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. 1997 Jun 24;94(13):6706-11.
doi: 10.1073/pnas.94.13.6706.

The recombination hotspot Chi is recognized by the translocating RecBCD enzyme as the single strand of DNA containing the sequence 5'-GCTGGTGG-3'

P R Bianco  1 , S C Kowalczykowski
Affiliations

The recombination hotspot Chi is recognized by the translocating RecBCD enzyme as the single strand of DNA containing the sequence 5'-GCTGGTGG-3'

P R Bianco et al. Proc Natl Acad Sci U S A. .

Abstract

The RecBCD enzyme of Escherichia coli functions in the seemingly disparate roles of homologous recombination and the degradation of DNA. Which of these two roles it assumes is regulated by the 8-base recombination hotspot, Chi. Using double-stranded DNA substrates that are heteroduplex at the Chi locus we have established the determinants for Chi recognition. Our results show that an actively translocating RecBCD enzyme requires only the sequence information in the 5'-GCTGGTGG-3'-containing strand to recognize and to be regulated by Chi. Furthermore, the RecBCD enzyme can translocate through DNA heteroduplex bubbles as large as 22 bases, and still recognize a Chi sequence embedded in this region. This implies that recognition of Chi occurs following the unwinding of the DNA.

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Figures

Figure 1
Figure 1
Substrates used for χ-fragment production assays. (A) Homoduplexes. (B) Heteroduplexes. The χ+/χ° and χ°/χ+ DNA heteroduplexes contain a single base mismatch; χ+/M-8 and χ+/M-22, contain 8-and 22-base mismatches, respectively. The arrow above each substrate indicates the direction from which RecBCD enzyme must translocate for χ recognition to occur. Sequences in bold highlight both the χ+ sequence and the positions where relevant differences with respect to χ+ occur. For M-22, only 8 bases out of 22 of the mismatch, corresponding to χ-complement are highlighted. ∗, 32P.
Figure 2
Figure 2
Recognition of χ by RecBCD enzyme requires sequence information in the top strand. The substrates and expected products are shown in A; the results of the χ-specific fragment production assays using these substrates are shown in B. χ fragment production assays were performed as described.
Figure 3
Figure 3
The sequence determinant, 5′-GCTGGTGG-3′, can be recognized by a translocating RecBCD enzyme as ssDNA. The substrates and expected products are shown in A; the gel showing χ-specific fragment production is shown in B. Assays were conducted as for Fig. 2. M-y, M-8 or M-22.
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