Dennis W. Schultz

Structure of chi hotspots of generalized recombination

Chi recombinational hotspots are sites around which the rate of Rec-promoted recombination in bacteriophage lambda is elevated. Examination of a derivative of lambda into which the plasmid pBR322 was inserted reveals that pBR322 lacks Chi sites. Using this lambda-pBR322 hybrid, we obtained mutations creating Chi sites at three widely separated loci within pBR322. Nucleotide sequence analysis reveals that the mutations are single base-pair changes creating the octamer 5 GCTGGTGG 3. This sequence is present at three previously analyzed Chi sites in lambda, and all analyzed mutations creating or inactivating these Chi sites occur within this octamer. We conclude that Chi is 5 GCTGGTGG 3, or its complement, or both.

Chi-dependent DNA strand cleavage by RecBC enzyme

Chi sites enhance in their vicinity homologous recombination by the E. coli RecBC pathway. We report here that RecBC enzyme catalyzes Chi-dependent cleavage of one DNA strand, that containing the Chi sequence 5G-C-T-G-G-T-G-G3. Chi-specific cleavage is greatly reduced by single base pair changes within the Chi sequence and by mutations within the E. coli recC gene, coding for a RecBC enzyme subunit. Although cleavage occurs preferentially with double-stranded DNA, the product of the reaction is single-stranded DNA. These results demonstrate the direct interaction of RecBC enzyme with Chi sites that was inferred from the genetic properties of Chi and recBC, and they support models of recombination in which Chi acts before the initiation of strand exchange.

RecBC enzyme nicking at chi sites during DNA unwinding: Location and orientation-dependence of the cutting

Homologous recombination by the E. coli RecBC pathway occurs at elevated frequency near Chi sites. We reported previously that Chi induces RecBC enzyme to cleave one DNA strand--that containing the Chi sequence 5G-C-T-G-G-T-G-G3. We report here that the Chi-dependent cleavage occurs four, five, or six nucleotides to the 3 side of the Chi octamer and produces nicks with 3-OH and 5-PO4 groups. Chi-dependent cleavage occurs if RecBC enzyme approaches the Chi sequence from the right, but not if it approaches only from the left, during unwinding of the duplex DNA substrate. A single RecBC enzyme molecule appears to cleave the DNA and to release part of it as a single-stranded fragment. These and previous results indicate that Chi-dependent cleavage is concomitant with DNA unwinding by RecBC enzyme and provide an enzymatic basis for the orientation-dependence of Chi recombinational hotspot activity. These observations demonstrate a key step of a proposed model of recombination in which RecBC enzyme produces a potentially invasive single-stranded DNA tail extending from Chi to its left. We discuss the relation between the action of Chi sites and that of special sites enhancing eukaryotic recombination.

Nucleotide sequence of the lysozyme gene of bacteriophage T4. Analysis of mutations involving repeated sequences.

The nucleotide sequence of the lysozyme (e) gene of bacteriophage T4 and approximately 130 additional nucleotides on each side has been determined. The 5-end of the gene for internal protein III appears to be located about 70 base-pairs from the 3-end of the lysozyme gene. Nucleotide sequence analysis of mutant e genes confirmed that three identified hotspots of frameshift mutations are runs of five A nucleotides in the wild-type gene. The endpoints of two deletions are direct repeats of eight base-pairs in the wild-type gene. Two frameshift mutations with high reversion frequencies are duplications of five or seven base-pairs. The cloning and nucleotide sequence determination of the lysozyme gene will facilitate further study of the molecular biology of T4 lysozyme.

Clustering of mutations inactivating a Chi recombinational hotspot

Chi sites promote Rec-mediated recombination in bacteriophage λ. Nine independent, nitrous acid-induced mutations were obtained within one of these sites, χ+C. Eight of the mutations completely inactivated the Chi site, while one mutation left partial activity. Nucleotide sequence analysis showed that the mutations were located at four different sites one to four base-pairs from the site of the χ+C mutation that created the active Chi locus. This interval is within a region of homology common to the χ+C locus and another sequenced Chi locus, χ+B. These results support the view that Chi is a unique nucleotide sequence and suggest the extent of the Chi sequence.

Generalized Recombination: Nucleotide Sequence Homology between Chi Recombinational Hotspots

Chi sites stimulate generalized recombination catalyzed by the RecA-RecBC-dependent system of E. coli. This stimulation occurs over a region of several thousand base pairs surrounding the Chi site. These sites arise by mutation at four distinct loci in bacteriophage lambda. We report here the nucleotide sequence surrounding one of these loci, chi B, located between the xis and reda genes. Alteration of a single GC base pair, by deletion or by transversion to a CG base pair, creates the Chi recombinational hotspot chi + B. In a section of 30 bp, the chi + B sequence has 23 bp in common with the chi + C sequence determined previously. We presume that some part of this common sequence is the recognition sequence for a protein which acts at a rate-limiting step of generalized recombination.

Conservation of Chi cutting activity in terrestrial and marine enteric bacteria

Homologous recombination in Escherichia coli occurs at increased frequency near Chi sites, 5G-C-T-G-G-T-G-G3. Cutting of DNA close to the Chi sequence by the E. coli RecBC enzyme is essential to Chis stimulation of recombination. We have detected Chi-dependent cutting activity in extracts of several genera of terrestrial enteric bacteria (family Enterobacteriaceae) and of two genera of marine enteric bacteria (family Vibrionaceae). More distantly related bacteria had no detectable Chi-dependent cutting activity. These results support the view that recognition of this specific nucleotide sequence as a signal activating recombination has been maintained during the evolution of certain groups of bacteria. We discuss the possibility that other sequences play a similar role in other groups of bacteria.

NUCLEOTIDE SEQUENCE OF CHI RECOMBINATIONAL HOTSPOTS

ABSTRACT Chi is one of a class of sites which affect general recombination. In phage λ, Chi stimulates in its vicinity recombination promoted by the RecBC pathway of Escherchia coli . Our current analysis of Chi sites suggests that Chi is a unique nucleotide sequence 5′ G-C-T-G-G-T-G-G 3′ (or its complement, or both). This result supports the hypothesis that Chi is a recognition sequence for a protein involved in general recombination.