Role of Exonucleolytic Degradation in Group I Intron Homing in Phage T4

Abstract

Abstract Homing of the phage T4 td intron is initiated by the intron-encoded endonuclease I-TevI, which cleaves the intronless allele 23 and 25 nucleotides upstream of the intron insertion site (IS). The distance between the I-TevI cleavage site (CS) and IS implicates endo- and/or exonuclease activities to resect the DNA segment between the IS and CS. Furthermore, 3′ tails must presumably be generated for strand invasion by 5′-3′ exonuclease activity. Three experimental approaches were used to probe for phage nucleases involved in homing: a comparative analysis of in vivo homing levels of nuclease-deficient phage, an in vitro assay of nuclease activity and specificity, and a coconversion analysis of flanking exon markers. It was thereby demonstrated that T4 RNase H, a 5′-3′ exonuclease, T4 DNA exonuclease A (DexA) and the exonuclease activity of T4 DNA polymerase (43Exo), 3′-5′ exonucleases, play a role in intron homing. The absence of these functions impacts not only homing efficiency but also the extent of degradation and flanking marker coconversion. These results underscore the critical importance of the 3′ tail in intron homing, and they provide the first direct evidence of a role for 3′ single-stranded DNA ends as intermediates in T4 recombination. Also, the involvement of RNase H, DexA, and 43Exo in homing provides a clear example of the harnessing of functions variously involved in phage nucleic acid metabolism for intron propagation.