Extended Data Fig. 8: Models and density of D-loop^DG and D-loop^DH.

Discussion of RecA–DNA contacts. The L2 loop-duplex contacts involve non-equivalent RecA protomers owing to the different duplex orientations. Thus, duplex^D abuts the L2 loop of the adjacent RecA^C and additionally stacks with Gly204 backbone atoms, whereas duplex^G abuts the L2 loop of RecA^E, two RecAs over, and packs with the Met202 side chain (Fig. 3b, c). The CTD–dsDNA interactions are very similar in the two duplexes, except those at duplex^G consistently have slightly longer distances. The contacts from the loop–helix and hairpin motifs expand the minor groove of the DNA to 15.2 Å for duplex^D and 14.6 Å for duplex^G. The loop and the amino terminus of the loop–helix motif (residues 297 to 302) make a set of hydrogen bonds to backbone phosphate groups of both strands (backbone amide of Lys302 and side chain of Lys297) while the side chain amide group of Gln300 hydrogen bonds to a thymidine O2 (duplex^D) or guanidine N3 (duplex^G) groups. Crucially, the Gln300 side chain is also in a π–π stack with the Trp290 side chain from the hairpin motif (residues 286 to 290 with the sequence Lys-Ala-Gly-Ala-Trp). With both side chains thus rigidified, they fit snugly in the minor groove and make multiple van der Waals contacts to the ribose groups, with the amino group of the Trp290 side chain also hydrogen-bonding to an N3 group of an adenine (duplex^D) or guanine (duplex^G). The tip of the hairpin (Ala-Gly-Ala portion) partially inserts in the minor groove as well, with the preceding Lys286 within contact distance of the phosphodiester backbone. A second hairpin at the end of the long β6 strand of the helicase core is positioned above the adjacent major groove, and Lys232 contacts the phosphodiester backbone of duplex^G, but the corresponding contact is not made to duplex^D, which is farther away owing to its 5′ tilt. Among the CTD–duplex contacts, the K286N and K302N mutations were shown to cause defects in UV-damage repair in vivo and in binding to and pairing with dsDNA, although they were interpreted as affecting the secondary DNA-binding activity³⁸. The S2 site contacts to the homologous strand are overall more extensive and the density stronger at the duplex^D-proximal two thirds of the strand than near duplex^G. The contacts start immediately after the opening of duplex^D by L2^C (these Ade28 contacts are discussed in the main text). The base group of the next residue, Cyt27, is sandwiched between the L2^C Met202 and L2^D Pro206 side chains, while its ribose group packs against the E207^D–R226^D salt bridge (Fig. 3g, bottom). Cyt26 then packs on one side with the L2^D Pro206 side chain and Gly204 and Asn205 backbone groups, and on the other side with Cyt25. The Cyt25 phosphodiester group in turn hydrogen-bonds to the Ala230 backbone amide and Arg227 side-chain groups, both from β6^E (Fig. 3g, middle). The next two nucleotides stack together and as a pair fit snugly into a tight gap between the backbones of β6^E and L2^D, as if they are pinched (Fig. 3g, middle). Here, β6^E side chain and backbone groups of Ile228 and Gly229 pack with Cyt24, while L2^D backbone groups from Phe203 and Gly204 pack with Cyt23. In addition, the phosphodiester group of Cyt24 hydrogen-bonds to the backbone amide of L2^D Asn205, and that of Cyt23 to the side chain of β6^E Arg226. Thereafter, Cyt22 has RecA contacts and relative position very similar to that of Cyt27, five nucleotides away in the 3′ filament direction. The one difference is the Cyt27–L2^C Met202 packing is replaced by Cyt22–L2^D Phe203, owing to the alternate conformation that L2^C adopts as it book-ends duplex^D (Fig. 3g, top). The Cyt22 base is also within contact distance of β6^E Lys245, where the K245N mutation was reported to affect homologous pairing¹¹. The D-loop^DH structure recapitulates the key aspects of D-loop^DG. These include the conformations of the L2^C and L2^F loops and their stacking with their respective duplex^D and duplex^H, the overall S2 ssDNA backbone conformation, and the β6-L2 pinch of a nucleotide pair, of which it has two (Fig. 4b). One, at Thy26-Thy27, is essentially identical to that of D-loop^DG, whereas the other, at Thy21-Thy22, has Thy21 in a slightly different conformation, as it is next to the 3-nt spacer that transitions from S2-binding to duplex^H. The D-loop^DH structure also exhibits the same pattern of contacts at the transitions from the duplexes to the opened up homologous strands. The first-opened up base immediately after duplex^D (Ade31) packs with L2^C, while its phosphodiester backbone is contacted by Arg226 of β6^D. The opposing, flipped-out base of the complementary strand has poor density and does not seem to make any RecA contacts. And, as with D-loop^DG, the three nucleotides just before duplex^H (Thy20-Thy19-Ade18) are poorly ordered, and make few contacts as they follow an alternative path around their respective L2^F. a, On dsDNA binding, the CTD domains undergo a rotation about an axis (red stick) near residue 269 and roughly perpendicular to the direction of the filament. The 9 RecA protomers of the 9-RecA fusion protein were superimposed by aligning their helicase core domains. The RecA^A, RecA^B, RecA^C, RecA^E, RecA^F, RecA^H, and RecA^I CTD domains (grey) do not exhibit any conformational changes, whereas those of RecA^D and RecA^G rotate (curved arrow) in opposite directions, by −3° and 13° respectively. b, Cartoon representation showing the superposition of RecA^D and RecA^G on their CTD domains to highlight the different tilts of duplex^D and duplex^G relative to their already rotated CTD domains. Coloured as in Fig. 3d (which shows the superposition of the RecA protomers on their helicase domains). The rest of the RecAs are coloured yellow and grey. c, d, Density of duplex^D and duplex^G from the D-loop^DG maps used in REFMAC5, as described in Methods, in the same orientation as Fig. 3b, c. e, f, Density of the interactions of duplex^D and duplex^G with their respective CTDs from the D-loop^DG REFMAC5 refinement the same orientation as Fig. 3e, f. g, Density of the S2 site structural elements and all DNA from the D-loop^DG maps used in REFMAC5. Orientations as in Fig. 3g. h, Density of the DNA only from D-loop^DH maps used in REFMAC5 in the same orientation as Fig. 4b. RecA protomers and their density are omitted for clarity.