The FANCJ helicase unfolds DNA-protein crosslinks to promote their repair.

Endogenous and exogenous agents generate DNA-protein crosslinks (DPCs), whose replication-dependent degradation by the SPRTN protease suppresses aging and liver cancer. SPRTN is activated after the replicative CMG helicase bypasses a DPC and polymerase extends the nascent strand to the adduct. Here, we identify a role for the 5'-to-3' helicase FANCJ in DPC repair. In addition to supporting CMG bypass, FANCJ is essential for SPRTN activation. FANCJ binds ssDNA downstream of the DPC and uses its ATPase activity to unfold the protein adduct, which exposes the underlying DNA and enables cleavage of the adduct. FANCJ-dependent DPC unfolding is also essential for translesion DNA synthesis past DPCs that cannot be degraded. In summary, our results show that helicase-mediated protein unfolding enables multiple events in DPC repair.

FANCJ DNA helicase is recruited to the replisome by AND-1 to ensure genome stability.

FANCJ, a DNA helicase linked to Fanconi anemia and frequently mutated in cancers, counteracts replication stress by dismantling unconventional DNA secondary structures (such as G-quadruplexes) that occur at the DNA replication fork in certain sequence contexts. However, how FANCJ is recruited to the replisome is unknown. Here, we report that FANCJ directly binds to AND-1 (the vertebrate ortholog of budding yeast Ctf4), a homo-trimeric protein adaptor that connects the CDC45/MCM2-7/GINS replicative DNA helicase with DNA polymerase α and several other factors at DNA replication forks. The interaction between FANCJ and AND-1 requires the integrity of an evolutionarily conserved Ctf4-interacting protein (CIP) box located between the FANCJ helicase motifs IV and V. Disruption of the CIP box significantly reduces FANCJ association with the replisome, causing enhanced DNA damage, decreased replication fork recovery and fork asymmetry in cells unchallenged or treated with Pyridostatin, a G-quadruplex-binder, or Mitomycin C, a DNA inter-strand cross-linking agent. Cancer-relevant FANCJ CIP box variants display reduced AND-1-binding and enhanced DNA damage, a finding that suggests their potential role in cancer predisposition.

DNA repair protein RAD52 is required for protecting G-quadruplexes in mammalian cells.

G-quadruplex (G4)-forming DNA sequences are abundant in the human genome, and they are hot spots for inducing DNA double-strand breaks (DSBs) and genome instability. The mechanisms involved in protecting G4s and maintaining genome stability have not been fully elucidated. Here, we demonstrated that RAD52 plays an important role in suppressing DSB accumulation at G4s, and RAD52-deficient cells are sensitive to G4-stabilizing compounds. Mechanistically, we showed that RAD52 is required for efficient homologous recombination repair at G4s, likely due to its function in recruiting structure-specific endonuclease XPF to remove G4 structures at DSB ends. We also demonstrated that upon G4 stabilization, endonuclease MUS81 mediates cleavage of stalled replication forks at G4s. The resulting DSBs recruit RAD52 and XPF to G4s for processing DSB ends to facilitate homologous recombination repair. Loss of RAD52 along with G4-resolving helicase FANCJ leads to a significant increase of DSB accumulation before and after treatment with the G4-stabilizing compound pyridostatin, and RAD52 exhibits a synthetic lethal interaction with FANCJ. Collectively, our findings reveal a new role of RAD52 in protecting G4 integrity and provide insights for new cancer treatment strategies.

FANCJ suppresses microsatellite instability and lymphomagenesis independent of the Fanconi anemia pathway.

Microsatellites are short tandem repeat sequences that are highly prone to expansion/contraction due to their propensity to form non-B-form DNA structures, which hinder DNA polymerases and provoke template slippage. Although error correction by mismatch repair plays a key role in preventing microsatellite instability (MSI), which is a hallmark of Lynch syndrome, activities must also exist that unwind secondary structures to facilitate replication fidelity. Here, we report that Fancj helicase-deficient mice, while phenotypically resembling Fanconi anemia (FA), are also hypersensitive to replication inhibitors and predisposed to lymphoma. Whereas metabolism of G4-DNA structures is largely unaffected in Fancj(-/-) mice, high levels of spontaneous MSI occur, which is exacerbated by replication inhibition. In contrast, MSI is not observed in Fancd2(-/-) mice but is prevalent in human FA-J patients. Together, these data implicate FANCJ as a key factor required to counteract MSI, which is functionally distinct from its role in the FA pathway.

A comprehensive molecular study identified 12 complementation groups with 56 novel FANC gene variants in Indian Fanconi anemia subjects.

Fanconi anemia (FA) is a rare autosomal or X-linked genetic disorder characterized by chromosomal breakages, congenital abnormalities, bone marrow failure (BMF), and cancer. There has been a discovery of 22 FANC genes known to be involved in the FA pathway. This wide number of pathway components makes molecular diagnosis challenging for FA. We present here the most comprehensive molecular diagnosis of FA subjects from India. We observed a high frequency (4.42 ± 1.5 breaks/metaphase) of chromosomal breakages in 181 FA subjects. The major clinical abnormalities observed were skin pigmentation (70.2%), short stature (46.4%), and skeletal abnormalities (43.1%), along with a few minor clinical abnormalities. The combination of Sanger sequencing and Next Generation Sequencing could molecularly characterize 164 (90.6%) FA patients and identified 12 different complementation groups [FANCA (56.10%), FANCG (16.46%), FANCL (12.80%), FANCD2 (4.88%), FANCJ (2.44%), FANCE (1.22%), FANCF (1.22%), FANCI (1.22%), FANCN (1.22%), FANCC (1.22%), FANCD1 (0.61%) and FANCB (0.61%)]. A total of 56 novel variants were identified in our cohort, including a hotspot variant: a deletion of exon 27 in the FANCA gene and a nonsense variant at c.787 C>T in the FANCG gene. Our comprehensive molecular findings can aid in the stratification of molecular investigation in the diagnosis and management of FA patients.

Crosstalk between BRCA-Fanconi anemia and mismatch repair pathways prevents MSH2-dependent aberrant DNA damage responses.

Several proteins in the BRCA-Fanconi anemia (FA) pathway, such as FANCJ, BRCA1, and FANCD2, interact with mismatch repair (MMR) pathway factors, but the significance of this link remains unknown. Unlike the BRCA-FA pathway, the MMR pathway is not essential for cells to survive toxic DNA interstrand crosslinks (ICLs), although MMR proteins bind ICLs and other DNA structures that form at stalled replication forks. We hypothesized that MMR proteins corrupt ICL repair in cells that lack crosstalk between BRCA-FA and MMR pathways. Here, we show that ICL sensitivity of cells lacking the interaction between FANCJ and the MMR protein MLH1 is suppressed by depletion of the upstream mismatch recognition factor MSH2. MSH2 depletion suppresses an aberrant DNA damage response, restores cell cycle progression, and promotes ICL resistance through a Rad18-dependent mechanism. MSH2 depletion also suppresses ICL sensitivity in cells deficient for BRCA1 or FANCD2, but not FANCA. Rescue by Msh2 loss was confirmed in Fancd2-null primary mouse cells. Thus, we propose that regulation of MSH2-dependent DNA damage response underlies the importance of interactions between BRCA-FA and MMR pathways.

FANCJ promotes DNA synthesis through G-quadruplex structures.

Our genome contains many G-rich sequences, which have the propensity to fold into stable secondary DNA structures called G4 or G-quadruplex structures. These structures have been implicated in cellular processes such as gene regulation and telomere maintenance. However, G4 sequences are prone to mutations particularly upon replication stress or in the absence of specific helicases. To investigate how G-quadruplex structures are resolved during DNA replication, we developed a model system using ssDNA templates and Xenopus egg extracts that recapitulates eukaryotic G4 replication. Here, we show that G-quadruplex structures form a barrier for DNA replication. Nascent strand synthesis is blocked at one or two nucleotides from the G4. After transient stalling, G-quadruplexes are efficiently unwound and replicated. In contrast, depletion of the FANCJ/BRIP1 helicase causes persistent replication stalling at G-quadruplex structures, demonstrating a vital role for this helicase in resolving these structures. FANCJ performs this function independently of the classical Fanconi anemia pathway. These data provide evidence that the G4 sequence instability in FANCJ(-/-) cells and Fancj/dog1 deficient C. elegans is caused by replication stalling at G-quadruplexes.

Mutational analysis of FANCJ helicase.

FANCJ is a superfamily 2 DNA helicase, which also belongs to the iron-sulfur domain containing helicases that include XPD, ChlR1 (DDX11), and RTEL1. Mutations in FANCJ are genetically linked to Fanconi anemia (FA), breast cancer, and ovarian cancer. FANCJ plays a critical role in genome stability and participates in DNA interstrand crosslink and double-strand break repair. Enormous sequence alterations in exons and introns of FANCJ have been identified in patients, including 15 mutations in the coding region which are linked to breast cancer, 12 to FA, and two to ovarian cancer. We and other groups have characterized several FANCJ missense mutations, including M299I, A349P, R251C, and Q255H. As an increasing number of clinically relevant FANCJ mutations are identified, understanding the mechanism whereby FANCJ mutation leads to diseases is critical. Mutational analysis of FANCJ will help us elucidate the pathogenesis and potentially lead to therapeutic strategies by targeting FANCJ.

Single-molecule sorting of DNA helicases.

DNA helicases participate in virtually all aspects of cellular DNA metabolism by using ATP-fueled directional translocation along the DNA molecule to unwind DNA duplexes, dismantle nucleoprotein complexes, and remove non-canonical DNA structures. Post-translational modifications and helicase interacting partners are often viewed as determining factors in controlling the switch between bona fide helicase activity and other functions of the enzyme that do not involve duplex separation. The bottleneck in developing a mechanistic understanding of human helicases and their control by post-translational modifications is obtaining sufficient quantities of the modified helicase for traditional structure-functional analyses and biochemical reconstitutions. This limitation can be overcome by single-molecule analysis, where several hundred surface-tethered molecules are sufficient to obtain a complete kinetic and thermodynamic description of the helicase-mediated substrate binding and rearrangement. Synthetic oligonucleotides site-specifically labeled with Cy3 and Cy5 fluorophores can be used to create a variety of DNA substrates that can be used to characterize DNA binding, as well as helicase translocation and duplex unwinding activities. This chapter describes "single-molecule sorting", a robust experimental approach to simultaneously quantify, and distinguish the activities of helicases carrying their native post-translational modifications. Using this technique, a DNA helicase of interest can be produced and biotinylated in human cells to enable surface-tethering for the single-molecule studies by total internal reflection fluorescence microscopy. The pool of helicases extracted from the cells is expected to contain a mixture of post-translationally modified and unmodified enzymes, and the contributions from either population can be monitored separately, but in the same experiment providing a direct route to evaluating the effect of a given modification.

HP1 regulates the localization of FANCJ at sites of DNA double-strand breaks.

The breast and ovarian cancer predisposition protein BRCA1 forms three mutually exclusive complexes with Fanconi anemia group J protein (FANCJ, also called BACH1 or BRIP1), CtIP, and Abraxas/RAP80 through its BRCA1 C terminus (BRCT) domains, while its RING domain binds to BRCA1-associated RING domain 1 (BARD1). We recently found that the interaction between heterochromatin protein 1 (HP1) and BARD1 is required for the accumulation of BRCA1 and CtIP at sites of DNA double-strand breaks. Here, we investigated the importance of HP1 and BARD1-HP1 interaction in the localization of FANCJ together with the other BRCA1-BRCT binding proteins to clarify the separate role of the HP1-mediated pathway from the RNF8/RNF168-induced ubiquitin-mediated pathway for BRCA1 function. FANCJ interacts with HP1γ in a BARD1-dependent manner, and this interaction was enhanced by ionizing radiation or irinotecan hydrochloride treatment. Simultaneous depletion of all three HP1 isoforms with shRNAs disrupts the accumulation of FANCJ and CtIP, but not RAP80, at double-strand break sites. Replacement of endogenous BARD1 with a mutant BARD1 that is incapable of binding to HP1 also disrupts the accumulation of FANCJ and CtIP, but not RAP80. In contrast, RNF168 depletion disrupts the accumulation of only RAP80, but not FANCJ or CtIP. Consequently, the accumulation of conjugated ubiquitin was only inhibited by RNF168 depletion, whereas the accumulation of RAD51 and sister chromatid exchange were only inhibited by HP1 depletion or disruption of the BARD1-HP1 interaction. Taken together, the results suggest that the BRCA1-FANCJ and BRCA1-CtIP complexes are not downstream of the RNF8/RNF168/ubiquitin pathway, but are instead regulated by the HP1 pathway that precedes homologous recombination DNA repair.

Insight into the roles of helicase motif Ia by characterizing Fanconi anemia group J protein (FANCJ) patient mutations.

Helicases are molecular motors that couple the energy of ATP hydrolysis to the unwinding and remodeling of structured DNA or RNA, which is coordinated by conserved helicase motifs. FANCJ is a DNA helicase that is genetically linked to Fanconi anemia, breast cancer, and ovarian cancer. Here, we characterized two Fanconi anemia patient mutations, R251C and Q255H, that are localized in helicase motif Ia. Our genetic complementation analysis revealed that both the R251C and Q255H alleles failed to rescue cisplatin sensitivity of a FANCJ null cell line as detected by cell survival or γ-H2AX foci formation. Furthermore, our biochemical assays demonstrated that both purified recombinant proteins abolished DNA helicase activity and failed to disrupt the DNA-protein complex. Intriguingly, R251C impaired DNA binding ability to single-strand DNA and double-strand DNA, whereas Q255H retained higher binding activity to these DNA substrates compared with wild-type FANCJ protein. Consequently, R251C abolished its DNA-dependent ATP hydrolysis activity, whereas Q255H retained normal ATPase activity. Physically, R251C had reduced ATP binding ability, whereas Q255H had normal ATP binding ability and could translocate on single-strand DNA. Although both proteins were recruited to damage sites in our laser-activated confocal assays, they lost their DNA repair function, which explains why they exerted a domain negative effect when expressed in a wild-type background. Taken together, our work not only reveals the structural function of helicase motif Ia but also provides the molecular pathology of FANCJ in related diseases.

The Fanconi anemia proteins FANCD2 and FANCJ interact and regulate each other's chromatin localization.

Fanconi anemia is a genetic disease resulting in bone marrow failure, birth defects, and cancer that is thought to encompass a defect in maintenance of genomic stability. Mutations in 16 genes (FANCA, B, C, D1, D2, E, F, G, I, J, L, M, N, O, P, and Q) have been identified in patients, with the Fanconi anemia subtype J (FA-J) resulting from homozygous mutations in the FANCJ gene. Here, we describe the direct interaction of FANCD2 with FANCJ. We demonstrate the interaction of FANCD2 and FANCJ in vivo and in vitro by immunoprecipitation in crude cell lysates and from fractions after gel filtration and with baculovirally expressed proteins. Mutation of the monoubiquitination site of FANCD2 (K561R) preserves interaction with FANCJ constitutively in a manner that impedes proper chromatin localization of FANCJ. FANCJ is necessary for FANCD2 chromatin loading and focus formation in response to mitomycin C treatment. Our results suggest not only that FANCD2 regulates FANCJ chromatin localization but also that FANCJ is necessary for efficient loading of FANCD2 onto chromatin following DNA damage caused by mitomycin C treatment.

Novel function of the Fanconi anemia group J or RECQ1 helicase to disrupt protein-DNA complexes in a replication protein A-stimulated manner.

Understanding how cellular machinery deals with chromosomal genome complexity is an important question because protein bound to DNA may affect various cellular processes of nucleic acid metabolism. DNA helicases are at the forefront of such processes, yet there is only limited knowledge how they remodel protein-DNA complexes and how these mechanisms are regulated. We have determined that representative human RecQ and Fe-S cluster DNA helicases are potently blocked by a protein-DNA interaction. The Fanconi anemia group J (FANCJ) helicase partners with the single-stranded DNA-binding protein replication protein A (RPA) to displace BamHI-E111A bound to duplex DNA in a specific manner. Protein displacement was dependent on the ATPase-driven function of the helicase and unique properties of RPA. Further biochemical studies demonstrated that the shelterin proteins TRF1 and TRF2, which preferentially bind the telomeric repeat found at chromosome ends, effectively block FANCJ from unwinding the forked duplex telomeric substrate. RPA, but not the Escherichia coli single-stranded DNA-binding protein or shelterin factor Pot1, stimulated FANCJ ejection of TRF1 from the telomeric DNA substrate. FANCJ was also able to displace TRF2 from the telomeric substrate in an RPA-dependent manner. The stimulation of helicase-catalyzed protein displacement is also observed with the DNA helicase RECQ1, suggesting a conserved functional interaction of RPA-interacting helicases. These findings suggest that partnerships between RPA and interacting human DNA helicases may greatly enhance their ability to dislodge proteins bound to duplex DNA, an activity that is likely to be highly relevant to their biological roles in DNA metabolism.

Purification and biochemical characterization of the G4 resolvase and DNA helicase FANCJ.

G-quadruplex (G4) DNA or RNA poses a unique nucleic acid structure in genomic transactions. Because of the unique topology presented by G4, cells have exquisite mechanisms and pathways to metabolize G4 that arise in guanine-rich regions of the genome such as telomeres, promoter regions, ribosomal DNA, and other chromosomal elements. G4 resolvases are often represented by a class of molecular motors known as helicases that disrupt the Hoogsteen hydrogen bonds in G4 by harnessing the chemical energy of nucleoside triphosphate hydrolysis. Of special interest to researchers in the field, including us, is the human FANCJ DNA helicase that efficiently resolves G4 DNA structures. Notably, FANCJ mutations are linked to Fanconi Anemia and are prominent in breast and ovarian cancer. Since our discovery that FANCJ efficiently resolves G4 DNA structures 15 years ago, we and other labs have characterized mechanistic aspects of FANCJ-catalyzed G4 resolution and its biological importance in genomic integrity and cellular DNA replication. In addition to its G4 resolvase function, FANCJ is also a classic DNA helicase that acts on conventional duplex DNA structures, which are relevant to the enzyme's role in interstrand cross link repair, double-strand break repair via homologous recombination, and response to replication stress. Here, we describe detailed procedures for the purification of recombinant FANCJ protein and characterization of its G4 resolvase and duplex DNA helicase activity.

Landscape of BRIP1 molecular lesions in gastrointestinal cancers from published genomic studies.

BACKGROUND: BRIP1 is a helicase that partners with BRCA1 in the homologous recombination (HR) step in the repair of DNA inter-strand cross-link lesions. It is a rare cause of hereditary ovarian cancer in patients with no mutations of BRCA1 or BRCA2. The role of the protein in other cancers such as gastrointestinal (GI) carcinomas is less well characterized but given its role in DNA repair it could be a candidate tumor suppressor similarly to the two BRCA proteins. AIM: To analyze the role of helicase BRIP1 (FANCJ) in GI cancers pathogenesis. METHODS: Publicly available data from genomic studies of esophageal, gastric, pancreatic, cholangiocarcinomas and colorectal cancers were interrogated to unveil the role of BRIP1 in these carcinomas and to discover associations of lesions in BRIP1 with other more common molecular defects in these cancers. RESULTS: Molecular lesions in BRIP1 were rare (3.6% of all samples) in GI cancers and consisted almost exclusively of mutations and amplifications. Among mutations, 40% were possibly pathogenic according to the OncoKB database. A majority of BRIP1 mutated GI cancers were hyper-mutated due to concomitant mutations in mismatch repair or polymerase ε and δ1 genes. No associations were discovered between amplifications of BRIP1 and any mutated genes. In gastroesophageal cancers BRIP1 amplification commonly co-occurs with ERBB2 amplification. CONCLUSION: Overall BRIP1 molecular defects do not seem to play a major role in GI cancers whereas mutations frequently occur in hypermutated carcinomas and co-occur with other HR genes mutations. Despite their rarity, BRIP1 defects may present an opportunity for therapeutic interventions similar to other HR defects.

Loss of Mitochondrial Localization of Human FANCG Causes Defective FANCJ Helicase.

Fanconi anemia (FA) is a unique DNA damage repair pathway. To date, 22 genes have been identified that are associated with the FA pathway. A defect in any of those genes causes genomic instability, and the patients bearing the mutation become susceptible to cancer. In our earlier work, we identified that Fanconi anemia protein G (FANCG) protects the mitochondria from oxidative stress. In this report, we have identified eight patients having a mutation (C.65G>C), which converts arginine at position 22 to proline (p.Arg22Pro) in the N terminus of FANCG. The mutant protein, hFANCGR22P, is able to repair the DNA and able to retain the monoubiquitination of FANCD2 in the FANCGR22P/FGR22P cell. However, it lost mitochondrial localization and failed to protect mitochondria from oxidative stress. Mitochondrial instability in the FANCGR22P cell causes the transcriptional downregulation of mitochondrial iron-sulfur cluster biogenesis protein frataxin (FXN) and the resulting iron deficiency of FA protein FANCJ, an iron-sulfur-containing helicase involved in DNA repair.

The ZGRF1 Helicase Promotes Recombinational Repair of Replication-Blocking DNA Damage in Human Cells.

Replication-blocking DNA lesions are particularly toxic to proliferating cells because they can lead to chromosome mis-segregation if not repaired prior to mitosis. In this study, we report that ZGRF1 null cells accumulate chromosome aberrations following replication perturbation and show sensitivity to two potent replication-blocking anticancer drugs: mitomycin C and camptothecin. Moreover, ZGRF1 null cells are defective in catalyzing DNA damage-induced sister chromatid exchange despite accumulating excessive FANCD2, RAD51, and γ-H2AX foci upon induction of interstrand DNA crosslinks. Consistent with a direct role in promoting recombinational DNA repair, we show that ZGRF1 is a 5'-to-3' helicase that catalyzes D-loop dissociation and Holliday junction branch migration. Moreover, ZGRF1 physically interacts with RAD51 and stimulates strand exchange catalyzed by RAD51-RAD54. On the basis of these data, we propose that ZGRF1 promotes repair of replication-blocking DNA lesions through stimulation of homologous recombination.

Clinical importance of FANCD2, BRIP1, BRCA1, BRCA2 and FANCF expression in ovarian carcinomas.

OBJECTIVE: DNA repair pathways are potential targets of molecular therapy in cancer patients. The FANCD2, BRIP1, BRCA1/2, and FANCF genes are involved in homologous recombination DNA repair, which implicates their possible role in cell response to DNA-damaging agents. We evaluated a clinical significance of pre-treatment expression of these genes at mRNA level in 99 primary, advanced-stage ovarian carcinomas from patients, who later received taxane-platinum (TP) or platinum-cyclophosphamide (PC) treatment. METHODS: Gene expression was determined with the use of Real-Time PCR. The BRCA2 and BRIP1 gene sequence was investigated with the use of SSCP, dHPLC, and PCR-sequencing. RESULTS: Increased FANCD2 expression occurred to be a negative prognostic factor for all patients (PC+TP:HR 3.85, p = 0.0003 for the risk of recurrence; HR 1.96, p = 0.02 for the risk of death), and this association was even stronger in the TP-treated group (HR 6.7, p = 0.0002 and HR 2.33, p = 0.01, respectively). Elevated BRIP1 expression was the only unfavorable molecular factor in the PC-treated patients (HR 8.37, p = 0.02 for the risk of recurrence). Additionally, an increased FANCD2 and BRCA1/2 expression levels were associated with poor ovarian cancer outcome in either TP53-positive or -negative subgroups of the TP-treated patients, however these groups were small. Sequence analysis identified one protein truncating variant (1/99) in BRCA2 and no mutations (0/56) in BRIP1. CONCLUSIONS: Our study shows for the first time that FANCD2 overexpression is a strong negative prognostic factor in ovarian cancer, particularly in patients treated with TP regimen. Moreover, increased mRNA level of the BRIP1 is a negative prognostic factor in the PC-treated patients. Next, changes in the BRCA2 and BRIP1 genes are rare and together with other analyzed FA genes considered as homologous recombination deficiency may not affect the expression level of analyzed genes.

Assembly of a G-Quadruplex Repair Complex by the FANCJ DNA Helicase and the REV1 Polymerase.

The FANCJ helicase unfolds G-quadruplexes (G4s) in human cells to support DNA replication. This action is coupled to the recruitment of REV1 polymerase to synthesize DNA across from a guanine template. The precise mechanisms of these reactions remain unclear. While FANCJ binds to G4s with an AKKQ motif, it is not known whether this site recognizes damaged G4 structures. FANCJ also has a PIP-like (PCNA Interacting Protein) region that may recruit REV1 to G4s either directly or through interactions mediated by PCNA protein. In this work, we measured the affinities of a FANCJ AKKQ peptide for G4s formed by (TTAGGG)4 and (GGGT)4 using fluorescence spectroscopy and biolayer interferometry (BLI). The effects of 8-oxoguanine (8oxoG) on these interactions were tested at different positions. BLI assays were then performed with a FANCJ PIP to examine its recruitment of REV1 and PCNA. FANCJ AKKQ bound tightly to a TTA loop and was sequestered away from the 8oxoG. Reducing the loop length between guanine tetrads increased the affinity of the peptide for 8oxoG4s. FANCJ PIP targeted both REV1 and PCNA but favored interactions with the REV1 polymerase. The impact of these results on the remodeling of damaged G4 DNA is discussed herein.

Helicases FANCJ, RTEL1 and BLM Act on Guanine Quadruplex DNA in Vivo.

Guanine quadruplex (G4) structures are among the most stable secondary DNA structures that can form in vitro, and evidence for their existence in vivo has been steadily accumulating. Originally described mainly for their deleterious effects on genome stability, more recent research has focused on (potential) functions of G4 structures in telomere maintenance, gene expression, and other cellular processes. The combined research on G4 structures has revealed that properly regulating G4 DNA structures in cells is important to prevent genome instability and disruption of normal cell function. In this short review we provide some background and historical context of our work resulting in the identification of FANCJ, RTEL1 and BLM as helicases that act on G4 structures in vivo. Taken together these studies highlight important roles of different G4 DNA structures and specific G4 helicases at selected genomic locations and telomeres in regulating gene expression and maintaining genome stability.