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Figure 1.
Figure 1: Pathways of IntI-mediated cassette excision.
Figure 1 : Pathways of IntI-mediated cassette excision.
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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).
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