Initial state of DNA-Dye complex sets the stage for protein induced fluorescence modulation

Rashid, Fahad; Raducanu, Vlad-Stefan; Zaher, Manal S.; Tehseen, Muhammad; Habuchi, Satoshi; Hamdan, Samir M.

doi:10.1038/s41467-019-10137-9

Cited by 64 publications

(79 citation statements)

References 49 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…And this may explain the rather limited number of published smFRET studies on ABC transporters labeled in the NBDs or TMDs [145], [146], [147], [148]. In the future, successful characterization of conformational dynamics may depend on alternative assays based on approaches that make quality sample preparation more readily achievable, such as the single label methods, protein induced fluorescence enhancement [149], [150] and quenching [151].…”

Section: Discussion and Outlookmentioning

confidence: 99%

An integrated transport mechanism of the maltose ABC importer

Mächtel

Narducci

Griffith

et al. 2019

Research in Microbiology

View full text Add to dashboard Cite

ATP-binding cassette (ABC) transporters use the energy of ATP hydrolysis to transport a large diversity of molecules actively across biological membranes. A combination of biochemical, biophysical, and structural studies has established the maltose transporter MalFGK2 as one of the best characterized proteins of the ABC family. MalF and MalG are the transmembrane domains, and two MalKs form a homodimer of nucleotide-binding domains. A periplasmic maltose-binding protein (MalE) delivers maltose and other maltodextrins to the transporter, and triggers its ATPase activity. Substrate import occurs in a unidirectional manner by ATP-driven conformational changes in MalK2 that allow alternating access of the substrate-binding site in MalF to each side of the membrane. In this review, we present an integrated molecular mechanism of the transport process considering all currently available information. Furthermore, we summarize remaining inconsistencies and outline possible future routes to decipher the full mechanistic details of transport by MalEFGK2 complex and that of related importer systems.

show abstract

Section: Discussion and Outlookmentioning

confidence: 99%

An integrated transport mechanism of the maltose ABC importer

Mächtel

Narducci

Griffith

et al. 2019

Research in Microbiology

View full text Add to dashboard Cite

show abstract

“…The bound proteins were eluted with a linear gradient of buffer A + 250 mM Imidazole and 150 mM NaCl. From this step onward the purification followed the one described previously 63 with few modifications as following. The collected fractions containing all the three RPA subunits were then loaded onto a HiTrap Blue 5 mL column (GE healthcare) pre-equilibrated with buffer A + 150 mM NaCl and washed extensively with buffer A + 1 M NaCl.…”

Section: Methodsmentioning

confidence: 99%

“…FEN1-D181A was expressed and purified as described previously 63 . Briefly, the plasmid was transformed into E. coli BL21 (DE3) cells and cultured in 2YT media.…”

Section: Methodsmentioning

confidence: 99%

Structure of the processive human Pol δ holoenzyme

et al. 2020

Self Cite

View full text Add to dashboard Cite

show abstract

“…D181A was expressed and purified as described previously62 . Briefly, the plasmid was transformed into E. coli BL21 (DE3) cells and cultured in 2YT media.…”

mentioning

confidence: 99%

Structure of the processive human Pol δ holoenzyme

Lancey

Tehseen

Raducanu

et al. 2019

Preprint

Self Cite

View full text Add to dashboard Cite

In eukaryotes, DNA polymerase δ (Pol δ) bound to the proliferating cell nuclear antigen (PCNA) replicates the lagging strand and cooperates with flap endonuclease 1 (FEN1) to process the Okazaki fragments for their ligation. We present the high-resolution cryo-EM structure of the human processive Pol δ-DNA-PCNA complex in the absence and presence of FEN1. Pol δ is anchored to one of the three PCNA monomers through the C-terminal domain of the catalytic subunit. The catalytic core sits on top of PCNA in an open configuration while the regulatory subunits project laterally. This arrangement allows PCNA to thread and stabilize the DNA exiting the catalytic cleft and recruit FEN1 to one unoccupied monomer in a toolbelt fashion. Alternative holoenzyme conformations reveal important functional interactions that maintain PCNA orientation during synthesis. This work sheds light on the structural basis of Pol δ's activity in replicating the human genome.Three DNA polymerases (Pols), a, d and e replicate the genomic DNA in eukaryotes, with the latter two possessing the proofreading exonuclease activity required for high fidelity DNA synthesis 1,2 . Pol d replicates the lagging strand and may share a role with Pol e in replicating the leading strand [3][4][5][6][7][8][9][10] . In contrast to the continuous leading strand synthesis, the lagging strand is synthesized discontinuously in ~200 nucleotide (nt)-long Okazaki fragments, which are then ligated to form the contiguous lagging strand 2 .Synthesis of each Okazaki fragment starts with the low fidelity Pol a synthesizing a ~30 nt RNA/DNA initiator primer. Replication factor C (RFC) then loads the homotrimeric clamp PCNA at the primer/template (P/T) junction 11 . PCNA encircles the duplex DNA 3 and tethers Pol d to the DNA enhancing its processivity from few nucleotides to hundreds of nucleotides per DNA binding event [12][13][14] . PCNA has also been shown to increase the nucleotide incorporation rate of Pol d 15 . Additionally, the interaction of PCNA with Pol d is critical for coordinating its transient replacement by other PCNA partner proteins. In the maturation of Okazaki fragments, Pol d invades the previously synthesized Okazaki fragments to gradually displace the RNA-DNA primers for their removal by the PCNA-bound FEN1 16 . In translesion DNA synthesis, Pol d is transiently replaced by a PCNA-bound translesion DNA polymerase to ensure the continuation of DNA replication 17 .Mammalian Pol d consists of a catalytic subunit and three regulatory subunits ( Figure 1a). The catalytic subunit (p125) harbours the polymerase and exonuclease activities, and a metal-binding C-terminal domain (CTD). The regulatory subunits (p50, also referred to as the B-subunit, p66 and p12) are required for optimal activity of the holoenzyme 18 and there is evidence of different context-specific subassemblies of Pol d in vivo [19][20][21] . In particular, DNA damage or replication stress triggers the degradation of the p12 subunit, resulting in the formation of a three-subunit enzyme with an...

show abstract

Initial state of DNA-Dye complex sets the stage for protein induced fluorescence modulation

Cited by 64 publications

References 49 publications

An integrated transport mechanism of the maltose ABC importer

An integrated transport mechanism of the maltose ABC importer

Structure of the processive human Pol δ holoenzyme

Structure of the processive human Pol δ holoenzyme

Contact Info

Product

Resources

About