Two recombination-dependent DNA replication pathways of bacteriophage T4, and their roles in mutagenesis and horizontal gene transfer

Abstract

Two major pathways of recombination-dependent DNA replication, "join-copy" and "join-cut-copy," can be distinguished in phage T4: join-copy requires only early and middle genes, but two late proteins, endonuclease VII and terminase, are uniquely important in the join-cut-copy pathway. In wild-type T4, timing of these pathways is integrated with the developmental program and related to transcription and packaging of DNA. In primase mutants, which are defective in origin-dependent lagging-strand DNA synthesis, the late pathway can bypass the lack of primers for lagging-strand DNA synthesis. The exquisitely regulated synthesis of endo VII, and of two proteins from its gene, explains the delay of recombination-dependent DNA replication in primase (as well as topoisomerase) mutants, and the temperature-dependence of the delay. Other proteins (e.g., the single-stranded DNA binding protein and the products of genes 46 and 47) are important in all recombination pathways, but they interact differently with other proteins in different pathways. These homologous recombination pathways contribute to evolution because they facilitate acquisition of any foreign DNA with limited sequence homology during horizontal gene transfer, without requiring transposition or site-specific recombination functions. Partial heteroduplex repair can generate what appears to be multiple mutations from a single recombinational intermediate. The resulting sequence divergence generates barriers to formation of viable recombinants. The multiple sequence changes can also lead to erroneous estimates in phylogenetic analyses.

Figure 1

Different ways to initiate DNA replication from intermediates of homologous recombination. The ss end of parent 2 DNA invades homologous ds DNA of parent 1. Details are explained in the text.

Figure 2

(A) Cumulative T4 DNA synthesis, measured by incorporation of ³H-labeled thymidine (45) at 30°C in E. coli B of a primase-defective mutant (E219), alone or in combination with endo VII (C9)- or terminase (NG178)- defective mutations. The E219-C9 incorporation relative to wt and E219 represents the average of three experiments done on different days. At first (14) we had mistakenly assumed that only endo VII produces the nicks required for the join-cut-copy pathway. Subsequently we found that the primase-endo VII double mutant used in those studies had acquired a third mutation. (B and C) Cumulative phage DNA synthesis at 25°C in E. coli B. (B) The primase mutant E219, the gene 46 mutant N130, and the double mutant are compared with wild-type T4. (C) The primase mutant E219, three gene 32 ts mutants (L171, P7, and G26), and the corresponding double mutants were compared with wild-type T4.

Figure 3

A recombination model for acquisition of a DNA (gene 69 in T4 or soc.1 and soc.2 in T2) by a T-even phage (e.g., RB15) lacking a gene between genes 56 and soc. Details are explained in the text.

Figure 4

Comparison of dCTPases and adjacent genes of different T-even phages. (A) Positions of genes 56 and soc in classes A, B, and C. (B) Alignment of the gene 56 sequences of T2 and RB15. The −10 region of the promoter P_M and the initiation and termination codons are underlined. (C) dCTPase sequences of different phages. Green, different in T4 from all others; red, different in RB15 from all others; purple, identical among class B (T2, LZ5, and T6), but different in T4 and RB15; blue, different in only one phage.