FANCJ Helicase Defective in Fanconia Anemia and Breast Cancer Unwinds G-Quadruplex DNA To Defend Genomic Stability

Abstract

FANCJ mutations are associated with breast cancer and genetically linked to the bone marrow disease Fanconi anemia (FA). The genomic instability of FA-J mutant cells suggests that FANCJ helicase functions in the replicational stress response. A putative helicase with sequence similarity to FANCJ in Caenorhabditis elegans (DOG-1) and mouse (RTEL) is required for poly(G) tract maintenance, suggesting its involvement in the resolution of alternate DNA structures that impede replication. Under physiological conditions, guaninerich sequences spontaneously assemble into four-stranded structures (G quadruplexes [G4]) that influence genomic stability. FANCJ unwound G4 DNA substrates in an ATPase-dependent manner. FANCJ G4 unwinding is specific since another superfamily 2 helicase, RECQ1, failed to unwind all G4 substrates tested under conditions in which the helicase unwound duplex DNA. Replication protein A stimulated FANCJ G4 unwinding, whereas the mismatch repair complex MSH2/MSH6 inhibited this activity. FANCJ-depleted cells treated with the G4-interactive compound telomestatin displayed impaired proliferation and elevated levels of apoptosis and DNA damage compared to small interfering RNA control cells, suggesting that G4 DNA is a physiological substrate of FANCJ. Although the FA pathway has been classically described in terms of interstrand cross-link (ICL) repair, the cellular defects associated with FANCJ mutation extend beyond the reduced ability to repair ICLs and involve other types of DNA structural roadblocks to replication.The identification of FANCJ mutations in early-onset breast cancer patients (3, 34) and Fanconi anemia (FA) group J patients (22,23,25) implicates FANCJ as a tumor suppressor caretaker that ensures genomic stability. FANCJ interacts with the tumor suppressor BRCA1 (3) and is a DNA helicase that catalytically unwinds duplex DNA in an ATP hydrolysis-dependent manner (2, 14). FA-J cells are hypersensitive to interstrand cross-linking (ICL) agents (1, 25); untreated FA-J cells exhibit diminished BRCA1 foci, and the cells show delayed formation of ionizing radiation-induced BRCA1 foci (31). In response to DNA damage or replicational stress, FANCJ colocalizes with the single-stranded DNA (ssDNA) binding protein replication protein A (RPA), which serves as an auxiliary factor for the unwinding function of FANCJ (15). FANCJ interacts with the mismatch repair complex MutL␣, and this interaction is required for the correction of the ICL response in FA-J cells (32). The activation of FANCJ helicase activity is required for timely progression through S phase of the cell cycle (20); however, the precise functions of the FANCJ helicase in S-phase progression remain to be understood.Although a role for FANCJ in DNA replication has been proposed previously, definitive evidence for a role of the FANCJ helicase in preventing genomic instability is lacking. One source of genomic instability is alternate DNA structures that may impede the replication fork. Guanine-rich nucleic acids have the potential to f...

The Fanconi Anemia Proteins FANCD2 and FANCJ Interact and Regulate Each Other's Chromatin Localization

“…FANCJ has also been demonstrated to unwind guanine quadruplex structures (G4-DNA) that can form in tracts of guanines, and a FA-J patient-derived mutant FANCJ protein is unable to unwind these structures in vitro (48,49). It has been speculated that certain interstrand cross-links may preferentially form in these quadruplex structures and that FANCJ is required for their resolution.…”

A Polymerase Theta-dependent repair pathway suppresses extensive genomic instability at endogenous G4 DNA sites

“…An evolutionary conserved role for DOG-1/ FANCJ in preventing G4 DNA-induced genome alteration was suggested by the mapping of large genomic deletions accumulating in a FANCJ-deficient human patient cell line to G4 DNA-containing regions 18 . In line with a proposed role for this helicase in resolving G-quadruplexes in vivo to help the fork replicate the affected strand 16 , the purified human FANCJ protein was shown to behave as a structure-specific DNA helicase that can unwind G4 DNA with 5 0 to 3 0 polarity 19 . While the molecular nature of genomic alterations in C. elegans demonstrated a strikingly atypical mutation profile 16,17 , little is known about the molecular mechanisms that underlie G4 DNA-induced genome rearrangements.…”

DNA Sequences Proximal to Human Mitochondrial DNA Deletion Breakpoints Prevalent in Human Disease Form G-quadruplexes, a Class of DNA Structures Inefficiently Unwound by the Mitochondrial Replicative Twinkle Helicase

“…The four-stranded (tetramolecular) parallel TP-G4 substrate (20), two-stranded (bimolecular) anti-parallel OX-1-G2Ј DNA substrate (20), four-stranded TelR2-G4 (20), four-stranded 22), and unimolecular poly(A) Zic1-G4 substrate with 5Ј single-stranded DNA (ssDNA) tail lengths of 20, 30, or 40 nt were prepared from gel-purified oligonucleotides as described previously (23). The 19-bp forked duplex DNA substrate was prepared by annealing DC26 and TSTEM25 as described earlier (24).…”