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Genetics. Dec 1999; 153(4): 1501–1512.
PMCID: PMC1460841

Role of exonucleolytic degradation in group I intron homing in phage T4.

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.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.
  • Hill SA, Stahl MM, Stahl FW. Single-strand DNA intermediates in phage lambda's Red recombination pathway. Proc Natl Acad Sci U S A. 1997 Apr 1;94(7):2951–2956. [PMC free article] [PubMed]
  • Ito J, Braithwaite DK. Compilation and alignment of DNA polymerase sequences. Nucleic Acids Res. 1991 Aug 11;19(15):4045–4057. [PMC free article] [PubMed]
  • Hollingsworth HC, Nossal NG. Bacteriophage T4 encodes an RNase H which removes RNA primers made by the T4 DNA replication system in vitro. J Biol Chem. 1991 Jan 25;266(3):1888–1897. [PubMed]
  • Huang KN, Symington LS. A 5'-3' exonuclease from Saccharomyces cerevisiae is required for in vitro recombination between linear DNA molecules with overlapping homology. Mol Cell Biol. 1993 Jun;13(6):3125–3134. [PMC free article] [PubMed]
  • Huang WM, Lehman IR. On the exonuclease activity of phage T4 deoxyribonucleic acid polymerase. J Biol Chem. 1972 May 25;247(10):3139–3146. [PubMed]
  • Jeong-Yu S, Carroll D. Effect of terminal nonhomologies on homologous recombination in Xenopus laevis oocytes. Mol Cell Biol. 1992 Dec;12(12):5426–5437. [PMC free article] [PubMed]
  • Kogoma T, Hong X, Cadwell GW, Barnard KG, Asai T. Requirement of homologous recombination functions for viability of the Escherichia coli cell that lacks RNase HI and exonuclease V activities. Biochimie. 1993;75(1-2):89–99. [PubMed]
  • Kreuzer KN, Alberts BM. Characterization of a defective phage system for the analysis of bacteriophage T4 DNA replication origins. J Mol Biol. 1986 Mar 20;188(2):185–198. [PubMed]
  • Belfort M, Roberts RJ. Homing endonucleases: keeping the house in order. Nucleic Acids Res. 1997 Sep 1;25(17):3379–3388. [PMC free article] [PubMed]
  • Bell-Pedersen D, Quirk SM, Aubrey M, Belfort M. A site-specific endonuclease and co-conversion of flanking exons associated with the mobile td intron of phage T4. Gene. 1989 Oct 15;82(1):119–126. [PubMed]
  • Kushner SR, Nagaishi H, Clark AJ. Isolation of exonuclease VIII: the enzyme associated with sbcA indirect suppressor. Proc Natl Acad Sci U S A. 1974 Sep;71(9):3593–3597. [PMC free article] [PubMed]
  • Bell-Pedersen D, Quirk S, Clyman J, Belfort M. Intron mobility in phage T4 is dependent upon a distinctive class of endonucleases and independent of DNA sequences encoding the intron core: mechanistic and evolutionary implications. Nucleic Acids Res. 1990 Jul 11;18(13):3763–3770. [PMC free article] [PubMed]
  • Lin TC, Karam G, Konigsberg WH. Isolation, characterization, and kinetic properties of truncated forms of T4 DNA polymerase that exhibit 3'-5' exonuclease activity. J Biol Chem. 1994 Jul 29;269(30):19286–19294. [PubMed]
  • Bell-Pedersen D, Quirk SM, Bryk M, Belfort M. I-TevI, the endonuclease encoded by the mobile td intron, recognizes binding and cleavage domains on its DNA target. Proc Natl Acad Sci U S A. 1991 Sep 1;88(17):7719–7723. [PMC free article] [PubMed]
  • Mickelson C, Wiberg JS. Membrane-associated DNase activity controlled by genes 46 and 47 of bacteriophage T4D and elevated DNase activity associated with the T4 das mutation. J Virol. 1981 Oct;40(1):65–77. [PMC free article] [PubMed]
  • Bryk M, Belisle M, Mueller JE, Belfort M. Selection of a remote cleavage site by I-tevI, the td intron-encoded endonuclease. J Mol Biol. 1995 Mar 24;247(2):197–210. [PubMed]
  • Miesel L, Roth JR. Evidence that SbcB and RecF pathway functions contribute to RecBCD-dependent transductional recombination. J Bacteriol. 1996 Jun;178(11):3146–3155. [PMC free article] [PubMed]
  • Chu FK, Maley GF, Maley F, Belfort M. Intervening sequence in the thymidylate synthase gene of bacteriophage T4. Proc Natl Acad Sci U S A. 1984 May;81(10):3049–3053. [PMC free article] [PubMed]
  • Morishima N, Nakagawa K, Shibata T. A sequence-specific endonuclease, Endo.SceI, can efficiently induce gene conversion in yeast mitochondria lacking a major exonuclease. Curr Genet. 1993 May-Jun;23(5-6):537–541. [PubMed]
  • Clyman J, Belfort M. Trans and cis requirements for intron mobility in a prokaryotic system. Genes Dev. 1992 Jul;6(7):1269–1279. [PubMed]
  • Cunningham RP, Berger H. Mutations affecting genetic recombination in bacteriophage T4D. I. Pathway analysis. Virology. 1977 Jul 1;80(1):67–82. [PubMed]
  • Mueller JE, Smith D, Bryk M, Belfort M. Intron-encoded endonuclease I-TevI binds as a monomer to effect sequential cleavage via conformational changes in the td homing site. EMBO J. 1995 Nov 15;14(22):5724–5735. [PMC free article] [PubMed]
  • Fiorentini P, Huang KN, Tishkoff DX, Kolodner RD, Symington LS. Exonuclease I of Saccharomyces cerevisiae functions in mitotic recombination in vivo and in vitro. Mol Cell Biol. 1997 May;17(5):2764–2773. [PMC free article] [PubMed]
  • Mueller JE, Clyman J, Huang YJ, Parker MM, Belfort M. Intron mobility in phage T4 occurs in the context of recombination-dependent DNA replication by way of multiple pathways. Genes Dev. 1996 Feb 1;10(3):351–364. [PubMed]
  • Frey MW, Nossal NG, Capson TL, Benkovic SJ. Construction and characterization of a bacteriophage T4 DNA polymerase deficient in 3'-->5' exonuclease activity. Proc Natl Acad Sci U S A. 1993 Apr 1;90(7):2579–2583. [PMC free article] [PubMed]
  • Mueller JE, Smith D, Belfort M. Exon coconversion biases accompanying intron homing: battle of the nucleases. Genes Dev. 1996 Sep 1;10(17):2158–2166. [PubMed]
  • Mueser TC, Nossal NG, Hyde CC. Structure of bacteriophage T4 RNase H, a 5' to 3' RNA-DNA and DNA-DNA exonuclease with sequence similarity to the RAD2 family of eukaryotic proteins. Cell. 1996 Jun 28;85(7):1101–1112. [PubMed]
  • George JW, Kreuzer KN. Repair of double-strand breaks in bacteriophage T4 by a mechanism that involves extensive DNA replication. Genetics. 1996 Aug;143(4):1507–1520. [PMC free article] [PubMed]
  • Gruber H, Kern G, Gauss P, Gold L. Effect of DNA sequence and structure on nuclease activity of the DexA protein of bacteriophage T4. J Bacteriol. 1988 Dec;170(12):5830–5836. [PMC free article] [PubMed]
  • Pâques F, Haber JE. Two pathways for removal of nonhomologous DNA ends during double-strand break repair in Saccharomyces cerevisiae. Mol Cell Biol. 1997 Nov;17(11):6765–6771. [PMC free article] [PubMed]
  • Hercules K, Wiberg JS. Specific suppression of mutations in genes 46 and 47 by das, a new class of mutations in bacteriophage T4D. J Virol. 1971 Nov;8(5):603–612. [PMC free article] [PubMed]
  • Parker MM, Court DA, Preiter K, Belfort M. Homology requirements for double-strand break-mediated recombination in a phage lambda-td intron model system. Genetics. 1996 Jul;143(3):1057–1068. [PMC free article] [PubMed]
  • Hershfield MS, Nossal NG. Hydrolysis of template and newly synthesized deoxyribonucleic acid by the 3' to 5' exonuclease activity of the T4 deoxyribonucleic acid polymerase. J Biol Chem. 1972 Jun 10;247(11):3393–3404. [PubMed]
  • Parker MM, Belisle M, Belfort M. Intron homing with limited exon homology. Illegitimate double-strand-break repair in intron acquisition by phage t4. Genetics. 1999 Dec;153(4):1513–1523. [PMC free article] [PubMed]
  • Prashad N, Hosoda J. Role of genes 46 and 47 in bacteriophage T4 reproduction. II. Formation of gaps on parental DNA of polynucleotide ligase defective mutants. J Mol Biol. 1972 Oct 14;70(3):617–635. [PubMed]
  • Wang CC, Yeh LS, Karam JD. Modular organization of T4 DNA polymerase. Evidence from phylogenetics. J Biol Chem. 1995 Nov 3;270(44):26558–26564. [PubMed]
  • Razavy H, Szigety SK, Rosenberg SM. Evidence for both 3' and 5' single-strand DNA ends in intermediates in chi-stimulated recombination in vivo. Genetics. 1996 Feb;142(2):333–339. [PMC free article] [PubMed]
  • Warner HR, Snustad DP, Koerner JF, Childs JD. Identification and genetic characterization of mutants of bacteriophage T4 defective in the ability to induce exonuclease A. J Virol. 1972 Mar;9(3):399–407. [PMC free article] [PubMed]
  • West DK, Belfort M, Maley GF, Maley F. Cloning and expression of an intron-deleted phage T4 td gene. J Biol Chem. 1986 Oct 15;261(29):13446–13450. [PubMed]
  • Reha-Krantz LJ, Nonay RL. Genetic and biochemical studies of bacteriophage T4 DNA polymerase 3'-->5'-exonuclease activity. J Biol Chem. 1993 Dec 25;268(36):27100–27108. [PubMed]
  • Wilson GG, Young KY, Edlin GJ, Konigsberg W. High-frequency generalised transduction by bacteriophage T4. Nature. 1979 Jul 5;280(5717):80–82. [PubMed]
  • Selick HE, Kreuzer KN, Alberts BM. The bacteriophage T4 insertion/substitution vector system. A method for introducing site-specific mutations into the virus chromosome. J Biol Chem. 1988 Aug 15;263(23):11336–11347. [PubMed]
  • Woodworth DL, Kreuzer KN. Bacteriophage T4 mutants hypersensitive to an antitumor agent that induces topoisomerase-DNA cleavage complexes. Genetics. 1996 Jul;143(3):1081–1090. [PMC free article] [PubMed]
  • Zassenhaus HP, Denniger G. Analysis of the role of the NUC1 endo/exonuclease in yeast mitochondrial DNA recombination. Curr Genet. 1994 Feb;25(2):142–149. [PubMed]
  • Shimizu K, Sekiguchi M. 5' leads to 3'-Exonucleases of bacteriophage T4. J Biol Chem. 1976 May 10;251(9):2613–2619. [PubMed]

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