G-quadruplex and G-rich sequence stimulate Pif1p-catalyzed downstream duplex DNA unwinding through reducing waiting time at ss/dsDNA junction

Published online 28 July 2016 Nucleic Acids Research, 2016, Vol. 44, No. 17 8385–8394 doi: 10.1093/nar/gkw669 G-quadruplex and G-rich sequence stimulate Pif1p-catalyzed downstream duplex DNA unwinding through reducing waiting time at ss/dsDNA junction Bo Zhang1,† , Wen-Qiang Wu1,† , Na-Nv Liu1 , Xiao-Lei Duan1 , Ming Li2 , Shuo-Xing Dou2 , Xi-Miao Hou1,* and Xu-Guang Xi1,3,* 1 College of Life Sciences, Northwest A&F University, Yangling, Shaanxi 712100, China, 2 Beijing National Laboratory for Condensed Matter Physics and CAS Key Laboratory of Soft Matter Physics, Institute of Physics, Chinese Academy of Sciences, Beijing 100190, China and 3 Laboratoire de Biologie et Pharmacologie Appliquée, Ecole Normale Supérieure de Cachan, Centre National de la Recherche Scientifique, 61 Avenue du Président Wilson, 94235 Cachan, France Received April 5, 2016; Revised July 18, 2016; Accepted July 19, 2016 ABSTRACT Alternative DNA structures that deviate from B-form double-stranded DNA such as G-quadruplex (G4) DNA can be formed by G-rich sequences that are widely distributed throughout the human genome. We have previously shown that Pif1p not only unfolds G4, but also unwinds the downstream duplex DNA in a G4-stimulated manner. In the present study, we further characterized the G4-stimulated duplex DNA unwinding phenomenon by means of single-molecule fluorescence resonance energy transfer. It was found that Pif1p did not unwind the partial duplex DNA immediately after unfolding the upstream G4 structure, but rather, it would dwell at the ss/dsDNA junction with a ‘waiting time’. Further studies revealed that the waiting time was in fact related to a protein dimerization process that was sensitive to ssDNA sequence and would become rapid if the sequence is G-rich. Furthermore, we identified that the G-rich sequence, as the G4 structure, equally stimulates duplex DNA unwinding. The present work sheds new light on the molecular mechanism by which G4-unwinding helicase Pif1p resolves physiological G4/duplex DNA structures in cells. INTRODUCTION G-quadruplex (G4) DNA is a four-stranded non-canonical structure held together by Hoogsteen base pairs and further stabilized by monovalent cations K+ or Na+ (1–3). Stable G4 structures were found in sub-telomeres, intron-extron splicing junction sites, untranslated regions, gene bodies and gene expression regulatory regions such as promoters (4). The existence of G4 in living cells has been confirmed using an engineered antibody that can recognize G4 structure with high affinity and specificity (5,6). Initial computational analyses have revealed that there are >375 000 G4 motifs in the human genome (7,8). Recent high-resolution sequencing based method has identified >2 times higher than the previous prediction (9), highlighting the importance of G4 in genome integrity. Indeed, G4s have been shown to be implicated in critical cellular processes including initiation of DNA replication at the origin, restart of the collapsed replication fork, DNA recombination and telomere maintenance (10). At each cell division in human, 30 000–50 000 DNA replication origins are activated, and it remains unclear how they are selected and recognized by replication factors (11). The recent advances have shown that G-rich repeated elements are present in 67–90% of the DNA replication origins from Drosophila to human cells (12). More importantly, it appears that it is G4 and its orientation that determine the precise position of the replication start site (13). These observations raise the question: how G4 influences its downstream duplex DNA unwinding for providing a platform for replicon assembly? Similarly, the same question may also be raised for G4induced replication fork stalling and restart of the collapsed replication fork after G4 unfolding. During DNA replication, replicative helicases separate the two strands to form a two-pronged replication fork. The synthesis along the leading-strand template is continuous and that along the lagging strand is discontinuous, leading to the formation of long single-stranded regions termed as Okazaki fragments * To whom correspondence should be addressed. Tel: +86 29 8708 1664; Fax: +86 29 87081664; Email: Correspondence may also be addressed to Xu-Guang Xi. Tel: +33 1 4740 7754; Fax: +33 1 4740 7754; Email: † These authors contributed equally to the paper as first authors.  C The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact 8386 Nucleic Acids Research, 2016, Vol. 44, No. 17 (14). If these regions contain G-rich sequences, formation of G4s along the lagging-strand template will slow down replication and increase the likelihood of chromosomal breakage and genomic rearrangement (15,16). Similarly, G4 may also be formed along the leading-strand template in regions of G-rich single-stranded DNA due to transient discordance between the replicative helicase and leading-strand DNA polymerase (17). The cell must face the serious problem of handling these obstacles for an ongoing synthesis of lagging or leading strand by unfolding G4 (18). In this regard, there are at least two fundamental questions: (i) how these G4s are resolved by special helicase? (ii) After unfolding a G4, the helicase immediately meets the downstream duplex DNA, then how the helicase toggles its function or activity from G4 unfolding to duplex unwinding? Concerning the first question, there are many studies showing that as formation of such stable G4 structures may threaten genomic stability, cells have evolved a special family of helicases to unfold G4 and remove those obstacles. A number of G4-helicases have been identified, illustrated by, but not limited to, RecQ family (19), Pif1 family DNA helicases (20,21) and adenosine triphosphate (ATP)-dependent DExH/D family RNA helicases (22). The Saccharomyces cerevisiae Pif1 helicase (Pif1p), has been shown to suppress genome instability at G4 motifs by means of its potent G4 unwinding activity and to keep cells from replication fork impairment, unusual epigenetic silencing and gross chromosomal rearrangement (23–25), which were otherwise observed in a Pif1p deficient strain (Pif1Δ) (23,26). We and others have studied the molecular mechanism of Pif1pmediated G4 unfolding (27,28) and found that Pif1p unfolds G4 in two large steps, and then halts at the ss/dsDNA junction, followed by rapid reformation of G4 and re-initiation of unfolding by the same monomer (27). As to the second question, we have previously studied, using stopped-flow method, how the duplex DNA downstream of G4 was unwound by Pif1p helicase and found that G4 greatly (...truncated)