodimer, acted as a 3'-to-5' exonuclease on cross-linked DNA in the presence of RPA. Collectively, these observations shed some light on the cellular processing of DNA cross-links and reveal that cross-links induce a futile DNA synthesis cycle that may constitute a signal for specific cellular responses to cross-linked DNA.     

Replication Protein A (RPA) deficiency activates the Fanconi anemia DNA repair pathway

The Fanconi anemia (FA) pathway regulates DNA inter-strand crosslink (ICL) repair. Despite our greater understanding of the role of FA in ICL repair, its function in the preventing spontaneous genome instability is not well understood. Here, we show that depletion of replication protein A (RPA) activates the FA pathway. RPA1 deficiency increases chromatin recruitment of FA core complex, leading to FANCD2 monoubiquitination (FANCD2-Ub) and foci formation in the absence of DNA damaging agents. Importantly, ATR depletion, but not ATM, abolished RPA1 depletion-induced FANCD2-Ub, suggesting that ATR activation mediated FANCD2-Ub. Interestingly, we found that depletion of hSSB1/2-INTS3, a single-stranded DNA-binding protein complex, induces FANCD2-Ub, like RPA1 depletion. More interestingly, depletion of either RPA1 or INTS3 caused increased accumulation of DNA damage in FA pathway deficient cell lines. Taken together, these results indicate that RPA deficiency induces activation of the FA pathway in an ATR-dependent manner, which may play a role in the genome maintenance.     

Fanconi anemia: at the Crossroads of DNA repair

Fanconi anemia (FA) is an autosomal disorder that causes genome instability. FA patients suffer developmental abnormalities, early-onset bone marrow failure, and a predisposition to cancer. The disease is manifested by defects in DNA repair, hypersensitivity to DNA crosslinking agents, and a high degree of chromosomal aberrations. The FA pathway comprises 13 disease-causing genes involved in maintaining genomic stability. The fast pace of study of the novel DNA damage network has led to the constant discovery of new FA-like genes involved in the pathway that when mutated lead to similar disorders. A majority of the FA proteins act as signal transducers and scaffolding proteins to employ other pathways to repair DNA. This review discusses what is known about the FA proteins and other recently linked FA-like proteins. The goal is to clarify how the proteins work together to carry out interstrand crosslink repair and homologous recombination-mediated repair of damaged DNA.     

Diepoxybutane interstrand cross-links induce DNA bending

The bifunctional alkylating agent 1,2,3,4-diepoxybutane (DEB) is thought to be a major contributor to the carcinogenicity of 1,3-butadiene, from which it is derived in vivo. DEB forms DNA interstrand cross-links primarily between distal deoxyguanosine residues at the duplex sequence 5′-GNC. In order for the short butanediol tether to span this distance, distortion of the DNA target has been postulated. We determined that the electrophoretic mobility of ligated DNA oligomers containing DEB cross-links was retarded in comparison with control, uncross-linked DNA. Our data are consistent with DNA bending of ∼34° per lesion towards the major groove.     

The Fanconi anemia pathway and ICL repair: implications for cancer therapy

Fanconi anemia (FA) is an inherited disease caused by mutations in at least 13 genes and characterized by genomic instability. In addition to displaying strikingly heterogenous clinical phenotypes, FA patients are exquisitely sensitive to treatments with crosslinking agents that create interstrand crosslinks (ICL). In contrast to bacteria and yeast, in which ICLs are repaired through replication-dependent and -independent mechanisms, it is thought that ICLs are repaired primarily during DNA replication in vertebrates. However, recent data indicate that replication-independent ICL repair also operates in vertebrates. While the precise role of the FA pathway in ICL repair remains elusive, increasing evidence suggests that FA proteins function at different steps in the sensing, recognition and processing of ICLs, as well as in signaling from these very toxic lesions, which can be generated by a wide variety of cancer chemotherapeutic drugs. Here, we discuss some of the recent findings that have shed light on the role of the FA pathway in ICL repair, with special emphasis on the implications of these findings for cancer therapy since disruption of FA genes have been associated with cancer predisposition.     

Monoubiquitylation in the Fanconi anemia DNA damage response pathway

The hereditary genetic disorder Fanconi anemia (FA) belongs to the heterogeneous group of diseases associated with defective DNA damage repair. Recently, several reviews have discussed the FA pathway and its molecular players in the context of genome maintenance and tumor suppression mechanisms [H. Joenje, K.J. Patel, The emerging genetic and molecular basis of Fanconi anaemia, Nat. Rev. Genet. 2 (2001) 446–457; W. Wang, Emergence of a DNA-damage response network consisting of Fanconi anaemia and BRCA proteins, Nat. Rev. Genet. 8 (2007) 735–748; L.J. Niedernhofer, A.S. Lalai, J.H. Hoeijmakers, Fanconi anemia (cross)linked to DNA repair, Cell 123 (2005) 1191–1198; K.J. Patel, Fanconi anemia and breast cancer susceptibility, Nat. Genet. 39 (2007) 142–143]. This review assesses the influence of post-translational modification by ubiquitin. We review and extract the key features of the enzymatic cascade required for the monoubiquitylation of the FANCD2/FANCI complex and attempt to include recent findings into a coherent mechanism. As this part of the FA pathway is still far from fully understood, we raise several points that must be addressed in future studies.     

DNA polymerase II (polB) is involved in a new DNA repair pathway for DNA interstrand cross-links in Escherichia coli

DNA-DNA interstrand cross-links are the cytotoxic lesions for many chemotherapeutic agents. A plasmid with a single nitrogen mustard (HN2) interstrand cross-link (inter-HN2-pTZSV28) was constructed and transformed into Escherichia coli, and its replication efficiency (RE = [number of transformants from inter-HN2-pTZSV28]/[number of transformants from control]) was determined to be approximately 0.6. Previous work showed that RE was high because the cross-link was repaired by a pathway involving nucleotide excision repair (NER) but not recombination. (In fact, recombination was precluded because the cells do not receive lesion-free homologous DNA.) Herein, DNA polymerase II is shown to be in this new pathway, since the replication efficiency (RE) is higher in a polB+ ( approximately 0. 6) than in a DeltapolB (approximately 0.1) strain. Complementation with a polB+-containing plasmid restores RE to wild-type levels, which corroborates this conclusion. In separate experiments, E. coli was treated with HN2, and the relative sensitivity to killing was found to be as follows: wild type < polB < recA < polB recA approximately uvrA. Because cells deficient in either recombination (recA) or DNA polymerase II (polB) are hypersensitive to nitrogen mustard killing, E. coli appears to have two pathways for cross-link repair: an NER/recombination pathway (which is possible when the cross-links are formed in cells where recombination can occur because there are multiple copies of the genome) and an NER/DNA polymerase II pathway. Furthermore, these results show that some cross-links are uniquely repaired by each pathway. This represents one of the first clearly defined pathway in which DNA polymerase II plays a role in E. coli. It remains to be determined why this new pathway prefers DNA polymerase II and why there are two pathways to repair cross-links.     

A Rad50-dependent pathway of DNA repair is deficient in Fanconi anemia fibroblasts

Fanconi anemia (FA) is a fatal genetic disorder associated with pancytopenia and cancer. Cells lacking functional FA genes are hypersensitive to bifunctional alkylating agents, and are deficient in DNA double-strand break repair. Multiple genes with FA-causing mutations have been cloned, however, the molecular basis for FA remains obscure. The results presented herein indicate that a Rad50-dependent end-joining process is non-functional in diploid fibroblasts from FA patients. Introduction of anti-Rad50 antibody into normal fibroblasts sensitized them to DNA damaging agents, whereas this treatment had no effect on fibroblasts from FA patients. The DNA end-joining process deficient in FA cells also requires the Mre11, Nbs1 and DNA ligase IV proteins. These data reveal the existence of a previously uncharacterized Rad50-dependent DNA double-strand break repair pathway in mammalian somatic cells, and suggest that failure to activate this pathway is responsible, at least in part, for the defective DNA end-joining observed in FA cells.     

Ubiquitylation and the Fanconi anemia pathway

The Fanconi anemia (FA) pathway maintains genome stability through co-ordination of DNA repair of interstrand crosslinks (ICLs). Disruption of the FA pathway yields hypersensitivity to interstrand crosslinking agents, bone marrow failure and cancer predisposition. Early steps in DNA damage dependent activation of the pathway are governed by monoubiquitylation of FANCD2 and FANCI by the intrinsic FA E3 ubiquitin ligase, FANCL. Downstream FA pathway components and associated factors such as FAN1 and SLX4 exhibit ubiquitin-binding motifs that are important for their DNA repair function, underscoring the importance of ubiquitylation in FA pathway mediated repair. Importantly, ubiquitylation provides the foundations for cross-talk between repair pathways, which in concert with the FA pathway, resolve interstrand crosslink damage and maintain genomic stability.     

The Pso4 mRNA splicing and DNA repair complex interacts with WRN for processing of DNA interstrand cross-links

DNA interstrand cross-links (ICLs) are perhaps the most formidable lesion encountered by the cellular DNA repair machinery, and the elucidation of the process by which they are removed in eukaryotic cells has proved a daunting task. In particular, the early stages of adduct recognition and uncoupling of the cross-link have remained elusive principally because genetic studies have not been highly revealing. We have developed a biochemical assay in which processing of a DNA substrate containing a site-specific psoralen ICL can be monitored in vitro. Using this assay we have shown previously that the mismatch repair factor MutSbeta, the nucleotide excision repair heterodimer Ercc1-Xpf, and the replication proteins RPA and PCNA are involved in an early stage of psoralen ICL processing. Here, we report the identification of two additional factors required in the ICL repair process, a previously characterized pre-mRNA splicing complex composed of Pso4/Prp19, Cdc5L, Plrg1, and Spf27 (Pso4 complex), and WRN the protein deficient in Werner syndrome. Analysis of the WRN protein indicates that its DNA helicase function, but not its exonuclease activity, is required for ICL processing in vitro. In addition, we show that WRN and the Pso4 complex interact through a direct physical association between WRN and Cdc5L. A putative model for uncoupling of ICLs in mammalian cells is presented.     

The Fanconi anemia/BRCA pathway: a coordinator of cross-link repair

Fanconi anemia (FA) is a rare inherited disease characterized by genomic instability and markedly increased cancer risk. Efforts to elucidate the molecular basis of FA have unearthed a novel DNA damage response pathway, the integrity of which is critical for cellular resistance to DNA cross-linking agents. Despite significant progress in uncovering the molecular events underlying FA, the precise function of this pathway in DNA repair is unknown. This article will review evidence implicating FA proteins in multiple aspects of DNA cross-link repair and propose a model to explain the selectivity of the FA pathway toward DNA cross-linking agents.