PDBsum entry 2a3v

Recombination

PDB id: 2a3v

Contents

Protein chains

313 a.a. *

DNA/RNA

Waters ×170

* Residue conservation analysis

PDB id:

2a3v

Name:

Recombination

Title:

Structural basis for broad DNA-specificity in integron recombination

Structure:

DNA (31-mer). Chain: e, g. Engineered: yes. DNA (34-mer). Chain: f, h. Engineered: yes. Site-specific recombinase inti4. Chain: a, b, c, d. Engineered: yes

Source:

Synthetic: yes. Other_details: sythetic construct. Vibrio cholerae o1 biovar eltor str. N16961. Organism_taxid: 243277. Strain: o1 biovar eltor str. N16961. Gene: inti4. Expressed in: escherichia coli. Expression_system_taxid: 562.

Biol. unit:

Octamer (from PQS)

Resolution:

2.80Å R-factor: 0.234 R-free: 0.262

Authors:

D.Macdonald,G.Demarre,M.Bouvier,D.Mazel,D.N.Gopaul

Key ref:

D.MacDonald et al. (2006). Structural basis for broad DNA-specificity in integron recombination. Nature, 440, 1157-1162. PubMed id: 16641988 DOI: 10.1038/nature04643

Date:

27-Jun-05 Release date: 02-May-06

Protein chains

O68847  (O68847_VIBCL) -  Integron integrase from Vibrio cholerae

Seq: 320 a.a.

Struc: 313 a.a.*

* PDB and UniProt seqs differ at 1 residue position (black cross)

DNA/RNA chains

C-C-G-G-T-T-A-T-A-A-C-G-C-C-C-G-C-C-T-A-A-G-G-G-G-C-T-G-A-C-A 31 bases

T-G-A-C-A-G-T-C-C-C-T-C-T-T-G-A-G-G-C-G-T-T-T-G-T-T-A-T-A-A-C-C-G-G 34 bases

C-G-G-T-T-A-T-A-A-C-G-C-C-C-G-C-C-T-A-A-G-G-G-G-C-T-G-A-C 29 bases

G-A-C-A-G-T-C-C-C-T-C-T-T-G-A-G-G-C-G-T-T-T-G-T-T-A-T-A-A-C-C-G 32 bases

Enzyme reactions

• Enzyme class: E.C.?

DOI no: 10.1038/nature04643

Nature 440:1157-1162 (2006)

PubMed id: 16641988

Structural basis for broad DNA-specificity in integron recombination.

D.MacDonald, G.Demarre, M.Bouvier, D.Mazel, D.N.Gopaul.

ABSTRACT

Lateral DNA transfer--the movement of genetic traits between bacteria--has a profound impact on genomic evolution and speciation. The efficiency with which bacteria incorporate genetic information reflects their capacity to adapt to changing environmental conditions. Integron integrases are proteins that mediate site-specific DNA recombination between a proximal primary site (attI) and a secondary target site (attC) found within mobile gene cassettes encoding resistance or virulence factors. The lack of sequence conservation among attC sites has led to the hypothesis that a sequence-independent structural recognition determinant must exist within attC. Here we report the crystal structure of an integron integrase bound to an attC substrate. The structure shows that DNA target site recognition and high-order synaptic assembly are not dependent on canonical DNA but on the position of two flipped-out bases that interact in cis and in trans with the integrase. These extrahelical bases, one of which is required for recombination in vivo, originate from folding of the bottom strand of attC owing to its imperfect internal dyad symmetry. The mechanism reported here supports a new paradigm for how sequence-degenerate single-stranded genetic material is recognized and exchanged between bacteria.

Selected figure(s)

Figure 1.
Figure 1: Pathways of IntI-mediated cassette excision.
Figure 1 : Pathways of IntI-mediated cassette excision. Unfortunately we are unable to provide accessible alternative text for this. If you require assistance to access this image, or to obtain a text description, please contact npg@nature.com-
a, Integrons contain a gene, IntI, encoding a tyrosine recombinase, and an adjacent recombination site, attI. Gene cassettes (open reading frames, ORFs) are flanked by secondary sites, attC sites. IntI recombines attI and attC during integration and two attC sites during excision. P[i] and P[c] are promoters for IntI and gene cassettes, respectively; DR1 and DR2 are directly repeated accessory binding sites; L and R are binding sites within the core region of attI; L' and L" are inner repeats; R' and R" are flanking repeats. b, Excision by the classic tyrosine recombinase model. Each duplex attC site (step 1) is bound by two IntI molecules to form an antiparallel recombination synapse (step 2). Tyr 302 cleavage forms covalent 3'-phosphotyrosine intermediates (step 3). The free 5'-hydroxyl groups attack their partner substrates yielding a Holliday junction (HJ) intermediate (step 4), which isomerizes (step 5) before undergoing a second round of cleavage and strand-exchange reactions to yield the recombinant products^5,6 (step 6). c, Proposed IntI excision through a single-stranded DNA substrate pathway. The bottom strand of the integron element, produced by conjugation or transformation, folds upon itself to yield an active stem-loop substrate (step 1). Two IntI molecules bind each folded attC site to form an antiparallel recombination synapse (step 2). The attack and strand exchange steps proceed in a similar fashion to steps 3–4 in panel b; however, the HJ intermediate requires cellular components in order to be resolved^12 (steps 5–6). The reaction intermediate shown in step 2 represents the VchIntIA–VCR[bs] structure described here. IntI molecules coloured green and magenta are potentially active or non-active for cleavage, respectively. d, DNA sequence of VCR[bs] used to form VchIntIA–DNA co-crystals. Yellow boxes highlight the inner (L' and L") and flanking (R' and R") repeats. The nucleotides T12" (red) and G20" (blue) have an extrahelical geometry upon folding of attC bottom strands (see also Supplementary Fig. 1).

Figure 2.
Figure 2: Architecture of the VchIntIA–VCR[bs] synapse.
Figure 2 : Architecture of the VchIntIA–VCRbs synapse. Unfortunately we are unable to provide accessible alternative text for this. If you require assistance to access this image, or to obtain a text description, please contact npg@nature.com-
a, N-terminal view of the complex. Four VchIntIA molecules bind two antiparallel VCR[bs] duplexes to form the active synapse. The extrahelical base T12" (red) is stabilized by cis interactions and is involved with DNA site recognition (Fig. 4a, b). The extrahelical base G20" (blue) is buried in subunits that are bound to the other VCR[bs] duplex forming a set of trans interactions (Fig. 4c, d). The non-symmetric interfaces between VchIntIA molecules yield a two-fold symmetric synapse. b, Orthogonal view with respect to a. The C-terminal helices (N) bury one face in a hydrophobic pocket of the adjacent subunit in a cyclic manner (N[A] arrow B, N[B] arrow C, and so on).

The above figures are reprinted by permission from Macmillan Publishers Ltd: Nature (2006, 440, 1157-1162) copyright 2006.

Figures were selected by an automated process.