Structure of a Holliday junction complex reveals mechanisms governing a highly regulated DNA transaction

Laxmikanthan, Gurunathan; Xu, Chen; Brilot, Axel F.; Warren, David J.; Steele, Lindsay; Seah, Nicole; Tong, Wenjun; Grigorieff, Nikolaus; Landy, Arthur; Duyne, Gregory D. Van

doi:10.7554/elife.14313

Cited by 24 publications

(31 citation statements)

References 67 publications

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“…DNase I footprints of Apl at its binding sites at pR-pL and at att P are indicative of Apl binding on the inside face of bent DNA ( 17 ). Our results and previous studies establish a number of features of Apl DNA binding, many or all of which are shared with other well-studied RDFs ( 1–2 , 5 , 9 , 12–14 , 20 , 41 ): (i) the operators are arranged as direct repeats, spaced roughly one turn of the DNA helix apart, presumably with one monomer bound per operator; (ii) binding to a single specific operator is weak; (iii) binding to adjacent operators is highly cooperative; (iv) binding causes DNA bending; and (v) the difference in affinity for specific and non-specific sites is small, presumably reflecting flexible sequence recognition. The presence of these features in Apl supports the idea that this basic mode of DNA binding is universal in this group of proteins ( 13 , 14 ).…”

Section: Discussionsupporting

confidence: 87%

“…Where examined, RDFs have been shown to cause large bends in attachment site ( att ) DNA (λ Xis ( 7–9 ), P2 Cox ( 10 ), L5 Xis ( 11 ), P4 Vis ( 12 ), Wϕ Cox ( 10 ), P22 Xis ( 13 ) and Pukovnik Xis ( 5 )). A cryo-EM structure of the Holliday junction intermediate of the λ excisive complex ( 9 ) revealed three key roles for λ Xis in formation of the complex, including promoting integrase (Int) binding, mediating an Xis/Int interface, and bending of att DNA to position the DNA for cooperative Int binding. A crystal structure of λ Xis, showed three Xis monomers bound to the X1-X1.5-X2 sites in att R causing a 72° non-planar bend in the DNA, leading to the hypothesis that a twisted microfilament forms ( 14 ), a hypothesis supported by DNA compaction studies on P2 Cox ( 15 ).…”

Section: Introductionmentioning

confidence: 99%

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A quantitative binding model for the Apl protein, the dual purpose recombination-directionality factor and lysis-lysogeny regulator of bacteriophage 186

Cutts

Egan

Dodd

et al. 2020

Nucleic Acids Research

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The Apl protein of bacteriophage 186 functions both as an excisionase and as a transcriptional regulator; binding to the phage attachment site (att), and also between the major early phage promoters (pR-pL). Like other recombination directionality factors (RDFs), Apl binding sites are direct repeats spaced one DNA helix turn apart. Here, we use in vitro binding studies with purified Apl and pR-pL DNA to show that Apl binds to multiple sites with high cooperativity, bends the DNA and spreads from specific binding sites into adjacent non-specific DNA; features that are shared with other RDFs. By analysing Apl's repression of pR and pL, and the effect of operator mutants in vivo with a simple mathematical model, we were able to extract estimates of binding energies for single specific and non-specific sites and for Apl cooperativity, revealing that Apl monomers bind to DNA with low sequence specificity but with strong cooperativity between immediate neighbours. This model fit was then independently validated with in vitro data. The model we employed here is a simple but powerful tool that enabled better understanding of the balance between binding affinity and cooperativity required for RDF function. A modelling approach such as this is broadly applicable to other systems.

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Section: Discussionsupporting

confidence: 87%

Section: Introductionmentioning

confidence: 99%

A quantitative binding model for the Apl protein, the dual purpose recombination-directionality factor and lysis-lysogeny regulator of bacteriophage 186

Cutts

Egan

Dodd

et al. 2020

Nucleic Acids Research

View full text Add to dashboard Cite

show abstract

“…The role played by IHF also represents a surprising variation on a feature sometimes seen in transposases. In both λ and μ phage mobilization pathways, IHF, or the related protein HU, are involved in bringing recognition sequences on the viral DNA into contact with the integrase (29, 30). Notably, in the phage pathways, IHF aids in the recognition of donor DNA, while in CRISPR acquisition it is important for recognition of the target DNA, highlighting the shift in substrate selectivity from donor to target that was essential for the “domestication” of Cas1 for use in immunity (25, 26).…”

Section: Discussionmentioning

confidence: 99%

Structures of the CRISPR genome integration complex

Wright

Liu

Knott

et al. 2017

Science

127

150

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CRISPR-Cas systems depend on the Cas1-Cas2 integrase to capture and integrate short foreign DNA fragments into the CRISPR locus, enabling adaptation to new viruses. We present crystal structures of Cas1-Cas2 bound to both donor and target DNA in intermediate and product integration complexes, as well as a cryo-electron microscopy structure of the full CRISPR locus integration complex including the accessory protein Integration Host Factor (IHF). The structures show unexpectedly that indirect sequence recognition dictates integration site selection by favoring deformation of the repeat and the flanking sequences. IHF binding bends the DNA sharply, bringing an upstream recognition motif into contact with Cas1 to increase both the specificity and efficiency of integration. These results explain how the Cas1-Cas2 CRISPR integrase recognizes a sequence-dependent DNA structure to ensure site-selective CRISPR array expansion during the initial step of bacterial adaptive immunity.

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“…Modeling of this intasome shows that, while the IHF-H' complex might need to flex slightly to allow synergetic binding of two domains of integrase to DNA 31 sequences flanking it, it can remain static throughout the integration reaction. 58 However, in the fully bent form of the IHF-H1 complex, the flanking DNA and the copy of integrase bound to it block incorporation of the bacterial attB DNA segment. Significant flexing of the H1-induced DNA bend as shown in Figure 9 is therefore required for its biological function.…”

Section: Discussionmentioning

confidence: 99%

Static Kinks or Flexible Hinges: Conformational Distributions of Bent DNA Bound to Integration Host Factor Mapped by Fluorescence Lifetime Measurements

Connolly

Arra

Zvoda

et al. 2018

Preprint

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Gene regulation depends on proteins that bind to specific DNA sites. Such specific recognition often involves severe DNA deformations including sharp kinks. It has been unclear how rigid or flexible these protein-induced kinks are. Here, we investigated the dynamic nature of DNA in complex with integration host factor (IHF), a nucleoid-associated architectural protein known to bend one of its cognate sites (35 base pair H') into a U-turn by kinking DNA at two sites. We utilized fluorescence lifetime based FRET spectroscopy to map the distribution of bent conformations in various IHF-DNA complexes. Our results reveal a surprisingly dynamic specific complex: while 80% of the IHF-H' population exhibited FRET efficiency consistent with the crystal structure, 20% exhibited FRET efficiency indicative of unbent or partially bent DNA. This conformational flexibility is modulated by sequence variations in the cognate site. In another site (H1) that lacks an A-tract of H' on one side of the binding site, the population in the fully U-bent conformation decreased to 36%, as did the extent of bending. A similar decrease in the U-bent population was observed with a single base mutation in H' in a consensus region on the other side. Taken together, these results provide important insights into the finely tuned interactions between IHF and its cognate sites that keep the DNA bent (or not), and yield quantitative data on the dynamic equilibrium between different DNA conformations (kinked or not kinked) that depend sensitively on DNA sequence and deformability. Notably, the difference in dynamics between IHF-H' and IHF-H1 reflects the different roles of these complexes in their natural context, in the phage lambda "intasome" (the complex that integrates phage lambda into the E. coli chromosome).

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Structure of a Holliday junction complex reveals mechanisms governing a highly regulated DNA transaction

Cited by 24 publications

References 67 publications

A quantitative binding model for the Apl protein, the dual purpose recombination-directionality factor and lysis-lysogeny regulator of bacteriophage 186

A quantitative binding model for the Apl protein, the dual purpose recombination-directionality factor and lysis-lysogeny regulator of bacteriophage 186

Structures of the CRISPR genome integration complex

Static Kinks or Flexible Hinges: Conformational Distributions of Bent DNA Bound to Integration Host Factor Mapped by Fluorescence Lifetime Measurements

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