scholarly journals Single-molecule visualization of human BLM helicase as it acts upon double- and single-stranded DNA substrates

2019 ◽  
Vol 47 (21) ◽  
pp. 11225-11237 ◽  
Author(s):  
Chaoyou Xue ◽  
James M Daley ◽  
Xiaoyu Xue ◽  
Justin Steinfeld ◽  
Youngho Kwon ◽  
...  
Keyword(s):  
Single Molecule ◽  
Molecular Mechanisms ◽  
Genetic Disorder ◽  
Protein A ◽  
Base Pairs ◽  
Single Stranded Dna ◽  
Double Stranded Dna ◽  
Blm Helicase ◽  
Dna Replication And Repair ◽  
Regulatory Effects

Abstract Bloom helicase (BLM) and its orthologs are essential for the maintenance of genome integrity. BLM defects represent the underlying cause of Bloom Syndrome, a rare genetic disorder that is marked by strong cancer predisposition. BLM deficient cells accumulate extensive chromosomal aberrations stemming from dysfunctions in homologous recombination (HR). BLM participates in several HR stages and helps dismantle potentially harmful HR intermediates. However, much remains to be learned about the molecular mechanisms of these BLM-mediated regulatory effects. Here, we use DNA curtains to directly visualize the activity of BLM helicase on single molecules of DNA. Our data show that BLM is a robust helicase capable of rapidly (∼70–80 base pairs per second) unwinding extensive tracts (∼8–10 kilobases) of double-stranded DNA (dsDNA). Importantly, we find no evidence for BLM activity on single-stranded DNA (ssDNA) that is bound by replication protein A (RPA). Likewise, our results show that BLM can neither associate with nor translocate on ssDNA that is bound by the recombinase protein RAD51. Moreover, our data reveal that the presence of RAD51 also blocks BLM translocation on dsDNA substrates. We discuss our findings within the context of potential regulator roles for BLM helicase during DNA replication and repair.

scholarly journals Polymerase theta-helicase promotes end joining by stripping single-stranded DNA-binding proteins and bridging DNA ends

2021 ◽  
Author(s):  
Jeffrey M Schaub ◽  
Michael M Soniat ◽  
Ilya J Finkelstein
Keyword(s):  
Single Molecule ◽  
Protein A ◽  
Strand Break ◽  
Helicase Domain ◽  
End Joining ◽  
Single Stranded Dna ◽  
Double Stranded Dna ◽  
Dna Strands ◽  
In Trans

Homologous recombination-deficient cancers rely on DNA polymerase Theta (Polθ)-Mediated End Joining (TMEJ), an alternative double-strand break repair pathway. Polθ is the only vertebrate polymerase that encodes an N-terminal superfamily 2 (SF2) helicase domain, but the role of this helicase domain in TMEJ remains unclear. Using single-molecule imaging, we demonstrate that Polθ-helicase (Polθ-h) is a highly processive single-stranded DNA (ssDNA) motor protein that can efficiently strip Replication Protein A (RPA) from ssDNA. Polθ-h also has a limited capacity for disassembling RAD51 filaments but is not processive on double- stranded DNA. Polθ-h can bridge two non-complementary DNA strands in trans. PARylation of Polθ-h by PARP-1 resolves these DNA bridges. We conclude that Polθ-h removes RPA and RAD51 filaments and mediates bridging of DNA overhangs to aid in polymerization by the Polθ polymerase domain.

scholarly journals Concentration-Dependent Exchange of Replication Protein A on Single-Stranded DNA Revealed by Single-Molecule Imaging

PLoS ONE ◽  
2014 ◽  
Vol 9 (2) ◽  
pp. e87922 ◽  
Author(s):  
Bryan Gibb ◽  
Ling F. Ye ◽  
Stephanie C. Gergoudis ◽  
YoungHo Kwon ◽  
Hengyao Niu ◽  
...  
Keyword(s):  
Single Molecule ◽  
Protein A ◽  
Replication Protein A ◽  
Replication Protein ◽  
Single Molecule Imaging ◽  
Single Stranded Dna

scholarly journals Mismatch sensing by nucleofilament deciphers mechanism of RecA-mediated homologous recombination

2020 ◽  
Vol 117 (34) ◽  
pp. 20549-20554
Author(s):  
Xingyuan Huang ◽  
Ying Lu ◽  
Shuang Wang ◽  
Mingyu Sui ◽  
Jinghua Li ◽  
...  
Keyword(s):  
Homologous Recombination ◽  
Single Molecule ◽  
Traveling Wave ◽  
Strand Exchange ◽  
Basic Unit ◽  
Base Pairs ◽  
Single Stranded Dna ◽  
Homologous Sequences ◽  
Transient Intermediates

Recombinases polymerize along single-stranded DNA (ssDNA) at the end of a broken DNA to form a helical nucleofilament with a periodicity of ∼18 bases. The filament catalyzes the search and checking for homologous sequences and promotes strand exchange with a donor duplex during homologous recombination (HR), the mechanism of which has remained mysterious since its discovery. Here, by inserting mismatched segments into donor duplexes and using single-molecule techniques to catch transient intermediates in HR, we found that, even though 3 base pairs (bp) is still the basic unit, both the homology checking and the strand exchange may proceed in multiple steps at a time, resulting in ∼9-bp large steps on average. More interestingly, the strand exchange is blocked remotely by the mismatched segment, terminating at positions ∼9 bp before the match–mismatch joint. The homology checking and the strand exchange are thus separated in space, with the strand exchange lagging behind. Our data suggest that the strand exchange progresses like a traveling wave in which the donor DNA is incorporated successively into the ssDNA–RecA filament to check homology in ∼9-bp steps in the frontier, followed by a hypothetical transitional segment and then the post-strand-exchanged duplex.

scholarly journals Switch-like control of helicase processivity by single-stranded DNA binding protein

2020 ◽  
Author(s):  
Barbara Stekas ◽  
Masayoshi Honda ◽  
Maria Spies ◽  
Yann R. Chemla
Keyword(s):  
Dna Binding ◽  
Single Molecule ◽  
Molecular Mechanisms ◽  
Binding Protein ◽  
Optical Trap ◽  
Dna Binding Protein ◽  
Single Stranded Dna ◽  
Associated Proteins ◽  
Underlying Mechanisms ◽  
Xpd Helicase

Helicases utilize the energy of NTP hydrolysis to translocate along single-stranded nucleic acids (NA) and unwind the duplex. In the cell, helicases function in the context of other NA-associated proteins which regulate helicase function. For example, single-stranded DNA binding proteins are known to enhance helicase activity, although the underlying mechanisms remain largely unknown. F. acidarmanus XPD helicase serves as a model for understanding the molecular mechanisms of Superfamily 2B helicases, and previous work has shown that its activity is enhanced by the cognate single-stranded DNA binding protein RPA2. Here, single-molecule optical trap measurements of the unwinding activity of a single XPD helicase in the presence of RPA2 reveal a mechanism in which XPD interconverts between two states with different processivities and transient RPA2 interactions stabilize the more processive state, activating a latent “processivity switch” in XPD. These findings provide new insights on mechanisms of helicase regulation by accessory proteins.

scholarly journals Molecular Mechanisms of Staphylococcus and Pseudomonas Interactions in Cystic Fibrosis

2022 ◽  
Vol 11 ◽  
Author(s):  
Lalitha Biswas ◽  
Friedrich Götz
Keyword(s):  
Cystic Fibrosis ◽  
Molecular Mechanisms ◽  
Genetic Disorder ◽  
Protein A ◽  
Chronic Infections ◽  
Transport Pathway ◽  
Small Colony Variant ◽  
Cooperative Interactions ◽  
Small Colony ◽  
Infancy And Early Childhood

Cystic fibrosis (CF) is an autosomal recessive genetic disorder that is characterized by recurrent and chronic infections of the lung predominantly by the opportunistic pathogens, Gram-positive Staphylococcus aureus and Gram-negative Pseudomonas aeruginosa. While S. aureus is the main colonizing bacteria of the CF lungs during infancy and early childhood, its incidence declines thereafter and infections by P. aeruginosa become more prominent with increasing age. The competitive and cooperative interactions exhibited by these two pathogens influence their survival, antibiotic susceptibility, persistence and, consequently the disease progression. For instance, P. aeruginosa secretes small respiratory inhibitors like hydrogen cyanide, pyocyanin and quinoline N-oxides that block the electron transport pathway and suppress the growth of S. aureus. However, S. aureus survives this respiratory attack by adapting to respiration-defective small colony variant (SCV) phenotype. SCVs cause persistent and recurrent infections and are also resistant to antibiotics, especially aminoglycosides, antifolate antibiotics, and to host antimicrobial peptides such as LL-37, human β-defensin (HBD) 2 and HBD3; and lactoferricin B. The interaction between P. aeruginosa and S. aureus is multifaceted. In mucoid P. aeruginosa strains, siderophores and rhamnolipids are downregulated thus enhancing the survival of S. aureus. Conversely, protein A from S. aureus inhibits P. aeruginosa biofilm formation while protecting both P. aeruginosa and S. aureus from phagocytosis by neutrophils. This review attempts to summarize the current understanding of the molecular mechanisms that drive the competitive and cooperative interactions between S. aureus and P. aeruginosa in the CF lungs that could influence the disease outcome.

scholarly journals Human RPA activates BLM’s bidirectional DNA unwinding from a nick

eLife ◽  
2020 ◽  
Vol 9 ◽  
Author(s):  
Zhenheng Qin ◽  
Lulu Bi ◽  
Xi-Miao Hou ◽  
Siqi Zhang ◽  
Xia Zhang ◽  
...  
Keyword(s):  
Dna Repair ◽  
Dna Replication ◽  
Single Molecule ◽  
Genome Stability ◽  
Protein A ◽  
Replication Protein A ◽  
Replication Protein ◽  
High Abundance ◽  
Dna Unwinding ◽  
Dna Replication And Repair

BLM is a multifunctional helicase that plays critical roles in maintaining genome stability. It processes distinct DNA substrates, but not nicked DNA, during many steps in DNA replication and repair. However, how BLM prepares itself for diverse functions remains elusive. Here, using a combined single-molecule approach, we find that a high abundance of BLMs can indeed unidirectionally unwind dsDNA from a nick when an external destabilizing force is applied. Strikingly, human replication protein A (hRPA) not only ensures that limited quantities of BLMs processively unwind nicked dsDNA under a reduced force but also permits the translocation of BLMs on both intact and nicked ssDNAs, resulting in a bidirectional unwinding mode. This activation necessitates BLM targeting on the nick and the presence of free hRPAs in solution whereas direct interactions between them are dispensable. Our findings present novel DNA unwinding activities of BLM that potentially facilitate its function switching in DNA repair.

scholarly journals Single-molecule visualization of human RECQ5 interactions with single-stranded DNA recombination intermediates

2020 ◽  
Author(s):  
Chaoyou Xue ◽  
Lucia Molnarova ◽  
Justin B Steinfeld ◽  
Weixing Zhao ◽  
Chujian Ma ◽  
...  
Keyword(s):  
Single Molecule ◽  
Atp Hydrolysis ◽  
Protein A ◽  
Dna Recombination ◽  
Binding Activity ◽  
Negative Regulator ◽  
Genome Maintenance ◽  
Single Stranded Dna ◽  
Ssdna Binding ◽  
Translocation Velocity

Abstract RECQ5 is one of five RecQ helicases found in humans and is thought to participate in homologous DNA recombination by acting as a negative regulator of the recombinase protein RAD51. Here, we use kinetic and single molecule imaging methods to monitor RECQ5 behavior on various nucleoprotein complexes. Our data demonstrate that RECQ5 can act as an ATP-dependent single-stranded DNA (ssDNA) motor protein and can translocate on ssDNA that is bound by replication protein A (RPA). RECQ5 can also translocate on RAD51-coated ssDNA and readily dismantles RAD51–ssDNA filaments. RECQ5 interacts with RAD51 through protein–protein contacts, and disruption of this interface through a RECQ5–F666A mutation reduces translocation velocity by ∼50%. However, RECQ5 readily removes the ATP hydrolysis-deficient mutant RAD51–K133R from ssDNA, suggesting that filament disruption is not coupled to the RAD51 ATP hydrolysis cycle. RECQ5 also readily removes RAD51–I287T, a RAD51 mutant with enhanced ssDNA-binding activity, from ssDNA. Surprisingly, RECQ5 can bind to double-stranded DNA (dsDNA), but it is unable to translocate. Similarly, RECQ5 cannot dismantle RAD51-bound heteroduplex joint molecules. Our results suggest that the roles of RECQ5 in genome maintenance may be regulated in part at the level of substrate specificity.

scholarly journals Molecular characteristics of reiterative DNA unwinding by the Caenorhabditis elegans RecQ helicase

2019 ◽  
Vol 47 (18) ◽  
pp. 9708-9720 ◽  
Author(s):  
Seoyun Choi ◽  
Seung-Won Lee ◽  
Hajin Kim ◽  
Byungchan Ahn
Keyword(s):  
Caenorhabditis Elegans ◽  
Single Molecule ◽  
Molecular Mechanisms ◽  
Protein A ◽  
Direct Interaction ◽  
Premature Aging ◽  
Molecular Characteristics ◽  
Dna Unwinding ◽  
Recq Helicase ◽  
Recq Helicases

Abstract The RecQ family of helicases is highly conserved both structurally and functionally from bacteria to humans. Defects in human RecQ helicases are associated with genetic diseases that are characterized by cancer predisposition and/or premature aging. RecQ proteins exhibit 3′-5′ helicase activity and play critical roles in genome maintenance. Recent advances in single-molecule techniques have revealed the reiterative unwinding behavior of RecQ helicases. However, the molecular mechanisms involved in this process remain unclear, with contradicting reports. Here, we characterized the unwinding dynamics of the Caenorhabditis elegans RecQ helicase HIM-6 using single-molecule fluorescence resonance energy transfer measurements. We found that HIM-6 exhibits reiterative DNA unwinding and the length of DNA unwound by the helicase is sharply defined at 25–31 bp. Experiments using various DNA substrates revealed that HIM-6 utilizes the mode of ‘sliding back’ on the translocated strand, without strand-switching for rewinding. Furthermore, we found that Caenorhabditis elegans replication protein A, a single-stranded DNA binding protein, suppresses the reiterative behavior of HIM-6 and induces unidirectional, processive unwinding, possibly through a direct interaction between the proteins. Our findings shed new light on the mechanism of DNA unwinding by RecQ family helicases and their co-operation with RPA in processing DNA.

scholarly journals Concentration-Dependent Exchange of Replication Protein a on Single-Stranded DNA Revealed by Single-Molecule Imaging

2014 ◽  
Vol 106 (2) ◽  
pp. 274a ◽  
Author(s):  
Bryan Gibb ◽  
Ling F. Ye ◽  
Stephanie C. Gergoudis ◽  
YoungHo Kwon ◽  
Hengyao Niu ◽  
...  
Keyword(s):  
Single Molecule ◽  
Protein A ◽  
Replication Protein A ◽  
Replication Protein ◽  
Single Molecule Imaging ◽  
Single Stranded Dna

scholarly journals Rtt105 promotes high-fidelity DNA replication and repair by regulating the single-stranded DNA-binding factor RPA

2021 ◽  
Vol 118 (25) ◽  
pp. e2106393118
Author(s):  
Xuejie Wang ◽  
Yang Dong ◽  
Xiaocong Zhao ◽  
Jinbao Li ◽  
Jordan Lee ◽  
...  
Keyword(s):  
Dna Replication ◽  
Protein A ◽  
High Fidelity ◽  
End Joining ◽  
Single Stranded Dna ◽  
Interacting Protein ◽  
Ssdna Binding ◽  
Single Strand Annealing ◽  
Dna Replication And Repair ◽  
Strand Annealing

Single-stranded DNA (ssDNA) covered with the heterotrimeric Replication Protein A (RPA) complex is a central intermediate of DNA replication and repair. How RPA is regulated to ensure the fidelity of DNA replication and repair remains poorly understood. Yeast Rtt105 is an RPA-interacting protein required for RPA nuclear import and efficient ssDNA binding. Here, we describe an important role of Rtt105 in high-fidelity DNA replication and recombination and demonstrate that these functions of Rtt105 primarily depend on its regulation of RPA. The deletion of RTT105 causes elevated spontaneous DNA mutations with large duplications or deletions mediated by microhomologies. Rtt105 is recruited to DNA double-stranded break (DSB) ends where it promotes RPA assembly and homologous recombination repair by gene conversion or break-induced replication. In contrast, Rtt105 attenuates DSB repair by the mutagenic single-strand annealing or alternative end joining pathway. Thus, Rtt105-mediated regulation of RPA promotes high-fidelity replication and recombination while suppressing repair by deleterious pathways. Finally, we show that the human RPA-interacting protein hRIP-α, a putative functional homolog of Rtt105, also stimulates RPA assembly on ssDNA, suggesting the conservation of an Rtt105-mediated mechanism.

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