Structural basis for DNA unwinding at forked dsDNA by two coordinating Pif1 helicases

Su, Nannan; Byrd, Alicia K.; Bharath, S.R.; Yang, Olivia; Yu, Jia; Tang, Xuhua; Ha, Taekjip; Raney, Kevin D.; Song, Haiwei

doi:10.1038/s41467-019-13414-9

Cited by 23 publications

(22 citation statements)

References 49 publications

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“…With the same analysis, the ToPif1 complexed with a DNA hairpin bearing a 15-nt 5′-ssDNA overhang (ToPif1-S 15 H 11 -ADP·AlF 4 ) fits well with a dimeric model ( χ 2 = 1.027), in which the 15-nt ssDNA is bound with a head-to-tail dimer while the dsDNA is located at the putative catalytic site (Figure 3E and F and Supplementary Figure S7A and B ). In addition, the comparison with BaPif1 bound to a DNA fork ( 35 ) shows that the two dimers are very different and different surfaces are involved ( Supplementary Figure S7C−E ).…”

Section: Resultsmentioning

confidence: 99%

“…The previous dimeric structure of BaPif1 in complex with a forked dsDNA (PDB code: 6L3G) has revealed how two BaPif1 molecules coordinate with each other to unwind a forked dsDNA. In this dimeric structure, the BaPif1 molecule bound to the 5′ arm and the other one bound to the 3′ ss/dsDNA junction dimerize through residue interactions between domain 2B of the former and domain 2A of the latter ( 35 ). Unlike ToPif1, the dimerization does not alter the domain orientations of the two BaPif1 molecules and each of which is an active helicase.…”

Section: Discussionmentioning

confidence: 99%

“…However, Pif1 family also exhibits domain variability as, for instance, a large insertion in domain 2B exists in ScPif1 ( 32 ) and a WYL domain at the C-terminal part is found in Pif1 helicases from thermophilic bacteria Thermotoga elfii ( 33 ) and Deferribacter desulfuricans ( Supplementary Figure S1 , alignment of Pif1 proteins from bacteria, yeast and human). In addition, a structure of BaPif1 (Pif1 form Bacteroides sp , PDB code: 6L3G) dimer in complex with a forked dsDNA has been observed, with the two interacting molecules binding to the 5′ arm and 3′ ss/dsDNA junction, respectively, and regulating each other’s helicase activity ( 35 ). Extensive biochemical studies have revealed characteristics of Pif1 helicases in duplex DNA unwinding.…”

Section: Introductionmentioning

confidence: 99%

See 2 more Smart Citations

Structural and functional studies of SF1B Pif1 from Thermus oshimai reveal dimerization-induced helicase inhibition

Dai

Chen

Liu

et al. 2021

Nucleic Acids Research

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Pif1 is an SF1B helicase that is evolutionarily conserved from bacteria to humans and plays multiple roles in maintaining genome stability in both nucleus and mitochondria. Though highly conserved, Pif1 family harbors a large mechanistic diversity. Here, we report crystal structures of Thermus oshimai Pif1 (ToPif1) alone and complexed with partial duplex or single-stranded DNA. In the apo state and in complex with a partial duplex DNA, ToPif1 is monomeric with its domain 2B/loop3 adopting a closed and an open conformation, respectively. When complexed with a single-stranded DNA, ToPif1 forms a stable dimer with domain 2B/loop3 shifting to a more open conformation. Single-molecule and biochemical assays show that domain 2B/loop3 switches repetitively between the closed and open conformations when a ToPif1 monomer unwinds DNA and, in contrast with other typical dimeric SF1A helicases, dimerization has an inhibitory effect on its helicase activity. This mechanism is not general for all Pif1 helicases but illustrates the diversity of regulation mechanisms among different helicases. It also raises the possibility that although dimerization results in activation for SF1A helicases, it may lead to inhibition for some of the other uncharacterized SF1B helicases, an interesting subject warranting further studies.

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Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Structural and functional studies of SF1B Pif1 from Thermus oshimai reveal dimerization-induced helicase inhibition

Dai

Chen

Liu

et al. 2021

Nucleic Acids Research

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“…Failure to bypass these barriers may result in genome instability, which can lead to cellular abnormalities and genetic disease. Cells contain various accessory helicases that help the replisome bypass these difficult barriers (10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20). A subset of these helicases act on the opposite strand of the replicative helicase (1,2,14,19).…”

mentioning

confidence: 99%

Replisome bypass of a protein-based R-loop block by Pif1

Schauer

Spenkelink

Lewis

et al. 2020

Proc. Natl. Acad. Sci. U.S.A.

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Efficient and faithful replication of the genome is essential to maintain genome stability. Replication is carried out by a multiprotein complex called the replisome, which encounters numerous obstacles to its progression. Failure to bypass these obstacles results in genome instability and may facilitate errors leading to disease. Cells use accessory helicases that help the replisome bypass difficult barriers. All eukaryotes contain the accessory helicase Pif1, which tracks in a 5′–3′ direction on single-stranded DNA and plays a role in genome maintenance processes. Here, we reveal a previously unknown role for Pif1 in replication barrier bypass. We use an in vitro reconstitutedSaccharomyces cerevisiaereplisome to demonstrate that Pif1 enables the replisome to bypass an inactive (i.e., dead) Cas9 (dCas9) R-loop barrier. Interestingly, dCas9 R-loops targeted to either strand are bypassed with similar efficiency. Furthermore, we employed a single-molecule fluorescence visualization technique to show that Pif1 facilitates this bypass by enabling the simultaneous removal of the dCas9 protein and the R-loop. We propose that Pif1 is a general displacement helicase for replication bypass of both R-loops and protein blocks.

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“…In general, SF 3–6 helicases form hexamers and are highly processive enzymes, while SF 1–2 helicases are moderately processive or distributive enzymes that can translocate along single‐ or double‐stranded DNA (dsDNA) as monomers [5]. However, several reports indicate that dimerization of SF 1–2 helicases may be required for optimal enzyme processivity or substrate specificity and could be important for DNA unwinding in vivo [6–11].…”

mentioning

confidence: 99%

Irc3 is a monomeric DNA branch point‐binding helicase in mitochondria of the yeast Saccharomyces cerevisiae

et al. 2020

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Irc3 is a superfamily II DNA helicase required for the maintenance of mitochondrial DNA stability in Saccharomyces cerevisiae. Here, we show that recombinant Irc3 is a monomeric protein and that it can form a binary complex with forked DNA. The catalytically active enzyme is a monomer as no positive cooperativity of ATP hydrolysis or DNA unwinding can be detected. Interestingly, we find that Irc3 prefers to unwind the nascent lagging strand at a replication fork. Using DNase I footprinting, we demonstrate that Irc3 captures DNA substrates by establishing a strong contact at the DNA branching point. Additional protections on the lagging strand template suggest a 3 0-to-5 0 polarity for Irc3 movement.

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Structural basis for DNA unwinding at forked dsDNA by two coordinating Pif1 helicases

Cited by 23 publications

References 49 publications

Structural and functional studies of SF1B Pif1 from Thermus oshimai reveal dimerization-induced helicase inhibition

Structural and functional studies of SF1B Pif1 from Thermus oshimai reveal dimerization-induced helicase inhibition

Replisome bypass of a protein-based R-loop block by Pif1

Irc3 is a monomeric DNA branch point‐binding helicase in mitochondria of the yeast Saccharomyces cerevisiae

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