FANCA is a component of the Fanconi anemia (FA) core complex that activates DNA interstrand crosslink repair by monoubiquitination of FANCD2. Here, we report that purified FANCA protein catalyzes bidirectional single-strand annealing (SA) and strand exchange (SE) at a level comparable to RAD52, while a disease-causing FANCA mutant, F1263Δ, is defective in both activities. FANCG, which directly interacts with FANCA, dramatically stimulates its SA and SE activities. Alternatively, FANCB, which does not directly interact with FANCA, does not stimulate this activity. Importantly, five other patient-derived FANCA mutants also exhibit deficient SA and SE, suggesting that the biochemical activities of FANCA are relevant to the etiology of FA. A cell-based DNA double-strand break (DSB) repair assay demonstrates that FANCA plays a direct role in the single-strand annealing sub-pathway (SSA) of DSB repair by catalyzing SA, and this role is independent of the canonical FA pathway and RAD52.

Persistent dysregulation of the DNA damage response and repair in cells causes genomic instability. The resulting genetic changes permit alterations in growth and proliferation observed in virtually all cancers. However, an unstable genome can serve as a double-edged sword by providing survival advantages in the ability to evade checkpoint signaling, but also creating vulnerabilities through dependency on alternative genomic maintenance factors. The Fanconi anemia pathway comprises an intricate network of DNA damage signaling and repair that are critical for protection against genomic instability. The importance of this pathway is underlined by the severity of the cancer predisposing syndrome Fanconi anemia which can be caused by biallelic mutations in any one of the 21 genes known thus far. This review delineates the roles of the Fanconi anemia pathway and the molecular actions of Fanconi anemia proteins in confronting replicative, oxidative, and mitotic stress.

Fanconi anemia (FA) is a recessive genetic disorder caused by biallelic mutations in at least one of 22 FA genes. Beyond its pathological presentation of bone marrow failure and congenital abnormalities, FA is associated with chromosomal abnormality and genomic instability, and thus represents a genetic vulnerability for cancer predisposition. The cancer relevance of the FA pathway is further established with the pervasive occurrence of FA gene alterations in somatic cancers and observations of FA pathway activation-associated chemotherapy resistance. In this article we describe the role of the FA pathway in canonical interstrand crosslink (ICL) repair and possible contributions of FA gene alterations to cancer development. We also discuss the perspectives and potential of targeting the FA pathway for cancer intervention.

FANCA is a key player in the canonical Fanconi anemia (FA) repair pathway. We have recently shown that FANCA also plays an important role in the single-strand annealing sub-pathway (SSA) of DNA double-strand break (DSB) repair by biochemically catalyzing single-strand annealing. Here, we report that a steroidal lactone withaferin A (WA) specifically impedes SSA repair by promoting FANCA downregulation at a sub-micromolar concentration range. We find that WA causes FANCA downregulation post-translationally in a proteasome-dependent manner. This WAmediated downregulation is achieved through HSP90 inhibition and disruption of the FANCA-HSP90 interaction. WA-mediated FANCA degradation significantly reduces cellular SSA repair, abolishes FANCD2 monoubiquitination, elevates sensitivity to mitomycin C, and results in accumulation of DSBs. Importantly, the WA-induced defect in SSA repair is highly dependent on the absence of FANCA protein and overexpression of exogenous WT-FANCA protein in WAtreated cells significantly complements the repair defect.

RAD52 rejoins resected broken DNA ends by mediating single-strand annealing. Our recent work elucidates that FANCA, a Fanconi anemia protein, also directly repairs double-strand breaks (DSBs) by catalyzing annealing of single-stranded DNA. FANCA and RAD52 likely play complementary roles to each other to prevent deleterious consequences of DSBs.

Background Reporter methods to quantitatively measure the efficiency and specificity of genome editing tools are important for the development of novel editing techniques and successful applications of available ones. However, the existing methods have major limitations in sensitivity, accuracy, and/or readiness for in vivo applications. Here, we aim to develop a straight-forward method by using nucleotide insertion/deletion resulted from genome editing. In this system, a target sequence with frame-shifting length is inserted after the start codon of a cerulean fluorescence protein (CFP) to inactivate its fluorescence. As such, only a new insertion/deletion event in the target sequence will reactivate the fluorescence. This reporter is therefore termed as “Insertion/deletion-activated frame-shift fluorescence protein”. To increase its traceability, an internal ribosome entry site and a red fluorescence protein mCherryFP are placed downstream of the reporter. The percentage of CFP-positive cells can be quantified by fluorescence measuring devices such as flow cytometer as the readout for genome editing frequency. Results To test the background noise level, sensitivity, and quantitative capacity of this new reporter, we applied this approach to examine the efficiency of genome editing of CRISPR/Cas9 on two different targeting sequences and in three different cell lines, in the presence or absence of guide-RNAs with or without efficiency-compromising mutations. We found that the insertion/deletion-activated frame-shift fluorescence protein has very low background signal, can detect low-efficiency genome editing events driven by mutated guideRNAs, and can quantitatively distinguish genome editing by normal or mutated guideRNA. To further test whether the positive editing event detected by this reporter indeed correspond to genuine insertion/deletion on the genome, we enriched the CFP-positive cells to examine their fluorescence under confocal microscope and to analyze the DNA sequence of the reporter in the genome by Sanger sequencing. We found that the positive events captured by this reporter indeed correlates with genuine DNA insertion/deletion in the expected genome location. Conclusion The insertion/deletion-activated frame-shift fluorescence protein reporter has very low background, high sensitivity, and is quantitative in nature. It will be able to facilitate the development of new genome editing tools as well as the application of existing tools. Electronic supplementary material The online version of this article (10.1186/s12864-019-5963-z) contains supplementary material, which is available to authorized users.

Amyotrophic lateral sclerosis (ALS) is a progressive nervous system disease that causes loss of muscle control. Over 30 mutated genes are associated with ASL. However, 90-95% of ASL cases have been found without a family history. Here, we have analyzed RNA-Seq data of NYGC ALS Consortium and identified fusion transcripts from ASL patients and non-neurologic controls (NNC). In this study, we combined previously-curated 1180 monozygotic (MZ) hereditary fusion genes (HFGs), and 204 HFGs discovered from NNC to analyze ASL fusion transcripts and identified 348 HFGs. Comparative analysis between ASL and GTEx shows that 139 HFGs are associated with ASL and ranged from 10.4% to 98.7% of 77 ASL patients. The most recurrent HFG is ZNF528-ZNF880, detected in 98.7% of 77 ASL patients and 4.5% of 133 GTEx brain cortexes. Alignments of HFG transcripts from ASL with fusion transcripts from mesial temporal lobe epilepsy (MTLE) and Alzheimer's disease (AD) showed that 43.9% and 11.6% of the ASL HFGs were present in MTLE and AD, respectively. The most recurrent and common HFG among ASL, MTLE, and AD was ADAMTSL3-SH3GL3, which behaves like ubiquitously-expressed SH3GL3-ADAMTSL3 epigenetic fusion gene (EFG) and shows that ADAMTSL3-SH3GL3 is a potential dormant or differentially-expressed HFG (dHFG), suggesting that they have common pathophysiological mechanisms. These HFGs associated with ASL have shown that HFGs are the missing genetic heritability and provide novel therapeutic targets for more efficient therapeutic drugs and methods to treat and cure many neurological diseases.

FANCA is one of the 22 known Fanconi anemia (FA) pathway genes and is indispensable for interstrand crosslink (ICL) repair. It is reported that FANCA is responsible for about 64% of FA cases and one important clinical characterization of FA patients is predisposition to cancer. However, analysis of TCGA database reveals that FANCA level is elevated in breast cancer patients, especially in ER- breast cancer, which is associated with the methylation status at the S-shore of CpG island ahead of FANCA gene. Moreover, the FANCA level is negatively correlated with the survival rate of breast cancer patients. To understand the implication of FANCA in breast cancer development, we have targeted FANCA by either CRISPR mediated knock-out (KO) or shRNA mediated knockdown (KD) in MDA-MB-231 breast cancer cells. Our data suggest that depletion of FANCA protein inhibits breast cancer growth both in vitro and in vivo. On the other hand, overexpressing FANCA in low-FANCA breast cancer cell line MDA-MB-468 promotes cancer cell proliferation. Cell cycle profiling reveals an inefficiency of MDA-MB-231 FANCA KO cells in G1 to S transition due to p21 and p27 upregulation in FANCA KO cells. In addition, the proliferation inefficiency in MDA-MB-231 FANCA KO cells is caused by Rb hypophosphorylation. Therefore, we conclude that FANCA contributes to the breast cancer development by promoting cell cycle progression through Rb/E2F signaling pathway. Our immunoprecipitation (IP) results indicate that in MDA-MB-231, FANCA is interacting with HES1, which is a transcription suppressor that binds to the promoter region of CDKN1A and CDKN1B to promote cell cycle progression, explaining how FANCA participates in cell cycle progression in breast cancer. Citation Format: Liang Luo, Wenjun Liu, Anna Palovcak, Fenghua Yuan, Fang Li, Daniel Calkins, Yan Li, Karoline Briegel, Daniel Bilbao, Evan Roberts, Christian Mason, Zhao-Jun Liu, Sylvia Daunert, Yanbin Zhang. Defining the role of FANCA in breast cancer development and cell cycle progression. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3983.