Copyright1999 by the Genetics Society of America
Intron Homing With Limited Exon Homology: Illegitimate Double-Strand-Break
Repair in Intron Acquisition by Phage T4
Monica M. Parker, Maureen Belisle and Marlene Belfort
Molecular Genetics Program, Wadsworth Center, New York State Department of Health and School of Public Health, State University of New York, Albany, New York 12201-2002
Manuscript received May 12, 1999 Accepted for publication August 30, 1999
ABSTRACT
The td intron of bacteriophage T4 encodes a DNA endonuclease that initiates intron homing to cognate intronless alleles by a double-strand-break (DSB) repair process. A genetic assay was developed to analyze the relationship between exon homology and homing efficiency. Because models predict exonucleolytic processing of the cleaved recipient leading to homologous strand invasion of the donor allele, the assay was performed in wild-type and exonuclease-deficient (rnh or dexA) phage. Efficient homing was supported by exon lengths of 50 bp or greater, whereas more limited exon lengths led to a precipitous decline in homing levels. However, extensive homology in one exon still supported elevated homing levels when the other exon was completely absent. Analysis of these “one-sided” events revealed recombination junctions at ectopic sites of microhomology and implicated nucleolytic degradation in illegitimate DSB repair in T4. Interestingly, homing efficiency with extremely limiting exon homology was greatly elevated in phage deficient in the 39-59exonuclease, DexA, suggesting that the length of 39tails is a major determinant of the efficiency of DSB repair. Together, these results suggest that illegitimate DSB repair may provide a means by which introns can invade ectopic sites.
S
EVERAL group I introns undergo a process termed mon to both models, but subsequent repair synthesis is mechanistically different. Each pathway implicated in homing, whereby the intron is efficiently insertedinto intronless cognates of the intron-containing allele T4 intron homing predicts a requirement for homology between donor and recipient molecules on both sides (Dujon 1989). Homing occurs by a gene conversion
process that depends on expression of a site-specific of the break. The efficiency of homing depends on exon length, suggesting that homing may require a minimal endonuclease encoded within the intron (reviewed in
Lambowitz and Belfort 1993; Mueller et al. 1993; length of homology (Quirket al. 1989a), as is the case in a phagel-td intron model system (Parkeret al. 1996). BelfortandPerlman1995). The intron endonuclease
makes a double-strand break (DSB) in the intronless Homing of the td intron occurs via the recombination-dependent mode of DNA replication of T4 (Mueller allele at or near the site of intron insertion. Intron
sequences are copied from the intron-containing allele et al. 1996a). Although the requisite UvsX protein, an analog of the Escherichia coli strand transferase, RecA, during repair of the DSB, resulting in inheritance of
has been well characterized (Kreuzerand Morrical the intron.
1994), several exonuclease activities that appear to be Homing of the intron in the td (thymidylate synthase)
involved in replication and recombination in T4 have gene of bacteriophage T4 is initiated by I-TevI, the td
poorly defined roles. Models of intron homing predict intron endonuclease, which makes a DSB in an
intron-a requirement for intron-a 59-39nucleolytic activity to generate less td allele 23 and 25 nucleotides (nt) upstream of the
invasive 39 ssDNA tails, as well as a 39-59 exonuclease intron insertion site (Bell-Pedersenet al. 1990; Figure
activity to degrade the “resection segment” produced 1A). Repair of the DSB results in precise acquisition of
by the eccentric cleavage of I-TevI (Figure 1A;Clyman the intron at the exon I-exon II junction. At least two
andBelfort1992). The gp46/47 complex, a required pathways of DSB repair have been implicated in td
function for T4 recombination and homing, has been intron homing (Muelleret al. 1996a), classic
double-implicated as a 59-39 exonuclease (Mickelson and strand-break repair (DSBR; Figure 1) and
synthesis-Wiberg 1981; Kreuzer et al. 1995; Mueller et al. dependent strand annealing (SDSA; Figure 1). The
ini-1996a). Although a specific role for this complex has tial steps of DSB formation, exonucleolytic processing
remained elusive, it likely makes a significant contribu-of the ends and homologous strand invasion, are
com-tion, albeit possibly indirect, to DNA end processing during recombination (Mosig 1998). T4 RNase H, which possesses 59-39 DNA exonuclease activity
(Hol-Corresponding author: Marlene Belfort, Wadsworth Center, New York
lingsworthand Nossal 1991), may also play a role
State Department of Health, P.O. Box 22002, Albany, NY 12201-2002.
E-mail: [email protected] in intron homing (Muelleret al. 1996a; Huanget al.
Figure 1.—Proposed pathways of td intron homing. The td homing site (A) comprises the intron insertion site (IS) at the junction of exon I and exon II, and the I-TevI cleavage site (CS) located 23 and 25 nt upstream. The recipient allele receives a DSB (B), which is processed by exonucleases (C). A 39 single-stranded end in-vades a donor allele and synapsis with homolo-gous sequences occurs (D). In DSBR, repair syn-thesis leads to formation of a D-loop (E) and, after ligation, double Holliday junctions can isomerize independently (F). Cleavage by resolvase results in noncrossover or crossover products. Only the crossover products are shown (G). In SDSA, a replication bubble proceeds along the donor tem-plate DNA (E9), and the newly synthesized strand anneals with complementary sequences and serves as template for repair synthesis (F9). Re-lease of the invading strand and ligation leads to gene conversion without crossing over of flanking markers (G9). Thick lines, homologous DNA in recipient and donor alleles; thin lines, flanking DNA of recipient; wavy lines, flanking DNA of donor; shaded lines, intron DNA; half arrow, 39 end of DNA; arrowheads, resolvase cleavage. Al-ternatives to these pathways have also been pro-posed (Luder and Mosig 1982; George and Kreuzer1996).
suppressing (Sup8) host, were provided by K. Kreuzer (Duke 1999). Although DexA, a 39-59exonuclease (Gruberet
University, Durham, NC). CR63cI857kanRcarrying a
tempera-al. 1988), and 43Exo, the 39-59 exonuclease activity of
ture-sensitive phagelrepressor was made by transduction of T4 DNA polymerase, are not required for homing, in strain CR63 with a P1 lysate containing galK::cI857kanR. E. coli
vivo and in vitro analyses suggest a role for these func- strain OK305 (E. coli B cd2sup1), a Sup8host lacking cytidine
tions in 39end processing (Muelleret al. 1996a;Huang and deoxycytidine deaminase activities (Hall et al. 1967), was provided by D. Hall (Georgia Institute of Technology, et al. 1999).
Atlanta). A sensitive genetic assay was developed to characterize
T4 38 (amB262) and T4 51 (amS29) were used to introduce events in which homology was limited, thereby
ad-38amand 51ammutations into our laboratory stock of wild-type
dressing the mechanism of illegitimate DSB repair in strain T4D, and T4D 38am51amwas confirmed by cross-streak
T4. Additionally, the results are relevant to the evolution complementation tests. The td intron was deleted from this strain through a two-step procedure (Bell-Pedersen et al. of mobile introns, for they suggest a means for intron
1989) by first crossing T4 38am51amwith a plasmid carrying an
transfer to ectopic sites.
intronless td variant, tdOP8, which is resistant to I-TevI cleav-age. T4 38am51amtdOP8 was then crossed with a plasmid carrying
the intronless allele, tdDIn, to generate T4 38am51amtdDIn. T4
MATERIALS AND METHODS
38am51amtdDIndexA and T4 38am51amtdOP8dexA containing a
short insertion in the dexA allele were constructed by marker
Bacterial and phage strains: E. coli strains CR63 (supD), the
rescue from pUC18dexA2provided by P. Gauss (Western State nonsense-suppressing suppressor-plus (Sup1) host, and MH1
TABLE 1
Intron donor plasmids
Exon I Exon II
Class Plasmid length (bp)a length (bp)
Wild type pMPtdSG2::supF 744 1016
DExonII pMPDEII-1 744 100
pMPDEII-2 744 50
pMPDEII-3 744 24
pMPDEII-4 744 8
pMPDEII-5 744 0
DExon I pMPDEI-6 100 1016
pMPDEI-7 50 1016
pMPDEI-8 25 1016
pMPDEI-9 10 1016
pMPDEI-10 0 1016
DExon I1 DExon II pMPDEIDEII-16 100 100
pMPDEIDEII-15 50 50
pMPDEIDEII-14 25 24
pMPDEIDEII-13 10 8
pMPDEIDEII-12 0 0
aLength reflects the amount of homology to exon I of the I-TevI-cleaved recipient allele where the cleavage
site is located 23 nt upstream of the exon I-exon II junction.
on an E. coli optA host (Gausset al. 1987). T4 38am51amtdOP8 was chloroform and phage lysates were titered on MH1 (Sup8) and
CR63 (Sup1) to determine the concentration of the suppressor-crossed with T4K10DrnhD10-777 (WoodworthandKreuzer
1996), provided by K. Kreuzer, and plated on CR63(l) to containing Su1and total phage, respectively.
Analysis of homing products:To distinguish homing events select against T4K10 (38am51amdenA denB) phage. T4 38am51am
td-OP8rnhD10-777 was confirmed by the polymerase chain re- (Td2) from plasmid integrants (Td1), lysates were plated on an OK305 (Sup8) host to select for nonsense-suppressed (Su1) action (PCR) and crossed with pBStdDIn to generate T4 38am
-51amtdDInrnhD10-777. phage and to screen for thymidylate synthase-negative (Td2)
phage using the halo phenotype (Hallet al. 1967). Su1Td2
Plasmids:Plasmid pMP is a derivative of pKK061, which has
a T4 origin of replication (ori34) in pBR322 (Selicket al. 1988). phage were plaque purified and homing events were confirmed by the absence of an intact copy of the tdDIn allele us-To generate pMP, the EcoRI site of pKK061 was filled in using
the Klenow fragment of E. coli DNA polymerase I, and the EcoRV- ing PCR. Recombination junction sequences were PCR ampli-fied using intron variant SG2::supF-specific oligonucleotides to-BamHI segment of the pBSKS1 polylinker (Stratagene, La
Jolla, CA) was inserted at these sites in pKK061 to introduce a new W165, 59-GCAGCTGGATATAATTCCGGGGTA-39, and W248, 59-TGGTGGTGGGGGAAGGATTCGA-39, and T4-specific oligo-EcoRI cloning site. The tdSG2::supF allele and its exon deletion
variants (Table 1) were amplified from phage T4K10 tdSG2::supF nucleotides W731, 59-CCTGAACTTAGTATCACAAGCG-39, and W657, 59-ATTCCATATCCCGTTCGTGC-39. Oligonucleotides by PCR and cloned into the EcoRI and BamHI sites of pMP to
generate pMPtdSG2::supF. The tdSG2::supF allele contains a td W731, W248, W165, and W657 are represented as P1, P2, P3, and P4, respectively, in Figure 4. PCR products were purified intron in which the I-TevI open reading frame has been replaced
with the XbaI-to-BanII fragment of pBSPLO1containing a supF by centrifugation through a Centricon 100 column (Amicon, Beverly, MA) and sequenced.
gene under control of a T4 gene 23 promoter (Selick et al. 1988). A point mutation, SC79, was introduced into the intron core to inhibit splicing and render the td gene thymidylate
syn-thase deficient (Td2;ChandryandBelfort1987). The intron RESULTS is flanked by the entire td coding sequence and adjacent T4
Genetic assay for intron homing in T4: An in vivo sequence extending to 766 bp upstream and 1016 bp
down-stream of the intron. The I-TevI expression plasmid pACYC-lPL- genetic assay was developed to quantitatively analyze exon
ORF has the HindIII-to-BamHI fragment of pKC30 (Rosenberg homology effects on intron homing during T4 infection et al. 1983), providing phagelregulatory elements, in pACYC184 (Figure 2). I-TevI was supplied from a plasmid (pACYC-(Chang and Cohen 1978). The I-TevI open reading frame l
PL-ORF) under control of the lPL promoter and a
(ORF) and flanking intron sequences are in the SmaI site of the
lcI857 repressor gene carried on the host chromosome. pKC30 fragment.
Intron homing assay:CR63cI857kanRcells carrying a wild-type The intron donor allele was on a compatible plasmid,
intron donor plasmid, pMPtdSG2::supF, or an exon deletion pMPtdSG2::supF. In this allele the td intron ORF has variant (Table 1), and pACYC-lPL-ORF were grown in tryptone- been replaced with a supF gene, encoding an
amber-yeast extract broth with 100mg/ml ampicillin and 25 mg/ml
suppressing tRNAtyr under control of the T4 gene 23
chloramphenicol at 308to an OD650of 0.2. Cultures were shifted
late promoter (Selicket al. 1988). The donor allele is to 428for 30 min, then infected with T4 phage at a multiplicity
Figure2.—Genetic selection scheme to monitor homing. The intron recipient, T4 38am51amtdDIn, was used to infect CR63cI857
carrying an I-TevI expression plasmid and an intron donor plasmid, pMPtdSG2::supF. The intron donor has a supF nonsense-sup-pressing gene in the intron, a mutation in the intron core (m) rendering an intron-containing td gene Td2, and varying lengths of exon I and/or exon II (n) (Table 1). Induction of I-TevI was followed by T4 infec-tion. Lysates were assayed on a Sup8 host to select for Su1phage that had inherited the intron and a Sup1 host to quantitate total phage. Open bars, td exons; shaded bar, intron variant tdSG2::supF; Apr,
ampicil-lin resistance; Cmr, chloramphenicol
resis-tance. Shaded background highlights hom-ing products. Td phenotypes:%, Td1; *, Td2.
rendering the intron splicing defective. The intron re- type” (Table 1). Intron homing with the wild-type donor was efficient, with an average ratio of Su1/total phage cipient was phage T4 38am51amtdDIn, which contains a
Td1intronless td allele (tdDIn). Additionally, the recipi- equaling 6.8 31021 (Figure 3, A and B). Background
recombination, which was determined with an isogenic ent phage harbored amber mutations in genes 38 and
51, encoding essential packaging functions. These muta- recipient phage, T4 38am51amtdOP8, containing an
I-TevI-resistant, intronless td allele (tdOP8), resulted in a mean tions prevent plaque formation on a nonsuppressing
(Sup8) host (Selicket al. 1988), resulting in a nonsup- ratio of 7.2 3 1023 Su1/total phage (Figure 3, A and
B). Thus, intron homing levels were two orders of mag-pressed Su2Td1 phenotype (Figure 2). Plasmid
integ-rants would contain the suppressing supF allele and two nitude above background, I-TevI-independent recom-bination levels when homology in both exons was non-copies of the td gene (the donor allele and an
uninter-rupted recipient allele) and would display a Su1Td1 limiting.
Analysis of homology effects on intron homing in T4: phenotype. In contrast, true homing events are
distin-guishable by their Su1Td2phenotype, because inheri- To determine the relationship between homing and length of homology, intron donor plasmids were con-tance of the splicing-deficient intron converts the
recipi-ent tdDIn gene to Td2(Figure 2). Td2plaques can be structed with truncated exons (Table 1). The length of homology to recipient exons ranged from 0 to 1016 bp readily distinguished from their Td1 counterparts by
white halos on OK305 cells (Hall et al. 1967). In our on one or both sides of the intron. Due to the eccentric position of the I-TevI cleavage site in the td homing assay, the ratio of Su1/total phage was used to quantitate
levels of intron inheritance. The Td phenotype was used site (Figure 1A), homology with exon I of the cleaved recipient occurs upstream of the DSB (Figure 1; Table only when homing approached background
recombina-tion and to isolate homing products for physical analysis. 1). When homology was limiting in one exon, intron homing levels remained elevated above background re-In our assay tdSG2::supF, containing full-length td
However, below 50 bp of homology on both sides a dramatic decrease in homing levels occurred (Figure 3B), whereas at 25 bp or less homing occurred at or below the level of background recombination, in con-trast with comparable donor variants in which only one exon was limiting in homology.
Analysis of one-sided heterology events:The apparent homing in the complete absence of either exon I or exon II (Figure 3A) prompted investigation of the na-ture of these events. Independent homing assays for donor plasmids lacking exon I or exon II were per-formed (Figure 4). Su1 phage were screened for Td2 plaques to distinguish true homing events from plasmid integrants, events that are detected increasingly when homology on one side of the break is eliminated (Figure 2). Intron homing was confirmed to have occurred by PCR analysis of plaque-purified phage (data not shown). A single homing product from each of 14 independent infections with theDEI donor pMPDEI-10 and 21 infec-tions with theDEII donor pMPDEII-5 was selected for further analysis.
Phage DNA from independent homing events was amplified by PCR using intron-specific and phage-spe-cific primers flanking the exon that was deleted from the donor (Figure 4A). In each event resulting from the exon I-minus or exon II-minus donor, the PCR product differed in size from the product generated by the wild-type donor (Figure 4B and data not shown). Sequence analysis of recombination junctions revealed that intron homing from the exon-minus donors resulted in dele-tions, representing loss in the recipient phage of the exon that was absent from the donor (Figure 4, C and D). In addition, various amounts of vector DNA from the donor plasmid were coinherited with the intron, Figure3.—Exon-homology dependence of intron homing.
(A) Homing for donors with one exon truncated. The mean explaining why in some cases a PCR product larger than ratios of Su1/total phage from three independent experiments that of the wild-type event was generated despite the were plotted against the length of the exon for donors with
occurrence of a deletion (Figure 4, A and B). deletions in either exon I (m,n) or exon II (d,s). Filled
When exon I was absent from the donor allele, intron symbols, infections with I-TevI-sensitive strain T4 38am 51am
homing was associated with deletions originating at the tdDIn; open symbols, infections with I-TevI-resistant strain T4
38am51amtdOP8. Error bars reflect the standard deviation from
cleavage site (CS) and extending upstream for 584–2119 the mean. The total phage burst was equivalent for all infec- bp (Figure 4C). The absence of exon II from the donor tions with the I-TevI-resistant and the I-TevI-sensitive phage.
allele resulted in deletions extending downstream from (B) Homing for donors with both exons truncated. Constructs
the CS, but overall these were markedly smaller com-are listed in Table 1. Plot and conventions as in A.
pared to those in sequences upstream of the CS, ranging from 18 to 776 bp (Figure 4, C and D). Because the CS is located 23 nt upstream of the 59end of exon II (Figure homing levels were.100-fold above background.
How-ever, when either exon was reduced below 50 bp, hom- 1A), the two events that had only 18 bp deleted resulted in recombinant phage that had a complete exon II. ing levels underwent a sharp decrease, but remained
elevated at least 10-fold above background levels, even Analysis of recombination junctions revealed that in each of the cases examined, a short region of homology when one exon was completely deleted from the donor.
When both exons were limiting, an exon length of existed between sequences in the recipient phage and sequences in the donor plasmid (Figure 5). Of the 14 50 bp was again found to be critical for intron homing
(Figure 3B). Although at 50 bp of homology on both events analyzed from theDexon I donor (Figure 4C), 9 different sequences on the recipient phage and the sides of the break the level of homing was at least
10-fold lower than when a single exon was reduced to this donor plasmid were utilized in the repair event, indicat-ing that the 10-fold elevation of homindicat-ing above back-length, it was still more than three orders of magnitude
Figure4.—Homing products from one-sided heterology events. (A) The proposed formation of homing products from an intron donor lacking exon I (left) or exon II (right). Cleavage and processing of the recipient leads to extensive degradation of the exon that is absent in the donor. The donor is invaded on one side of the intron by homologous exon sequences of the recipient, and on the other side by heterologous sequences of the recipient, leading to intron inheritence. Thin lines, td exons; wavy lines, flanking phage DNA; thick lines, vector sequences; shading, intron.nmarks the recombination junction and the point to which degradation occurs in the recipient. Small arrows, intron-specific (P2, P3) and phage-specific (P1, P4) oligonucleo-tide primers used for PCR. (B) Agarose gel analysis of PCR products. PCR amplification using primers P1 and P2 (lanes 1–6) and P3 and P4 (lanes 7–12) was performed on phage from homing assays with the wild-type donor (lanes 1 and 7), exon I-minus donor pMPDEI-10 (lanes 2–5), and exon II-minus donor pMPDEII-5 (lanes 8–11). Lanes 6 and 12 are control PCR reactions without template DNA. M is a 1-kb ladder with DNA lengths in kilobases indicated on the left. The product names shown below each lane correspond to those shown in C and D. (C) Deletions upstream of cleavage site. The size of the deletion accompanying intron inheritance from a donor allele lacking exon I was determined by sequencing across recombination junctions of 14 independent homing products. Independent events that had identical junctions are indicated by brackets. (D) Deletions downstream of cleavage site. The size of the deletion was determined for 21 independent homing products resulting from a donor allele lacking exon II.
homology. Rather, the phage appeared to be quite pro- ent and the donor to effect repair. In each of the events for which the sequence was determined, a short region miscuous in selecting a site for initiation of repair.
recipi-Figure5.—Recombination junctions re-sulting from one-sided heterology. Su1Td2 phage from intron donors lacking exon I (A) or exon II (B) were amplified with re-cipient phage-specific and donor plasmid-specific primers (see Figure 4). PCR prod-ucts were sequenced and recombination junctions of homing products (P) are shown along with corresponding donor (D) and recipient sequences (R). In some cases se-quencing indicated the size of the deletion, but the exact junction sequence could not be determined (Figure 4, I-a, II-b, -c, -g, -j, and -l). Donor bases are indicated by un-derlining. Recipient bases are indicated by shading. Boxed sequences indicate the in-ferred crossover site. Brackets indicate se-quences with three or more identical nucle-otides, separated from the boxed sequence by a single mismatch. The length of se-quence deleted (D) and the number of times each junction was isolated (n) are indicated above the sequence.
tions were comprised of 4–13 bp of perfect homology, to.700 bp. These levels are consistent with previous analyses of intron homing in rnh phage (Muelleret al. although some junctions had interrupted homologies
(Figure 5B). However, none of the junctions indicated 1996a;Huanget al. 1999). Below 25 bp of homology, homing levels for the rnh recipient phage were equiva-a joining of donor equiva-and recipient DNA in the complete
absence of homology. lent to those of the wild-type phage (Figure 6A), indicat-ing that the absence of this exonuclease does not affect Homology effects on homing in
exonuclease-defi-cient phage backgrounds:Degradation of the resection homing levels when homology is very limiting.
In contrast to the rnh phage, there was a striking segment and processing of DNA ends is required to
allow precise intron insertion (Figure 1A;Mueller et elevation in homing levels in the dexA phage when ho-mology was limiting (Figure 6B). When hoho-mology was al. 1996a,b;Huanget al. 1999). Homing was therefore
examined under conditions of limiting homology in rnh extensive, as in the wild-type donor, the level of homing by the dexA variant was similar to that of the parental mutant phage deficient in the 59-39DNA exonuclease
activity of RNase H and dexA mutant phage deficient in dexA1 phage, but as homology was decreased to 50 bp in both exons, the ratio of Su1/total phage in the dexA the 39-59single-strand DNA exonuclease, DexA.
Homing assays were performed using the wild-type recipient was almost 100-fold greater than that of the parental recipient. At 25 bp of exon homology, homing donor plasmid and plasmids carrying deletions in both
exons (Table 1) with rnh or dexA recipient phage vari- in the dexA recipient was 1000-fold higher than with the parental phage (Figure 6B). When homology in both ants (Figure 6). Homing levels in the rnh recipient were
1989a;ClymanandBelfort1992;Parkeret al. 1996). Because intron homing occurs naturally in the context of the T4 lytic cycle, a systematic analysis of homology dependence within this context was important, not only for mechanistic reasons, but to establish which phage factors might influence homing when homology is lim-iting. A genetic system was developed, which allowed quantitative analysis of intron homing levels under vari-ous conditions of exon homology and phage genotype. The use of a selectable, splicing-deficient intron pro-vided exquisite sensitivity, allowing for detection of rare intron acquisition events and a genetic means for distin-guishing between homing and plasmid integration in recipient parental and relevant mutant phage.
In phage T4, the occurrence of a DSB stimulates the initiation of recombination-dependent replication at the break site (Kreuzeret al. 1995;Georgeand Kreu-zer1996;Muelleret al. 1996a). Accordingly, infection of cells harboring an intron donor plasmid with full-length exons with phage carrying an intronless td allele resulted in high levels (.50%) of intron inheritance in the presence of I-TevI (Figure 3, A and B). Homing levels remained elevated with as little as 50 bp of homol-ogy on one or both sides of the break, after which they began to decline, although they remained at least 10-Figure 6.—Homology dependence of homing in exo- fold above background, even when one exon was com-nuclease-deficient phage. The in vivo homing assay was
per-pletely deleted from the donor allele (Figure 3A). How-formed with donors containing truncations in both exons and
ever, homology lengths of 25 bp and less on both sides either the parental recipient phage or exonuclease-deficient
variants rnh (A) and dexA (B) (see Figure 2, Table 1). Su1/ of the break resulted in homing levels that were equal total phage ratios were plotted against the length of homology. to or less than background recombination levels oc-(j) T4 38am51amtdDIn, (h) T4 38am51amtdOP8, (m) T4 38am51am
curring in the absence of an I-TevI cleavage site (Figure tdDInrnh, (n) T4 38am51amtdOP8rnh (d) T4 38am51amtdDIndexA,
3B). (s) T4 38am51amtdOP8dexA. The phage burst in the rnh
infec-tions was consistently 10-fold lower than that of the parental It appears that the critical length of homology for infections, as previously reported (HobbsandNossal1996). intron homing in wild-type T4 lies between 25 and 50 bp. This is in agreement with previous studies on sponta-neous recombination in T4, which indicated that 25 to events for donors with 25-bp and 10-bp exons were
con-50 bp of homology is required for efficient recombina-firmed by the halo assay and PCR analysis (data not
tion (BautzandBautz1967;Singeret al. 1982). Like-shown). However, when both exons were completely
wise, 35 to 50 bp of homology is required for efficient deleted from the donor allele, homing levels became
intron homing in a phagelmodel system (Parkeret al. indistinguishable from background recombination
lev-1996). This similarity in homology length requirements els. More than 1010 total phage from several
indepen-likely reflects the functional components of DSB-medi-dent infections were analyzed by the halo assay and/or
ated recombination in these systems. One possibility is PCR but no I-TevI-mediated intron acquisition events
that the strand transferase is limited by its ability to in the absence of flanking homology were observed. For
recognize or bind to short homologies. In vitro analysis all donor alleles, the level of background
recombina-of UvsX indicated that it does not catalyze pairing be-tion, as well as the phage burst, for the dexA and parental
tween molecules with,30 bp of homology (Salinaset phage recipients were similar, indicating that the
exo-al. 1995). However, RecA effects pairing in vitro between nuclease-deficient variants do not have a general
replica-DNA molecules sharing 15 bp of homology (Hsieh et tion advantage over the parental phage (Figure 6 and
molecules sharing only 13 bp of homology (Kingand uninterrupted homology (13 bp), and appeared in 4 out of 14 events (Figures 4C and 5A, I-i), it seems likely that Richardson1986).
Exon deletions and microhomology mark illegitimate the length of microhomology plays a role in determining the site of repair. However, computer analysis indicated homing events: Analysis of those homing events with
one-sided homology revealed that in each case deletions that numerous segments of microhomology exist in proxi-mal upstream regions of the donor and recipient, with at occurred in the recipient phage extending from the CS
to various positions in the exon sequence corresponding least five sites displaying six or seven perfect matches (data not shown). Another factor must therefore also be consid-to the exon that was absent from the donor. Similarly,
DSB repair in a phage T7 in vitro system was associated ered to account for the observed bias. Indeed, the up-stream and downup-stream ends produced by the cleavage with deletion of nonhomologous DNA occurring at the
ends (LaiandMasker1998). Sequence analysis of the reaction are not equally available for exonucleolytic degra-dation because I-TevI remains bound to the downstream recombination junctions indicated that numerous short
stretches of homology between the donor plasmid and cleavage product, providing protection (Mueller et al. 1996b). This bias in exonucleolytic processing is likely to the recipient phage ranging from 4 to 13 bp were used
in repair of the DSB (Figure 5). This differs from a account in large measure for the lengthier degradation tracts in exon I than exon II (Figure 4, C and D), much similar analysis conducted on one-sided repair events
occurring in a phage l-td intron model system where as it does for lengthier coconversion tracts in the upstream than the downstream exon (Muelleret al. 1996b). two out of four junctions examined appeared to form
in the complete absence of homology (Parker et al. Exonuclease activity influences homing levels with limiting homology: The gp46/47 complex and RNase 1996). Similarly, analysis of restriction
endonuclease-mediated illegitimate recombination events in E. coli re- H likely contribute the majority of 59-39end processing occurring during recombination and replication in T4. vealed that only 1–4 nt of homology were present at the
junctions, with only a single nucleotide of homology ap- Because the gp46/47 complex is required for phage viability and the homing reaction (Epsteinet al. 1963; pearing in 5 of 10 cases (Kusano et al. 1997). Indeed,
illegitimate DSB repair events occurring in E. coli, yeast, Wiberg 1966;Mueller et al. 1996a), we were unable to determine its influence on homing when homology and mammalian systems have been attributed to an
end-joining mechanism (for example, Moore and Haber was limiting. However, the essential nature of gp46/47 makes the complex less likely than nonessential exo-1996;Kusanoet al. 1997). The propensity of phage T4
to utilize short regions of homology to effect DSB repair nucleases to influence homing efficiency under subopti-mal conditions such as limiting homology. We therefore in the absence of extensive homology indicates that the
T4 recombination machinery can catalyze strand transfer examined T4 RNase H, which has 59-39 DNA exo-nuclease activity and has been implicated in DNA repli-between very short regions of homology. Correspondingly,
spontaneous deletions in T4 have been associated with cation, repair, and intron homing (Hollingsworth andNossal1991;Muelleret al. 1996a;Woodworth recombination between direct repeats ranging from 4 to
15 bp in length (Pribnowet al. 1981;Owenet al. 1983; andKreuzer1996;Huanget al. 1999). Homing levels in rnh phage infections were somewhat decreased com-Mosiget al. 1998). The interesting question of whether
this mechanism involves UvsX is difficult to address in vivo pared to the wild-type phage for the majority of homol-ogy lengths analyzed (Figure 6A). However, it remains because of the requisite role of this protein in intron
homing (Clyman and Belfort 1992; Mueller et al. unclear whether these subtle decreases in homing levels in rnh phage are directly related to loss of exonuclease 1996a).
A comparison of the deletions produced in several inde- activity or due to an indirect effect on replication or repair (WoodworthandKreuzer1996;Huanget al. pendent events indicated that the extent of degradation
was markedly greater on the upstream side of the cleavage 1999). If the effect of RNase H on intron homing is direct, rather than through DNA replication, the en-site than on the downstream side (Figure 4, C and D).
The two most likely interpretations of this result relate to zyme is likely to generate 39 invasive ends through its 59-39 DNA exonuclease activity (Huang et al. 1999). the distribution of microhomologies on one hand and to
access to exonucleolytic degradation on the other. First, Therefore, an rnh deficiency may lead to shorter 39tails available for the search for homology.
regions of microhomology between the phage and the
donor may be distributed such that on the downstream Conversely, when donor alleles with limiting homol-ogy in both exons were analyzed in phage deficient in side the more lengthy regions are located closer to the
break than on the upstream side. Indeed, hot spots of the 39-59exonuclease DexA, homing levels were signifi-cantly elevated above those generated with the parental spontaneous deletion formation in T4 display
microho-mology between direct repeats, and the frequency of recipient phage (Figure 6B). The most likely explana-tion for this result is that in the absence of DexA, the 39 events occurring at particular sites corresponds well with
their length of microhomology (Allgood and Silhavy tail, containing homologous td sequences, is not readily degraded and consequently is utilized more efficiently 1988). Because the most upstream junction, located.2
Prokaryotic introns and inteins: a panoply of form and function.
Nucleases and intron translocation:Group I introns
J. Bacteriol. 177: 3897–3903.
occupy a wide variety of genomic locations within a Bell-Pedersen, D., S. M. Quirk, M. AubreyandM. Belfort,1989
A site-specific endonuclease and co-conversion of flanking exons
phylogenetically diverse set of organisms (Lambowitz
associated with the mobile td intron of phage T4. Gene 82: 119–
and Belfort 1993; Belfort et al. 1995). Among the
126.
T-even phages, group I introns are variably distributed, Bell-Pedersen, D., S. Quirk, J. ClymanandM. Belfort,1990
In-tron mobility in phage T4 is dependent upon a distinctive class
indicating that they may have been acquired after the
of endonucleases and independent of DNA sequences encoding
divergence of these strains from a common ancestor
the intron core: mechanistic and evolutionary implications.
Nu-(Shubet al. 1988;Quirket al. 1989b). Several observa- cleic Acids Res. 18: 3763–3770.
Bryk, M., S. M. Quirk, J. E. Mueller, N. Loizos, C. Lawrenceet
tions have led to the hypothesis that intron acquisition
al., 1993 The td intron endonuclease makes extensive sequence
at new sites may be mediated by intron
endonuclease-tolerant contacts across the minor groove of its DNA target.
stimulated DSB repair (Mueller et al. 1993). Indeed, EMBO J. 12: 2141–2149.
Bryk, M., M. Belisle, J. E. MuellerandM. Belfort,1995 Selection
I-TevI is highly tolerant of perturbations within its target
of a remote cleavage site by I-TevI, the td intron-encoded
endonu-sequence, suggesting that cleavage events occur at
re-clease. J. Mol. Biol. 247: 197–210.
lated sequences (Bryket al. 1993, 1995). Furthermore, Chandry, P. S.,andM. Belfort,1987 Activation of a cryptic 59 splice site in the upstream exon of the phage T4 td transcript:
substantial phylogenetic and molecular evidence
sup-exon context, missplicing, and mRNA deletion in a fidelity
mu-ports DSB-mediated horizontal transfer of a group I
tant. Genes Dev. 1: 1028–1037.
intron to the mitochondrial genome of numerous plant Chang, A. C. Y.,andS. N. Cohen,1978 Construction and
character-ization of amplifiable multicopy DNA cloning vehicles derived
species (Choet al. 1998).
from the P15A cryptic miniplasmid. J. Bacteriol. 134: 1141–1156.
Considering that the recognition sequence of I-TevI
Cho, Y., Y. Qiu, P. KuhlmanandJ. D. Palmer, 1998 Explosive
spans 37 bp (Bryket al. 1995), it is reasonable to specu- invasion of plant mitochondria by a group I intron. Proc. Natl. Acad. Sci. USA 95: 14244–14249.
late that an ectopic, low-specificity recognition site is
Clyman, J.,andM. Belfort,1992 Trans and cis requirements for
likely to share some homology with the natural
recogni-intron mobility in a prokaryotic system. Genes Dev. 6: 1269–1279.
tion sequence and therefore with exon sequences flank- Dujon, B.,1989 Group I introns as mobile genetic elements: facts ing the intron. The proclivity of the DSB repair appara- and mechanistic speculations—a review. Gene 82: 91–114.
Epstein, R. H., A. Bolle, C. M. Steinberg, E. Kellenberger, E.
tus to seek very short homologous sequences, as in
Boy Dela Touret al., 1963 Physiological studies of conditional
phage T4 (Figure 5), suggests that these sequences may lethal mutants of bacteriophage T4D. Cold Spring Harbor Symp. be preferentially utilized for repair. We further propose Quant. Biol. 28: 375–394.
Gauss, P., M. Gayle, R. B. WinterandL. Gold,1987 The
bacterio-that genetic background or fluctuations in the levels of
phage T4 dexA gene: sequence and analysis of a gene conditionally
DNA nucleases may be influential factors in such DSB- required for DNA replication. Mol. Gen. Genet. 206: 24–34. mediated intron translocation. Thus, cleavage at a low George, J. W.,andK. N. Kreuzer,1996 Repair of double-strand
breaks in bacteriophage T4 by a mechanism that involves
exten-specificity site with reduced 39-59exonucleolytic
degra-sive DNA replication. Genetics 143: 1507–1520.
dation and in the presence of endonuclease-mediated Gruber, H., G. Kern, P. GaussandL. Gold,1988 Effect of DNA protection (Muelleret al. 1996b) may preserve 39tails sequence and structure on nuclease activity of the DexA protein
of bacteriophage T4. J. Bacteriol. 170: 5830–5836.
at the break site and allow the ends to participate in
Hall, D. H., I. TessmanandO. Karlstrom,1967 Linkage of T4
illegitimate DSB repair. Significantly, repair events were genes controlling a series of steps in pyrimidine biosynthesis. observed in the absence of homology on the exon II Virology 31: 442–448.
Hobbs, L. J.,andN. G. Nossal,1996 Either bacteriophage T4 RNase
side in which the complete td coding sequence was
main-H or Escherichia coli DNA polymerase I is essential for phage
tained on that side, because degradation did not extend
replication. J. Bacteriol. 178: 6772–6777.
beyond the resection segment (Figures 1A, 4D, and 5B, Hollingsworth, H. C.,andN. G. Nossal,1991 Bacteriophage T4 II-a). Such an event would sustain gene function by encodes an RNase H which removes RNA primers made by the T4 DNA replication system in vitro. J. Biol. Chem. 266: 1888–1897.
maintaining the proper coding sequence of the newly
Hsieh, P., C. S. Camerini-OteroandR. D. Camerini-Otero,1992
invaded gene after intron splicing. The synapsis event in the homologous pairing of DNAs: RecA
recognizes and pairs less than one helical repeat of DNA. Proc. We are grateful to Gregory Lopez, Yi-Jiun Huang, and Dilip Nag for
Natl. Acad. Sci. USA 89: 6492–6496. helpful discussions and comments on the manuscript. The Molecular
Huang, Y-J., M. M. Parker andM. Belfort,1999 Role of exo-Genetics Core facility provided oligonucleotides and automated DNA
nucleolytic degradation in group I intron homing in phage T4. sequencing. We thank Dorie Smith for technical assistance and
Maryel-Genetics 153: 1501–1512.
len Carl for preparing the manuscript. This work was supported by King, S. R., andJ. P. Richardson, 1986 Role of homology and National Institutes of Health grants GM-39422 and GM-44844 to M.B. pathway specificity for recombination between plasmids and
bac-teriophage lambda. Mol. Gen. Genet. 204: 141–147.
Kreuzer, K. N.,andS. W. Morrical,1994 Initiation of DNA replica-tion, pp. 28–42 in Molecular Biology of Bacteriophage T4, edited by
LITERATURE CITED
J. D. Karam.American Society for Microbiology Press, Washing-ton, DC.
Allgood, N. D.,andT. J. Silhavy,1988 Illegitimate recombination
1995 Recombination-dependent DNA replication stimulated by otide sequence of the lysozyme gene of bacteriophage T4. Analysis of mutations involving repeated sequences. J. Mol. Biol. 165: double-strand breaks in bacteriophage T4. J. Bacteriol. 177: 6844–
6853. 229–248.
Parker, M. M., D. A. Court, K. PreiterandM. Belfort,1996
Ho-Kusano, K., K. Sakagami, T. Yokochi, T. Naito, Y. Tokinagaet al.,
1997 A new type of illegitimate recombination is dependent on mology requirements for double-strand break-mediated recombi-nation in a phage lambda-td intron model system. Genetics 143: restriction and homologous interaction. J. Bacteriol. 179: 5380–
5390. 1057–1068.
Pribnow, D., D. C. Sigurdson, L. Gold, B. S. SingerandC. Napoli, Lai, Y.,andW. Masker,1998 In vitro repair of gaps in bacteriophage
1981 rII cistrons of bacteriophage T4. DNA sequence around T7 DNA. J. Bacteriol. 180: 6193–6202.
the intercistronic divide and positions of genetic landmarks. J.
Lambowitz, A. M.,andM. Belfort,1993 Introns as mobile genetic
Mol. Biol. 149: 337–376. elements. Annu. Rev. Biochem. 62: 587–622.
Quirk, S. M., D. Bell-Pedersen andM. Belfort,1989a Intron
Luder, A.,and G. Mosig,1982 Two alternative mechanisms for
mobility in the T-Even phages: high frequency inheritance of initiation of DNA replication forks in bacteriophage T4: priming
group I introns promoted by intron open reading frames. Cell by RNA polymerase and by recombination. Proc. Natl. Acad. Sci.
56:455–465. USA 79: 1101–1105.
Quirk, S. M., D. Bell-Pedersen, J. Tomaschewski, W. Rugerand
Mickelson, C.,andJ. S. Wiberg,1981 Membrane-associated DNase
M. Belfort,1989b The inconsistent distribution of introns in activity controlled by genes 46 and 47 of bacteriophage T4D and
T-even phages indicates recent genetic exchanges. Nucleic Acids elevated DNase activity associated with the T4 das mutation. J.
Res. 17: 301–315. Virol. 40: 65–77.
Rosenberg, M., Y. S. HoandA. Shatzman,1983 The use of pKC30
Moore, J. K.,andJ. E. Haber,1996 Cell cycle and genetic
require-and its derivatives for controlled expression of genes. Methods ments of two pathways of nonhomologous end-joining repair of
Enzymol. 101: 123–138. double-strand breaks in Saccharomyces cerevisiae. Mol. Cell. Biol.
Salinas, F., H. JiangandT. Kodadek,1995 Homology dependence 16:2164–2173.
of UvsX protein-catalyzed joint molecule formation. J. Biol.
Mosig, G., 1998 Recombination and recombination-dependent
Chem. 270: 5181–5186. DNA replication in bacteriophage T4. Annu. Rev. Genet. 32:
Selick, H. E., K. N. KreuzerandB. M. Alberts,1988 The bacterio-379–413.
phage T4 insertion/substitution vector system. A method for
Mosig, G., N. E. Colowickand B. C. Pietz, 1998 Several new
introducing site-specific mutations into the virus chromosome. bacteriophage T4 genes, mapped by sequencing deletion
end-J. Biol. Chem. 263: 11336–11347. points between genes 56 (dCTPase) and dda (a DNA-dependent
Shub, D. A., J. M. Gott, M.-Q. Xu, B. F. Lang, F. Michelet al., ATPase-helicase) modulate transcription. Gene 223: 143–155.
1988 Structural conservation among three homologous introns
Mueller, J. E., M. Bryk, N. LoizosandM. Belfort,1993 Homing of bacteriophage T4 and the group I introns of eukaryotes. Proc. endonucleases, pp. 111–143 in Nucleases, Ed. 2, edited byS. M. Natl. Acad. Sci. USA 85: 1151–1155.
Linn, R. S. LloydandR. J. Roberts.Cold Spring Harbor Labora- Singer, B. S., L. Gold, P. GaussandD. H. Doherty,1982
Determi-tory Press, Cold Spring Harbor, NY. nation of the amount of homology required for recombination
Mueller, J. E., J. Clyman, Y. Huang, M. M. ParkerandM. Belfort, in bacteriophage T4. Cell 31: 25–33.
1996a Intron mobility in phage T4 occurs in the context of Wiberg, J. S.,1966 Mutants of bacteriophage T4 unable to cause recombination-dependent DNA replication by way of multiple breakdown of host DNA. Proc. Natl. Acad. Sci. USA 55: 614–621.
pathways. Genes Dev. 10: 351–364. Woodworth, D. L., andK. N. Kreuzer,1996 Bacteriophage T4
Mueller, J. E., D. SmithandM. Belfort,1996b Exon coconversion mutants hypersensitive to an antitumor agent that induces topo-biases accompanying intron homing: battle of the nucleases. isomerase-DNA cleavage complexes. Genetics 143: 1081–1090. Genes Dev. 10: 2158–2166.
