Recruitment of host functions suggests a repair pathway for late steps in group II intron retrohoming

Abstract

Retrohoming of group II introns occurs by a mechanism in which the intron RNA reverse splices directly into one strand of a DNA target site and is then reverse transcribed by the associated intron-encoded protein. Host repair enzymes are predicted to complete this process. Here, we screened a battery of Escherichia coli mutants defective in host functions that are potentially involved in retrohoming of the Lactococcus lactis Ll.LtrB intron. We found strong (greater than threefold) effects for several enzymes, including nucleases directed against RNA and DNA, replicative and repair polymerases, and DNA ligase. A model including the presumptive roles of these enzymes in resection of DNA, degradation of the intron RNA template, traversion of RNA-DNA junctions, and second-strand DNA synthesis is described. The completion of retrohoming is viewed as a DNA repair process, with features that may be shared by other non-LTR retroelements.

Figure 1.

Retrohoming pathway for the Ll.LtrB intron in L. lactis and E. coli. The steps in the pathway are as follows: (1) Transcription from the donor template, splicing, translation and RNP formation. The RNP consists of the intron lariat and IEP having RT, maturase (M), and DNA endonuclease (E) activities. (2) Cleavage of recipient DNA by reverse splicing into the top strand and endonuclease cleavage of the bottom strand, at the sites marked ▴ and ▾, respectively. (3) cDNA synthesis. (4-7) Extended cDNA synthesis, RNA degradation, second-strand DNA synthesis, and repair. The order of steps 4-7 is unknown.

Figure 2.

Homing assay. (A) Schematic of assay. Plasmids are described in B and Supplemental Material. (IM) Intron marker; (PM1) donor plasmid marker; (PM2) recipient plasmid marker. ORI1 and ORI2 are compatible origins of replication. Homing products were selected for the IM and PM2. The pT7lac designation indicates that the T7 polymerase, which drives intron expression, is under control of a lac promoter. White rectangles are exons (E1 and E2), and dark-gray rectangles represent the group II intron. The black rectangle with a white I represents the group I td intron used to monitor retrohoming. Two homing products are shown, with the lower (RH) representing the verified retrohoming product lacking the group I intron. These products were detected by hybridization to a splice junction probe (short black bar). (B) Characteristics of intron donor, recipient, and homing products in different crosses. Plasmid donors and recipients are described in Materials and Methods and Supplemental Material. (IM) Intron marker, with resistance to kanamycin (Kan) and trimethoprim (Trp). (PM1) Donor plasmid marker with resistance to ampicillin (Amp) and chloramphenicol (Cam). (ORI1) Replication origin of donor plasmid. Intron length (nucleotides) depends on whether LtrA is expressed from within or outside of the intron. (PM2) Recipient plasmid marker with drug resistances as above, as well as tetracycline (Tet) and spectinomycin (Spc). (ORI2) Replication origin of recipient plasmid. Selection was for IM-PM2. Efficiency = (homing product)/recipient × 100.

Figure 3.

LtrA polymerization activity on RNA and DNA templates. DNA-dependent DNA polymerase and RT assays were done with four different template-primer substrates (S, I-IV; depicted below the graphs). Reactions contained the indicated enzymes and 10 μCi [α-³²P]dTTP (plus 200 μM dATP, dCTP, and dGTP for substrates I and III) in buffers (B) that contained high (H) or low (L) salt (450 and 100 mM NaCl, respectively; see Materials and Methods). In other experiments, LtrA showed no detectable activity with substrate I in reaction medium optimal for Klenow or M-MLV RT (REact2 and first-strand buffer, respectively; see Materials and Methods). The activity of LtrA with substrate II remained <5% of that with substrate IV in reaction medium containing lower salt (10 mM NaCl), and the ratio of activities with substrates II and IV remained the same with 10 μM unlabeled dTTP added or with different LtrA concentrations (20, 200, or 2000 nM). LtrA also displayed very low processive DNA polymerase activity with substrate I relative to RT activity with substrate III in PAGE assays for extension of 5′-³²P-labeled primer with 200 μM of each dNTP (<2.5% at 100 mM NaCl and 0 at 450 mM NaCl) (data not shown).

Figure 4.

DNA replication and retrohoming. (A) Real-time PCR assay for replication dependence of retrohoming. Cells containing intron-donor plasmid pACD2 and a ts recipient plasmid pB102B-ltrB, or the corresponding wild-type recipient plasmid pB101B-ltrB, were grown either at the permissive temperature (30°C) or the semirestrictive temperature (37°C). Plasmid DNAs were isolated at the indicated times and were used as the template in two independent real-time PCR reactions to quantify homing products and recipient plasmids. The relative mobility frequency was calculated as the ratio of homing product/(homing product + recipient). (B) Intron homing in a Pol III mutant. Cross 3 (shown in Fig. 2A,B) was performed in AB1157 (denoted wild type) and AB1157 dnaE at 30°C, 37°C, and 42°C. Mobility frequency was determined for three independent experiments and is expressed as the percentage of homing products per recipient.

Figure 5.

Roles of accessory functions in retrohoming. Facilitatory (+, teal) and inhibitory (-, red) functions are superimposed on a retrohoming pathway that is expanded from that in Figure 1. The order of steps 5 and 6 is arbitrary. Accessory functions are labeled in black on a teal or red background, with implicated E. coli enzymes in white. Their putative sites of action are indicated by dotted lines, as described in the text. Three possible sites of DNA synthesis by the repair polymerases are encircled and enlarged on the left (a-c).