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Helicases are molecular motors that couple the energy of ATP hydrolysis to the unwinding of structured DNA or RNA and chromatin remodeling. The conversion of energy derived from ATP hydrolysis into unwinding and remodeling is coordinated by seven sequence motifs (I, Ia, II, III, IV, V, and VI). The Q motif, consisting of nine amino acids (GFXXPXPIQ) with an invariant glutamine (Q) residue, has been identified in some, but not all helicases. Compared to the seven well-recognized conserved helicase motifs, the role of the Q motif is less acknowledged. Mutations in the human ChlR1 (DDX11) gene are associated with a unique genetic disorder known as Warsaw Breakage Syndrome, which is characterized by cellular defects in genome maintenance. To examine the roles of the Q motif in ChlR1 helicase, we performed site directed mutagenesis of glutamine to alanine at residue 23 in the Q motif of ChlR1. ChlR1 recombinant protein was overexpressed and purified from HEK293T cells. ChlR1-Q23A mutant abolished the helicase activity of ChlR1 and displayed reduced DNA binding ability. The mutant showed impaired ATPase activity but normal ATP binding. A thermal shift assay revealed that ChlR1-Q23A has a melting point value similar to ChlR1-WT. Partial proteolysis mapping demonstrated that ChlR1-WT and Q23A have a similar globular structure, although some subtle conformational differences in these two proteins are evident. Finally, we found ChlR1 exists and functions as a monomer in solution, which is different from FANCJ, in which the Q motif is involved in protein dimerization. Taken together, our results suggest that the Q motif is involved in DNA binding but not ATP binding in ChlR1 helicase.

Mutations in the human ChlR1 (DDX11) gene are associated with a unique genetic disorder known as Warsaw breakage syndrome characterized by cellular defects in genome maintenance. The DNA triplex helix structures that form by Hoogsteen or reverse Hoogsteen hydrogen bonding are examples of alternate DNA structures that can be a source of genomic instability. In this study, we have examined the ability of human ChlR1 helicase to destabilize DNA triplexes. Biochemical studies demonstrated that ChlR1 efficiently melted both intermolecular and intramolecular DNA triplex substrates in an ATP-dependent manner. Compared with other substrates such as replication fork and G-quadruplex DNA, triplex DNA was a preferred substrate for ChlR1. Also, compared with FANCJ, a helicase of the same family, the triplex resolving activity of ChlR1 is unique. On the other hand, the mutant protein from a Warsaw breakage syndrome patient failed to unwind these triplexes. A previously characterized triplex DNA-specific antibody (Jel 466) bound triplex DNA structures and inhibited ChlR1 unwinding activity. Moreover, cellular assays demonstrated that there were increased triplex DNA content and double-stranded breaks in ChlR1-depleted cells, but not in FANCJ(-/-) cells, when cells were treated with a triplex stabilizing compound benzoquinoquinoxaline, suggesting that ChlR1 melting of triple-helix structures is distinctive and physiologically important to defend genome integrity. On the basis of our results, we conclude that the abundance of ChlR1 known to exist in vivo is likely to be a strong deterrent to the stability of triplexes that can potentially form in the human genome., (© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.)

It has been proposed that guanine-rich DNA forms four-stranded structures in vivo called G-quadruplexes or G4 DNA. G4 DNA has been implicated in several biological processes, but tools to study G4 DNA structures in cells are limited. Here we report the development of novel murine monoclonal antibodies specific for different G4 DNA structures. We show that one of these antibodies designated 1H6 exhibits strong nuclear staining in most human and murine cells. Staining intensity increased on treatment of cells with agents that stabilize G4 DNA and, strikingly, cells deficient in FANCJ, a G4 DNA-specific helicase, showed stronger nuclear staining than controls. Our data strongly support the existence of G4 DNA structures in mammalian cells and indicate that the abundance of such structures is increased in the absence of FANCJ. We conclude that monoclonal antibody 1H6 is a valuable tool for further studies on the role of G4 DNA in cell and molecular biology.

BRCA1 promotes DNA repair through interactions with multiple proteins, including CtIP and FANCJ (also known as BRIP1/BACH1). While CtIP facilitates DNA end resection when de-acetylated, the function of FANCJ in repair processing is less well defined. Here, we report that FANCJ is also acetylated. Preventing FANCJ acetylation at lysine 1249 does not interfere with the ability of cells to survive DNA interstrand crosslinks (ICLs). However, resistance is achieved with reduced reliance on recombination. Mechanistically, FANCJ acetylation facilitates DNA end processing required for repair and checkpoint signaling. This conclusion was based on the finding that FANCJ and its acetylation were required for robust RPA foci formation, RPA phosphorylation, and Rad51 foci formation in response to camptothecin (CPT). Furthermore, both preventing and mimicking FANCJ acetylation at lysine 1249 disrupts FANCJ function in checkpoint maintenance. Thus, we propose that the dynamic regulation of FANCJ acetylation is critical for robust DNA damage response, recombination-based processing, and ultimately checkpoint maintenance., Competing Interests: The authors have declared that no competing interests exist.

Mutations in the human ChlR1 gene are associated with a unique genetic disorder known as Warsaw breakage syndrome characterized by cellular defects in sister chromatid cohesion and hypersensitivity to agents that induce replication stress. A role of ChlR1 helicase in sister chromatid cohesion was first evidenced by studies of the yeast homolog Chl1p; however, its cellular functions in DNA metabolism are not well understood. We carefully examined the DNA substrate specificity of purified recombinant human ChlR1 protein and the biochemical effect of a patient-derived mutation, a deletion of a single lysine (K897del) in the extreme C terminus of ChlR1. The K897del clinical mutation abrogated ChlR1 helicase activity on forked duplex or D-loop DNA substrates by perturbing its DNA binding and DNA-dependent ATPase activity. Wild-type ChlR1 required a minimal 5' single-stranded DNA tail of 15 nucleotides to efficiently unwind a simple duplex DNA substrate. The additional presence of a 3' single-stranded DNA tail as short as five nucleotides dramatically increased ChlR1 helicase activity, demonstrating the preference of the enzyme for forked duplex structures. ChlR1 unwound G-quadruplex (G4) DNA with a strong preference for a two-stranded antiparallel G4 (G2') substrate and was only marginally active on a four-stranded parallel G4 structure. The marked difference in ChlR1 helicase activity on the G4 substrates, reflected by increased binding to the G2' substrate, distinguishes ChlR1 from the sequence-related FANCJ helicase mutated in Fanconi anemia. The biochemical results are discussed in light of the known cellular defects associated with ChlR1 deficiency.

Alternate DNA structures that deviate from B-form double-stranded DNA such as G-quadruplex (G4) DNA can be formed by sequences that are widely distributed throughout the human genome. G-quadruplex secondary structures, formed by the stacking of planar quartets composed of four guanines that interact by Hoogsteen hydrogen bonding, can affect cellular DNA replication and transcription, and influence genomic stability. The unique metabolism of G-rich chromosomal regions that potentially form quadruplexes may influence a number of biological processes including immunoglobulin gene rearrangements, promoter activation and telomere maintenance. A number of human diseases are characterized by telomere defects, and it is proposed that G-quadruplex structures which form at telomere ends play an important role in telomere stability. Evidence from cellular studies and model organisms suggests that diseases with known defects in G4 DNA helicases are likely to be perturbed in telomere maintenance and cellular DNA replication. In this minireview, we discuss the connections of G-quadruplex nucleic acids to human genetic diseases and cancer based on the recent literature.

Alternate DNA structures other than double-stranded B-form DNA can potentially impede cellular processes such as transcription and replication. The DNA triplex helix and G4 tetraplex structures that form by Hoogsteen hydrogen bonding are two examples of alternate DNA structures that can be a source of genomic instability. In this study, we have examined the ability of human replication protein A (RPA), a single-stranded DNA binding protein that is implicated in all facets of DNA metabolism, to destabilize DNA triplexes and tetraplexes. Biochemical studies demonstrate that RPA efficiently melts an intermolecular DNA triple helix consisting of a pyrimidine motif third strand annealed to a 4 kb duplex DNA fragment at protein concentrations equimolar to the triplex substrate. Heterologous single-stranded DNA binding proteins ( Escherichia coli SSB, T4 gene 32) melt the triplex substrate very poorly or not at all, suggesting that the triplex destabilizing effect of RPA is specific. In contrast to the robust activity on DNA triplexes, RPA does not melt intermolecular G4 tetraplex structures. Cellular assays demonstrated increased triplex DNA content when RPA is transiently repressed, suggesting that RPA melting of triple helical structures is physiologically important. On the basis of our results, we suggest that the abundance of RPA known to exist in vivo is likely to be a strong deterrent to the stability of triplexes that can potentially form from human genomic DNA sequences.

DNA helicases are directly responsible for catalytically unwinding duplexDNAin an ATP-dependent and directionally specific manner and play essential roles in cellular nucleic acid metabolism. It has been conventionally thought that DNA helicases are inhibited by bulky covalent DNA adducts in a strandspecific manner. However, the effects of highly stable alkyl phosphotriester (PTE) lesions that are induced by chemical mutagens and refractory to DNA repair have not been previously studied for their effects on helicases. In this study, DNA repair and replication helicases were examined for unwinding a forked duplex DNA substrate harboring a single isopropyl PTE specifically positioned in the helicase-translocating or -nontranslocating strand within the double-stranded region. A comparison of SF2 helicases (RecQ, RECQ1, WRN, BLM, FANCJ, and ChlR1) with a SF1 DNA repair helicase (UvrD) and two replicative helicases(MCMand DnaB) demonstrates unique differences in the effect of the PTE on the DNA unwinding reactions catalyzed by these enzymes. All of the SF2 helicases tested were inhibited by the PTE lesion, whereas UvrD and the replication fork helicases were fully tolerant of the isopropyl backbone modification, irrespective of strand. Sequestration studies demonstrated that RECQ1 helicase was trapped by the PTE lesion only when it resided in the helicase-translocating strand. Our results are discussed in light of the current models for DNA unwinding by helicases that are likely to encounter sugar phosphate backbone damage during biological DNA transactions. [ABSTRACT FROM AUTHOR]