Strand invasion promoted by recombination protein beta of coliphage lambda

Abstract

Studies of phage lambda in vivo have indicated that its own recombination enzymes, beta protein and lambda exonuclease, are capable of catalyzing two dissimilar pathways of homologous recombination that are widely distributed in nature: single-strand annealing and strand invasion. The former is an enzymatic splicing of overlapping ends of broken homologous DNA molecules, whereas the latter is characterized by the formation of a three-stranded synaptic intermediate and subsequent strand exchange. Previous studies in vitro have shown that beta protein has annealing activity, and that lambda exonuclease, acting on branched substrates, can produce a perfect splice that requires only ligation for completion. The present study shows that beta protein can initiate strand invasion in vitro, as evidenced both by the formation of displacement loops (D-loops) in superhelical DNA and by strand exchange between colinear single-stranded and double-stranded molecules. Thus, beta protein can catalyze steps that are central to both strand annealing and strand invasion pathways of recombination. These observations add beta protein to a set of diverse proteins that appear to promote recognition of homology by a unitary mechanism governed by the intrinsic dynamic properties of base pairs in DNA.

Fig. 1.

The two paradigms of homologous recombination. (A) Single-strand annealing as exemplified by the activities in vitro of the exonuclease and β protein of phage λ. Small filled black circles represent 5′ ends. (B) Strand invasion is represented here by the exchange of base pairs between the “incoming” strand and the “invaded” duplex (see Discussion), but the complete pathway in vivo involves many additional and varied steps that lead to the final recombinant products (–28).

Fig. 5.

Effect of G+C content of substrates on formation of D-loops. Reactions were carried out as described in Materials and Methods. The concentration of ³²P-labeled oligonucleotides was 2.5 μM, and of β protein was 3 μM, except in the sample in lane 8 where β protein was omitted. The concentration of all superhelical plasmids was 0.2 mM. In lanes 9 and 10, heterologous controls were accomplished by mixing oligonucleotides G25(–) and G37(–), respectively, with plasmid pEG16, which contained the duplex insert corresponding to oligonucleotide G16(–) (see Materials and Methods).

Fig. 2.

β protein forms D-loops in supercoiled DNA. The concentrations of ³²P-labeled oligonucleotide and β protein were 2.5 μM and 1.5 μM, respectively. The concentration of heterologous superhelical DNA (pEG177; lanes 1–3), homologous linear duplex DNA (pEG47, predigested by HindIII; lanes 4 and 5), and homologous superhelical DNA (pEG47; lanes 6–10) was 0.15 mM. These concentrations of DNA correspond to a slight excess of the single-stranded oligonucleotide with respect to the target site in duplex DNA.

Fig. 3.

Stoichiometry of D-loop formation by β protein. The concentrations of [³²P]G16(–) ssDNA and plasmid pEG47 dsDNA were 2.5 μM and 0.15 mM, respectively. Reaction conditions were as described in Materials and Methods. All reactions were stopped 2 min after the plasmid was added.

Fig. 4.

Effect of MgCl₂ on the formation of D-loops. [³²P]G16(–) oligonucleotide and β protein were present at 2.5 μM. G16(–) was present in 1.7-fold excess over homologous sites in the pEG47 plasmid. MgCl_2, at the indicated concentrations, was added together with the plasmid. (MgCl₂ was omitted in all other experiments on the formation of D-loops described in this paper.)

Fig. 6.

Strand exchange promoted by β protein. The substrates were 83-mer oligonucleotides G16(–) or EG673, the latter a derivative of G16(–) containing 10 A/T transversion mismatches, to provide a heterologous control, and duplex [5′-³²P]G16(–)·G16(+), all at 2.5 μM. β protein and RecA protein were at 2.5 μM and 1 μM, respectively.

Fig. 7.

Strand exchange (S.E.) mediated by β protein is not due to helicase activity. Lanes 1 and 2, strand exchange at 0 and 40 min, respectively, was carried out as described in Materials and Methods. The concentration of dsDNA was 1.2 μM, ssDNA was 3 μM, and β protein was 3 μM. Lane 3, the mock melting reaction, in which [³²P]G16(–)·G16(+), 1.2 μM, was incubated in the presence of 3 μM β protein for 20 min at 37°C, after which a 7-fold excess of G16(–), 8.5 μM, was added with stop solution. Lane 4, dsDNA, used as a marker.

Fig. 8.

The kinetics of D-loop formation and strand exchange. In the reaction for D-loop formation, β protein was present at a ratio of one molecule of protein per nucleotide residue of ssDNA. The latter, [³²P]G16(–) 83-mer (final concentration, 2.5 μM) was present in 2-fold excess over homologous sites in pEG47 plasmid. Strand-exchange reactions were carried out with ssDNA G16(–) and dsDNA [5′-[³²P]G16(–)·G16(+), each at 2.5 μM. The concentration of β protein was 2.5 μM.