Human Werner Syndrome DNA Helicase Unwinds Tetrahelical Structures of the Fragile X Syndrome Repeat Sequence d(CGG)

Abstract

DNA helicases of the RecQ family unwind DNA with a 3Ј 3 5Ј directionality and require ATP and Mg 2ϩ for catalysis. All members of the RecQ family of proteins share seven sequence motifs common to helicases, including the characteristic DexH box (1). Prokaryotic and yeast helicases of this family include Escherichia coli RecQ, Saccharomyces cerevisiae Sgs-1p, and Schizosaccharomyces pombe Rqh-1p. Three RecQ homologues have been identified in human cells: BLM, a helicase that is mutated in cells of Bloom's syndrome patients (2); WRN, 1 a helicase mutated in Werner syndrome (3); and RecQL, a helicase of unknown function (4). The in vivo functions of most helicases of the RecQ family are not fully understood. It appears, however, that these enzymes take part in diverse DNA transactions such as replication, repair, and recombination. E. coli RecQ is believed to initiate homologous recombination and to suppress illegitimate recombination (5, 6). RecQ is also thought to be involved in the reassembly of replication forks after their disruption by UV irradiation (7,8). Phenotypes resulting from mutations in the human homologues of RecQ also indicate their involvement in multiple DNA transactions. Bloom's syndrome, caused by mutations in BLM, is associated with high spontaneous frequencies of lymphatic and other malignancies and a high frequency of somatic mutations (9, 10). Cells of Bloom's syndrome patients display increased sister chromatid exchanges, augmented sensitivity to DNA damaging agents, and defective DNA replication (11). Werner syndrome resulting from mutations that inactivate WRN is expressed by aging in early adulthood and genetic instability (12). Cells of Werner syndrome patients exhibit large DNA deletions, chromosomal rearrangements, prolongation of the S phase, and an increased sensitivity to the genotoxic agent 4NQO (13-17). The helicases responsible for the distinct pathologies of Bloom's and Werner syndromes also differ. Whereas both enzymes display 3Ј 3 5Ј DNA helicase activity, only WRN possesses an integral 3Ј 3 5Ј exonuclease activity (18 -20).The compound phenotypes of Bloom's and Werner syndromes and the multiplicity of pathologies associated with these diseases complicate the search for the in vivo roles of the responsible helicases. One possibility is that these helicases are involved in the resolution of secondary structures of DNA. Of special interest are tetrahelical structures of guanine-rich sequences (G-DNA) that readily form in vitro under physiological-like conditions (21-23). These tetraplex structures have at their core guanine quartets that are stabilized by non-WatsonCrick hydrogen bonds and are coordinated by alkali cations. Although the existence in vivo of these quadruplex structures has yet to be directly demonstrated, indirect evidence indicates that they might take part in homologous recombination (21,22) and in telomere transactions (23,24). One possible adverse effect of tetraplex G-DNA formation is in the perturbation of the progression of DNA polymerases. Readily forme...

WRN, the protein deficient in Werner syndrome, plays a critical structural role in optimizing DNA repair

“…In E-or H-cells, unbalanced WRN activity might increase the accessibility of sequences that facilitate HR. However, because the preferred substrates for WRN are complex DNA structures (Fry & Loeb, 1999;Constantinou et al, 2000;Huang et al, 2000), this hypothesis seems less likely.…”

“…WRN also exhibits intrinsic 3 ′ → 5 ′ exonuclease activity (Huang et al ., 1998), which is not found in the other RECQ helicases. The WRN helicase unwinds unusual DNA structures, such as double-stranded DNA with mismatched tails (Suzuki et al ., 1997), bimolecular G4 quartets (Fry & Loeb, 1999) and Holliday junctions (Constantinou et al ., 2000). The WRN exonuclease preferentially degrades doublestranded DNA with mismatched bubbles and staggered ends (Huang et al ., 2000;Shen & Loeb, 2000).…”

Stabilization of Telomere G-Quadruplexes Interferes with Human Herpesvirus 6A Chromosomal Integration

“…In addition to PML, these ALT-associated PML nuclear bodies (APBs) contain many other proteins that form ring-like structures around the telomeric DNA that may negatively influence DNA folding or affect BRACO-19 binding. Furthermore, some of these proteins present at telomeres of ALT ϩ cells include the BLM and WRN RecQ helicases that bind telomeric DNA (60-62) and can unwind G-quadruplex structures (63)(64)(65), thereby limiting the potential number of G-quadruplex structures. Considering that ligands that stabilize G-structures are also reported to inhibit the processivity of these enzymes (66), the net in vivo impact of G-quadruplex unfolding by helicases and stabilization by BRACO-19 remains to be determined.…”

Asymmetry of DNA replication fork progression in Werner's syndrome

“…Alternatively, forks may stall or collapse, either spontaneously or due to the presence of some lesion in the template, forming a four-way DNA junction (Cox et al, 2000). WRN may function at these junctions both as a 3′−5′ exonuclease (or an endonuclease at 5′ overhangs - Xue et al, 2002), degrading the overhang from the leading strand (in the presence of co-factors such as Ku -Cooper et al, 2000;Orren et al, 2001) and/or as a helicase (Gray et al, 1997;Fry & Loeb, 1999;Constantinou et al, 2000), to facilitate branch migration. These two activities in concert ) may allow a normal fork to be re-established (right panel).…”