Mechanistic Insights Into E.coli DNA Polymerase ? And RecQ Helicase Mediated G-quadruplex Disruption

G-quadruplexes(G4s)are non-canonical four-stranded DNA structures stabilized by Hoogesteen base pairs between Guanine residues that are widely distributed in the genome.Stable G4 structures have been found to generally cluster in specific regions of the genome including chromosome ends,DNA replication origins,gene transcriptional regulatory regions,promoters of certain oncogenes and immunoglobulin switch regions.Recent high-resolution sequencing based method has identified 716,310 G4 motifs in the human genome,highlighting their in vivo significances.Indeed,landmark studies using G4-specific engineered antibody or drug have confirmed the existence of G4 in living cells.It is generally believed that G4 formation can influence biological processes including DNA replication,translation and telomere integrity maintenance.Recent advances have confirmed that G4 structure will be encountered by DNA polymerase during genomic replication process,causing replication fork stalled.In the meanwhile,emerging evidence has also demonstrated that some translesion DNA synthesis(TLS)polymerases are implicated in G4 structures processing to promote fork progression,especially polymerase Rev1,polymerase ? and ?.While knowledge about how polymerase deal with G4 structure has been documented with eukaryote and mammalian cells,the genetic evidence that the present situation of G4 and polymerases involved in G4-resolving in E.coli has not been reported according to our knowledge.While five kinds of E.coli DNA polymerases participate in the elongation of DNA strand named polymerase I,II,III,IV and V,polymerase I is the most abundant DNA polymerase in E.coli.In the meanwhile,Pol I widely participates in the maturation of Okazaki fragments during DNA replication,DNA repair,DNA recombination and even TLS processes,in which the transiently occurring G-rich ssDNA has ample opportunity to form a G-quadruplex structure.Thus,Pol I must meet G4 challenge during the above-mentioned DNA transaction processes.In this report,we systematacially characterized the interaction between G4 and Pol I(and Klenow fragment)by combination of single-molecule fluorescence resonance energy transfer(smFRET),DNA polymerase stop assay and stopped-flow assay.The main results were shown as follows: 1)The analysis by bioinformatics indicated that G4 structures indeed existed in E.coli genome and two classes of highly conserved motifs were identified.What's more,the analysis of next generation sequencing(NGS)data from the E.coli genome implied that G4 structures will block DNA polymerase during genome sequencing.2)The replication process of G4 with low polymerase concentration was detected by single molecular FRET.It indicated that the presence of G4 structure in template strand will partially or completely block DNA synthesis depending on G4 stability.3)by using DNA polymerase stop assay,we found that,at physiological concentration of protein,Pol I in 100 mM KCl displays the ability to overcome some kinds of stable G4 obstacles and keep on synthesizing DNA without the involvement of other kinds of proteins.4)By using stopped-flow assay,we revealed the molecular mechanism of Pol I-mediated G4 disruption,in which multiple polymerase I may be implicated in G4 unfolding,and the disruption of G4 needs the energy derived from dNTPs hydrolysis.In the meanwhile,RecQ helicase can specifically bind and unwind G4 structure in E.coli.As the structure and function prototype of RecQ-family helicase,E.coli RecQ widely participated in the cell metabolisms,especially DNA damage repair and DNA homologous recombination.In this report,we systematically detected the DNA binding,dsDNA unwinding and G4 DNA unwinding processes by RecQ and its mutants with smFRET assay.In addition,the influence of HRDC domain on the activity of E.coli RecQ has also been investigated.The main results were shown as follows: 1)The DNA binding activity of RecQ was very strong and effected by HRDC domain remarkably.Further experiments indicated that the increase of DNA binding activity by HRDC domain was caused by the interaction between HRDC domain and the RecA core of RecQ,rather than the direct DNA binding activity of HRDC domain.2)There was a distinct ‘unwinding initiation time' before dsDNA unwinding process,which was also prominent prolonged in the presence of HRDC domain.In the meantime,strand-switch process was detected during RecQ unwinding process.3)RecQ occupied the activity of reeling in ssDNA occasionally,and HRDC domain significantly suppressed the reeling process of RecQ.4)Although G4 structure was an obstacle for RecQ,we found RecQ can catalyze G4 DNA unfolding with an ATP-dependent manner.By determining the degree of G4 disrupted and the duration time of each G4 unfolding,we concluded that HRDC domain of RecQ would take a positive effect on G4 disruption.Above all,our studies provided the first biochemical evidence for bacterial DNA polymerase to disrupt G4 structures,and the first real-time processes of the DNA binding,dsDNA unwinding and G4 DNA unwinding by RecQ and its mutants,which not only provided a meaningful reference on methods for the future studies of other polymerases or helicases interacting with G4 strucute,but also deepened our understanding about the DNA transaction processes in E.coli.