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Review
. 2020 Nov 4:18:3350-3360.
doi: 10.1016/j.csbj.2020.10.025. eCollection 2020.

The intrinsic ability of double-stranded DNA to carry out D-loop and R-loop formation

Takehiko Shibata  1 , Wakana Iwasaki  2 , Kouji Hirota  1
Affiliations
Review

The intrinsic ability of double-stranded DNA to carry out D-loop and R-loop formation

Takehiko Shibata et al. Comput Struct Biotechnol J. .

Abstract

Double-stranded (ds)DNA, not dsRNA, has an ability to form a homologous complex with single-stranded (ss)DNA or ssRNA of homologous sequence. D-loops and homologous triplexes are homologous complexes formed with ssDNA by RecA/Rad51-family homologous-pairing proteins, and are a key intermediate of homologous (genetic/DNA) recombination. R-loop formation independent of transcription (R-loop formation in trans) was recently found to play roles in gene regulation and development of mammals and plants. In addition, the crRNA-Cas effector complex in CRISPR-Cas systems also relies on R-loop formation to recognize specific target. In homologous complex formation, ssDNA/ssRNA finds a homologous sequence in dsDNA by Watson-Crick base-pairing. crRNA-Cas effector complexes appear to actively melt dsDNA to make its bases available for annealing to crRNA. On the other hand, in D-loop formation and homologous-triplex formation, it is likely that dsDNA recognizes the homologous sequence before the melting of its double helix by using its intrinsic molecular function depending on CH2 at the 2'-position of the deoxyribose, and that the major role of RecA is the extension of ssDNA and the holding dsDNA at a position suitable for homology search. This intrinsic dsDNA function would also play a role in R-loop formation. The dependency of homologous-complex formation on 2'-CH2 of the deoxyribose would explain the absence of homologous complex formation by dsRNA, and dsDNA as sole genome molecule in all cellular organisms.

Keywords: CH-pi interaction (CH-π interaction); CRISPR-Cas system; Deoxyribose; Entropy-driven reaction; Homologous pairing; Homologous triplex; Rad51; RecA; crRNA-Cas-effector complex; dsDNA; single-stranded DNA; ssRNA.

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Figures

Fig. 1
Fig. 1
Homologous complex A. Substrates of homologous-complex formation. A pair of blue arrows represent strands of dsDNA. Vertical gray lines represent W-C base pairs. Red arrow represents a strand of homologous ssDNA or ssRNA. Arrows indicate the polarity of the strand (5′ to 3′). Broken lines flanking the arrows represent the continuity of the strands. B. D-loop/R-loop. Red arrow represents the strand derived from ssDNA for a D-loop or the strand derived from ssRNA for an R-loop. D- or R-loop length in ccc-dsDNA, measured in base pairs, is about 10 times the number of negative supercoils relaxed by the formation of the D- or R-loop. When the ssDNA exceeds this length, its terminal region(s) forms a tail(s) (See panel E). C. Homologous triplex. As in B, the red arrow represents the strand derived from ssDNA. Broken vertical blue lines represent base-pair-specific hydrogen bonds stabilizing the replaced strand in the major groove of the hybrid duplex (See Fig. 2A). D. R-loop formed by Class 1 crRNA-Cas-effector complex (Cascade, etc.). This R-loop comprises 5 segments, each of which contains a 5 bp region followed by a broken base pair (short grey vertical bars). PAM and seed regions are indicated. E. Electron micrograph of D-loops formed from negatively supercoiled ccc-dsDNA and homologous ssDNA-fragment by RecA. Arrows and arrow heads indicate D-loops and tails, respectively. This figure is reproduced from Ref. and is copyrighted to the authors. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
Fig. 2
Fig. 2
Triplexes. A. Crystal structure of a parallel DNA triplex at G·GC . This parallel triplex is included in the homologous triplex formed between the invading ssDNA (pink; red strand in Fig. 1C) and the parental homologous dsDNA (sky blue; blue strand in Fig. 1C). This panel is an example of base parings in the triplex. All combinations of C·CG, A·AT and T·TA in addition to G·GC exists in the homologous triplex. Note that the pink strand forms a hybrid W-C duplex with the complementary strand of the parental dsDNA (sky blue), and has the same polarity and sequence as the replaced strand of the parental dsDNA (sky blue) during homologous-triplex formation (See Fig. 1C). B. Crystal structure of an antiparallel DNA triplex at G·GC . The top strand with G (pink) and the strand with G of the parental dsDNA (sky blue) are antiparallel and paired with Hoogsteen hydrogen bonds. C. Antiparallel triplex formed from polypyrimidine ssRNA and polypurine-polypyrimidine W-C dsDNA without disruption of the double-strand structure. D. Antiparallel triplex at U in ssRNA (pink) paired with the A:T bp in dsDNA (sky blue). E. Antiparallel triplex at C in ssRNA (pink) paired with the GC W-C base pair in dsDNA (sky blue). The polypyrimidine RNA strand is parallel to the polypurine DNA strand and is antiparallel to the polypyrimidine DNA strand. Thus, this type of triplex cannot form between the transcript and the template dsDNA (See Ref. [34]). Panels A and B were generated from PDB 272D , and panels D and E are from PDB 1R3X . All the figures of the structures in this article were prepared with PyMOL (PyMOL Molecular Graphics System, Schrödinger, LLC). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
Fig. 3
Fig. 3
Crystal structure of active RecA-DNA filament. These panels show the 5 RecA protomers of 6 protomers per a turn of an elongated (i.e., active) RecA spiral filament containing dsDNA, which is likely to represent the hybrid duplex after D-loop formation (PDB: 3CMX). The 3D structures of the RecA filament and the ssDNA in the RecA-ssDNA complex are almost identical to those of the RecA-filament and the red strand of the dsDNA in the RecA-dsDNA complex, respectively . The blue strand is another strand of the dsDNA. After homologous-complex formation, the blue strand represents a strand of dsDNA paired with invading ssDNA in the hybrid duplex. A. Side view from the inside of the spiral filament. Basic amino acid residues consisting of gateway are colored green. B. Close-up of base triplets and L2 loops of which top is colored in magenta. C. ATP (analogue) molecules bound between two adjacent RecA protomers. Non-hydrolysable ATP analogues and Mg2+ ions are shown in orange. L1 and L2 loops are shown in cyan and purple, respectively. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
Fig. 4
Fig. 4
Extended DNA structure induced by binding to RecA/Rad51 (ATP-dependent) and ATP-independent homologous-pairing proteins, and a model for homology recognition by RecA/Rad51. A. Stereo view of extended ssDNA structure stabilized by CH-π interaction, an attractive molecular force , upon binding to RecA and Rad51 in the presence of an ATP-analogue , and to ATP-independent homologous-pairing proteins . Van der Waals spheres of a 2′-methylene moiety in a 5′ side residue (thymine) and of the base in the 3′ side residue (adenine) are indicated, and the contact of these surfaces represent CH-π interaction. B. Side views of extended DNA by binding to RecA (left) and B-form DNA (right) for a comparison. Van der Waals spheres of adjacent residues are indicated. In the extended DNA (left), in stead of base-base stacking, CH-π interaction stabilizes the distance between the planes of the adjacent bases (or axial rise per base) at nearly 5 Å, approximately 1.5 fold larger than that (3.5 Å) of B-form DNA (right), in which the structure is stabilized by base-base stacking. C. Model for homology search and W-C base-pairing between dsDNA extended by CH-π interaction and ssDNA (extended by binding to RecA). The 2D model (on the right) is the view from the bottom of the 3D model (on the left). Bases of dsDNA (dark and light blue, bottom) are randomly flipped out by the interconversion of sugar puckers from the C3′ endo to the C2′ endo (middle). An ssDNA (red) approaches the minor groove of the dsDNA (bottom). Only if the flipped base (dark blue) is complementary to the base of the ssDNA, the base-pair switch occurs (top) and the replace base (light blue) is in the major groove of the hybrid base pair that is newly formed. When this base-pair switch takes place between complementary sequences of dsDNA and ssDNA, homologous recognition and homologous-complex-core formation are accomplished simultaneously, and the replaced strand is left in the major groove of the newly formed hybrid duplex (See Fig. 2A). Panels A and B were reproduced with minor modification from Ref. , with permission (Copyright 1997, National Academy of Sciences, U.S.A.). Panel C is the reproduction of the Fig. 4 in Ref. , with permission (Copyright 1998, National Academy of Sciences, U.S.A.), generated using the coordinates of PDB code 1I1V (Model Archive DOI: https://doi.org//10.2210/pdb1I1V/pdb [86]). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
Fig. 5
Fig. 5
Molecular structure of R-loop formation by crRNA-Cas-effector complexes. crRNA, target DNA strand and non-target DNA strand are shown in red, dark blue and light blue, respectively. A. Cryo-electron microscopic 3D structures of crRNA Cascade (Class 1) that contains a full R-loop formed between the crRNA and target dsDNA (PDB code 5U0A [99]). The wedge inserted at the point of strand separation of parental dsDNA is represented by magenta spheres. B. Crystal structure of crRNA Cas9 (Class 2) complex that contains an R-loop of the crRNA and target dsDNA (PDB code 5F9R [101]). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
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