Genetic and Biochemical Assays Reveal a Key Role for Replication Restart Proteins in Group II Intron Retrohoming
[['Mobile group II introns are non-long-terminal-repeat ( non-LTR ) retroelements that are commonly found in prokaryotes and in organellar genomes of eukaryotes and are thought to be evolutionary ancestors of splicesomal introns and retrotransposons in higher organisms [1] .', 'They consist of an autocatalytic intron RNA ( “ribozyme” ) and an intron-encoded protein ( IEP ) , which has reverse transcriptase ( RT ) activity .', 'These two components function together in a ribonucleoprotein complex ( RNP ) to promote intron mobility by a mechanism in which the excised intron lariat RNA uses its ribozyme activity to reverse splice directly into a DNA site and is then reverse transcribed by the IEP , yielding an intron cDNA that is integrated into the genome by host enzymes [2]–[5] .', 'By using this mechanism , group II introns insert at high frequency into specific DNA target sites in a process called “retrohoming” and at low frequency into ectopic sites that resemble the normal homing site in a process called “retrotransposition” or “ectopic retrohoming” [6] .', 'These processes enabled the dispersal of group II introns to a wide variety of bacteria and some archaea and likely into eukaryotic nuclear genomes , where ancestral group II introns are thought to have evolved into both spliceosomal introns and non-LTR-retrotransposons [3] , [7] , [8] .', 'Although the early reverse splicing and reverse transcription steps catalyzed by group II intron RNPs are common to retrohoming pathways in all organisms , the late host-mediated steps of second-strand DNA synthesis and cDNA integration can occur in different ways .', 'In Saccharomyces cerevisiae mitochondria , where retrohoming was studied initially , cDNA integration occurs largely by a recombination mechanism in which the nascent intron cDNA initiated at the recipient allele invades an intron-containing allele for completion of intron DNA synthesis before switching back to the recipient DNA in the upstream exon [9] , [10] .', 'In bacteria , however , the fully reverse spliced intron RNA is reverse transcribed to yield a full-length intron cDNA that is integrated directly into the recipient DNA by a RecA-independent mechanism hypothesized to involve host DNA repair enzymes [5] , [11] , [12] .', 'Recently , non-lariat , linear forms of the Lactococcus lactis Ll . LtrB intron RNA were found to retrohome in Drosophila melanogaster by using host non-homologous end-joining enzymes for cDNA integration [13] .', 'However , host factors that function in late steps in the retrohoming of group II intron lariat RNAs , the major retrohoming pathway used in nature , have not been identified conclusively in any organism , and consequently , the mechanisms used for these steps have remained poorly understood .', 'Figure 1 diagrams the major steps elucidated thus far in the retrohoming pathway of the L . lactis Ll . LtrB intron , which has been studied extensively as a model system for group II intron lariat RNA retrohoming in bacteria .', 'The Ll . LtrB intron was discovered in a relaxase gene ( ltrB ) in a conjugative element , where its splicing is required to produce functional relaxase for conjugation [14] , [15] .', 'Its IEP , denoted LtrA protein , is multifunctional , with RT , RNA splicing ( “maturase” ) , DNA binding , and DNA endonuclease activities [16] , [17] .', 'Transcription of the ltrB gene yields a precursor RNA , which contains the intron flanked by the 5′ and 3′ ltrB exons ( E1 and E2 , respectively ) .', 'The IEP , which is translated from within the intron , binds to the intron in the unspliced precursor RNA and promotes its splicing by stabilizing the catalytically active RNA structure [18] .', 'Splicing occurs via two sequential RNA-catalyzed transesterification reactions that yield ligated ltrB exons and an excised intron lariat RNA to which the IEP remains tightly bound in an RNP .', 'RNPs then initiate retrohoming by recognizing a DNA target sequence ( corresponding to the ligated ltrB E1–E2 DNA sequence ) , using both the IEP and base pairing of the intron RNA [19]–[21] .', 'After DNA target site recognition , the intron RNA fully reverse splices into the top strand of the DNA , leading to insertion of the intron RNA between the two DNA exons , while the IEP cleaves the bottom strand 9 nts downstream of the intron-insertion site and uses the 3′ end of the cleaved DNA strand for target DNA-primed reverse transcription ( TPRT ) of the inserted intron RNA [16] , [17] .', 'Finally , the resulting intron cDNA is integrated into the recipient DNA by host factors in late steps that minimally include the degradation or displacement of the intron RNA template strand , second ( top ) -strand DNA synthesis , resection of DNA overhangs , and ligation to seal DNA nicks [12] .', 'In addition to its native host , the Ll . LtrB intron splices and retrohomes efficiently in a wide variety of other bacteria , including Escherichia coli , where it has been studied by using the facile genetic and biochemical methods available for that organism [5] .', 'By screening E . coli mutants using two different plasmid-based retrohoming assays , we in collaboration with the Belfort laboratory previously identified candidate host factors that potentially function in the late steps in retrohoming , including RNase H1 and the 5′→3′ exonuclease activity of Pol I , both of which could contribute to degrading the intron RNA template strand; the host replicative polymerase Pol III , which may function in second-strand DNA synthesis; and DNA ligase A , which presumably seals strand nicks [12] .', 'Decreased retrohoming frequencies were also found in mutants deficient in host exo- and endonucleases activities [RecJ , DnaQ ( MutD ) , and SbcD] , which could function to resect overhangs or resolve intermediates , and increased retrohoming frequencies were found for mutants deficient in RNases I and E and exonuclease III , which in wild-type strains may suppress retrohoming by degrading the intron RNA or nascent cDNA [12] .', 'More recently , Coros et al . [22] , [23] extended this work by screening an E . coli transposon-insertion library for mutants defective in group II intron retrohoming into chromosomal sites , using a donor plasmid to express an Ll . LtrB-ΔORF intron carrying a kanR marker .', 'This screen identified additional host factors potentially involved in retrohoming , including polynucleotide phosphorylase ( PNPase ) , the DNA helicase Rep , and MnmE ( TrmE ) , which functions in tRNA modification , with additional host proteins ( CyaA , SpoT , and AtpA ) acting by affecting accessibility of chromosomal target sites or energy metabolism , and RNase E acting to impede retrohoming by degrading the intron RNA .', 'Although the genetic screens described above identified host genes in which mutations decrease retrohoming efficiency , it remains possible that some or many of these mutations affect retrohoming indirectly .', 'Such indirect effects could result from mutations that impair the propagation or expression of the intron-donor plasmid , decrease the intracellular levels or activity of group II intron RNPs , or impede the accessibility of group II intron RNPs to DNA target sites .', 'Additionally , all previous genetic screens relied upon the expression of an antibiotic-resistance marker to identify retrohoming events and are thus vulnerable to false positives arising from mutations that affect the expression of antibiotic-resistance ( e . g . , by affecting the expression of the antibiotic-resistance gene or cellular permeability to the antibiotic ) .', 'In some cases , studies of “indirect” effects revealed by the genetic screens have provided rich information about host responses and the regulation of group II intron mobility [22] , [23] .', 'However , further insight into the retrohoming mechanism requires the identification of host factors that function directly in this process .', 'Here we used an E . coli genetic screen and Taqman qPCR assay that mitigate indirect effects to identify candidate host factors for Ll . LtrB retrohoming , and we tested their function in retrohoming by using a newly developed biochemical assay that combines group II intron RNPs with cellular extracts to reconstitute the complete retrohoming reaction in vitro .', 'By using these multiple approaches , we confirmed some previously identified host factors but not others .', 'Additionally , we found that replication restart enzymes play a key role in intron retrohoming by initiating second-strand DNA synthesis .', 'Our findings indicate a novel mechanism for the major pathway of group II intron lariat retrohoming in bacteria , with features that may be shared by human LINE elements and other non-LTR retrotransposons .'], ['To identify host factors that function in retrohoming of the Ll . LtrB group II intron , we used two complementary approaches that mitigate weaknesses of previous genetic screens .', 'First , we used a plasmid-based group II intron retrohoming assay that controls for indirect effects to screen an E . coli mariner transposon-insertion library for mutants that have decreased or increased retrohoming efficiency .', 'This screen used the intron-donor plasmid pALG3 and recipient plasmid pBRR3-ltrB ( Figure 2A ) and was done in E . coli host strain HMS174 ( DE3 ) , which is RecA− and encodes an isopropyl β-D-1 thiogalactopyranoside ( IPTG ) -inducible T7 RNA polymerase for donor-plasmid transcription .', 'The donor plasmid pALG3 , which was newly constructed for this screen , uses a T7lac promoter to synthesize a precursor RNA , which contains an Ll . LtrB-ΔORF intron ( i . e . , an intron deleted for the LtrA ORF ) flanked by short 5′ and 3′ exons , with the 3′ exon linked in frame to an ORF encoding GFP .', 'The LtrA protein , which functions stoichiometrically to splice and mobilize the Ll . LtrB intron , is expressed from a position downstream of the GFP ORF , where it is co-transcribed with the intron RNA and translated using its own Shine-Dalgarno sequence .', 'Because the expression of the Ll . LtrB-ΔORF intron and its splicing to produce RNPs are linked to GFP expression , mutants with decreased group II intron RNP production as a result of defects in donor plasmid replication , LtrA protein expression , or the expression and splicing of the Ll . LtrB intron RNA , are identified readily by decreased GFP fluorescence after IPTG induction .', 'To enable direct selection for retrohoming events , the Ll . LtrB intron in pALG3 carries a trimethoprim-resistance retrotransposition-activated genetic marker ( TpR-RAM ) , which consists of a small trimethoprim-resistance gene inserted in the orientation opposite group II intron transcription , but interrupted by an efficiently self-splicing group I intron , the phage T4 td intron , in the forward orientation [24] .', 'During retrohoming via an RNA intermediate , the td intron is spliced , thereby reconstituting the TpR marker and enabling its expression after the intron retrohomes into the DNA target site .', 'By using this combination of TpR-RAM and GFP markers , mutants with transposon insertions that inhibit retrohoming without affecting Ll . LtrB expression or splicing are identified as TpS and GFP+ .', 'The screen was done in 96-well plates under calibrated selective conditions in which the cell density for log phase cells provides a measure of retrohoming efficiency ( Figure S1; Materials and Methods ) .', 'Screening of 9 , 200 colonies by two rounds of 96-well plate assays identified 61 transposon insertions that reproducibly gave a >4-fold decrease in retrohoming efficiency compared to a wild-type control and had no defect in RNP production , as judged by a fluorescence-activated cell sorter ( FACS ) assay of GFP synthesis ( Table S1 ) .', 'After mapping of transposon-insertion sites by thermal-asymmetric-interlaced ( TAIL ) PCR [25] , we identified 67 candidate protein-encoding genes , whose disruption or altered expression due to the proximity of the transposon insertion results in decreased retrohoming efficiency .', 'Six of these candidate genes were sites of multiple transposon insertions , and 12 were genes with nucleic acid-related functions found downstream of transposon-insertion sites within operons .', 'An additional transposon insertion ( C0719 ) that decreased retrohoming efficiency mapped to a site predicted to encode a small non-coding RNA ( sRNA ) ( Table S1 ) .', 'All the candidates were confirmed by Southern hybridization to contain a single mariner-transposon insertion at the indicated genomic location ( data not shown ) .', 'To complement the transposon-insertion screen , our second approach was to screen individual candidate strains for efficient integration into a chromosomal target site directly by using a Taqman qPCR assay to quantify both the 5′- and 3′-intron-integration junctions , thereby eliminating false positives that arise from mutations affecting expression of a drug-resistance phenotype ( Figure 2B ) .', 'Mobile group II introns can be retargeted to retrohome into different chromosomal DNA sites simply by modifying the intron RNA sequences that base pair to the DNA target sequence ( see Introduction ) , a gene targeting technology known as “targetron” [19] , [21] , [26] .', 'The Taqman qPCR assay uses an Ll . LtrB-ΔORF intron that was retargeted in this way to retrohome efficiently into a site in the rhlE gene , which encodes a non-essential DEAD-box protein whose disruption has no effect on cellular growth rate [21] , [27] .', 'The intron was expressed from the broad-host range donor plasmid pBL1 , which has a different DNA replication origin than pALG3 and employs an m-toluic acid-inducible promoter; the latter functions independently of host factors and is activated by a freely permeable inducer ( m-toluic acid ) that does not require cellular transporters to enter the cell [28] .', 'Additionally , the screen was carried out in mutant strains from the Keio collection in which deleted genes are replaced with a kanR marker , thereby mitigating polarity effects on downstream genes in operons [29] .', 'The Keio strains were supplemented by temperature-sensitive mutants to test the contribution of essential genes .', 'We used the Taqman qPCR assay to test all 68 candidate host factors identified in our initial transposon-insertion screen , as well as 30 additional candidate proteins that act on nucleic acids , including all 21 such candidates identified in previous mutant screens [12] , [22] , [30] .', 'Table 1 shows results of the Taqman qPCR assay for notable mutants , and Tables S1 and Table S2 show complete results for the mariner-transposon screen and Taqman qPCR assay , respectively .', 'Among the 68 candidates identified in the initial transposon library screen ( Table S1 ) , only ten ( dnaC , dnaT , gyrB , mdoB , paoD , rpoH , rpoN , tonB , ydcM , and yjjB ) had statistically significant decreases in retrohoming efficiency in the Taqman qPCR assay , and only four ( dnaC , dnaT , gyrB , and rpoH ) had substantial decreases ( 10–67% of wild type retrohoming efficiency; Table 1 and Table S2 ) .', 'This poor correlation highlights the difficulty of distinguishing direct and indirect effects and the necessity of using multiple approaches to identify host factors that function in retrohoming .', 'Among the candidates identified as potential retrohoming factors in previous screens [12] , [22] , [30] , the Taqman qPCR assay confirmed significant reductions in retrohoming efficiency in the Keio deletions of rnhA ( RNase H1 , the major cellular RNase H [31] ) ; seqA ( initiation of chromosomal DNA replication ) ; sbcC ( ATP-dependent exonuclease ) ; hns ( histone-like nucleoid structuring protein ) ; and tus ( DNA replication termination site-binding protein ) ; as well as at restrictive temperatures in the temperature-sensitive mutants polAexts , which is defective in the 5′→3′ exonuclease but not the DNA polymerase activity of Pol I [32]; and dnaEts in the catalytic ( α ) subunit of the host replicative DNA polymerase Pol III [33] .', 'Also in agreement with previous results , we found no strong decrease in retrohoming efficiency in a Keio deletion of rnhB ( RNase H2 [34] ) .', 'In contrast to results of genetic assays , the Taqman qPCR assays found no decrease in retrohoming efficiency for Keio deletions of recJ ( single-stranded DNA exonuclease [35] ) ; dnaQ ( Pol III ε subunit , which has the proofreading exonuclease activity [36] ) ; rep and recQ ( DNA helicases [37] , [38] ) ; recF ( RecA-dependent recombination ) ; stpA ( H-NS-like DNA- and RNA-binding protein with RNA chaperone activity [39] ) ; mnmE ( trmE; tRNA modification ) ; ligA and ligB ( DNA ligases [40] , [41] ) ; and pnp ( polynucleotide phosphorylase [42] ) .', 'Additionally , the Taqman qPCR assay found no decrease in retrohoming efficiency for Keio deletions of the genes encoding DNA repair polymerases polB ( Pol II [43] ) , dinB ( Pol IV [44] ) , and umuC or D ( Pol V [45] ) , whereas polB and dinB deletions in a different strain showed moderate decreases in previous genetic assays [12] .', 'The new candidates that were identified in the transposon-insertion screen ( Table S1 ) and confirmed to have substantial decreases in retrohoming efficiency in the Taqman qPCR assay ( 10–67% wild type; Table 1 ) were: DnaC and DnaT , which function in replication restart ( identified as genes downstream of the transposon insertion in the yjjB operon [46] , [47] ) ; GyrB ( DNA gyrase subunit B ) ; and RpoH ( RNA polymerase σ32 factor ) .', 'For several mutants in which inhibition of retrohoming was found in genetic assays but not in the Taqman qPCR assay , we subsequently found significant effects on top- or bottom-strand DNA synthesis in biochemical assays below ( e . g . , dinB , dnaQ , ligA , recJ , pnp , polB , and stpA ) .', 'The disagreement between the genetic and Taqman qPCR assays for these mutants may reflect:', '( i ) that qPCR monitors only short DNA regions at the intron-integration junctions;', '( ii ) the longer time of the Taqman qPCR assay , which may give alternative enzymes a greater chance to act; or', '( iii ) the different genetic backgrounds of the strains used in the two assays .', 'The results again emphasize the need to use multiple assays to identify retrohoming factors , with biochemical support for a genetic assay in our view providing the most definitive identification .', 'The transposon-library screen in E . coli HMS174 ( DE3 ) identified eight retrohoming-deficient mutants that are TpS/GFP− , potentially indicating a defect in the production of Ll . LtrB RNPs ( Table S3 ) .', 'Although we hoped that such mutants would identify host factors required for Ll . LtrB intron splicing , all eight of these mutants have transposon insertions that likely affect donor plasmid transcription ( four in the lacUV5 promoter of the λDE3 prophage , one in the T7 RNA polymerase gene , and three in genes encoding membrane transporters that could affect IPTG induction or trimethoprim uptake: ugpA ( glycerol-3-phosphate uptake transporter subunit ) ; xylF ( xylose transporter subunit ) ; and yjbB ( a putative transporter ) .', 'Consistent with an effect on transcription , all eight mutants showed decreased GFP fluorescence when counter-screened with the control plasmid pALE , which lacks the Ll . LtrB intron and contains the ligated ltrB exons fused directly to GFP ( Figure S2 , Figure S3 , and Table S3 ) .', 'A direct screen of the transposon library for splicing-defective mutants using plasmid pALG2 , in which an Ll . LtrB-ΔORF intron lacking the TpR-RAM marker is linked to GFP expression [48] , also identified only mutants that are defective in GFP expression with both the reporter construct containing the intron and the control reporter construct lacking the intron [T7 RNA polymerase of the λDE3 prophage; malE ( maltose ABC transporter subunit ) ; kdsD ( arabinose-5-phosphate isomerase ) ; dppD ( dipeptide ABC transporter subunit ) ; clcA ( H+/Cl− exchange transporter ) ; yfeN ( conserved outer membrane protein ) ; pgi ( phosphoglucose isomerase; and yjbE ( predicted protein ) ] ( Table S4 ) .', 'These findings suggest either that splicing of the Ll . LtrB intron in vivo requires only the LtrA protein as it does in vitro [17] or that host-encoded splicing factors are essential proteins that are not readily identified in a transposon screen .', 'The TpR-RAM screen also identified five transposon insertions that give increased retrohoming efficiencies and potentially encode host factors that function to suppress retrohoming .', 'Surprisingly , all five of these transposon-insertions mapped to three closely linked genes , rnlA ( RNase LS ) , yfjK ( DExH/D-box protein ) , and yfjL ( protein of unknown function ) , which are part of a cryptic prophage ( CP4-57 ) in E . coli K12 ( Figure S4 , Table S5; [49] , [50] ) .', 'In addition to higher group II intron retrohoming efficiencies indicated by high levels of TpR , all five disruptants showed increased levels of GFP fluorescence both with the intron-donor plasmid pALG3 , in which splicing of the Ll . LtrB-ΔORF intron is required for GFP expression , and with the control plasmid pALE , which has ligated ltrB exons fused directly to GFP ( Figure S4 ) .', 'Given the identity of the affected genes , these findings suggest that the increased retrohoming efficiencies and GFP fluorescence result from decreased rates of RNA degradation , leading to elevated levels of group II intron RNPs and GFP mRNA .', 'The degradosome , which functions in mRNA turnover in E . coli , is a multiprotein complex consisting of RNase E ( an endoribonuclease ) , PNPase ( an exoribonuclease ) , RhlB ( a DEAD-box RNA helicase ) , and enolase [51] .', 'The close linkage of the three genes potentially involved in intron RNA degradation in our screen suggests that they may function together in a previously unknown RNA degradation pathway , possibly a second degradosome .', 'The decreased retrohoming efficiency resulting from a transposon insertion in the yjjB operon containing dnaC and dnaT in the TpR-RAM screen ( see above; Table S1 ) focused our attention on replication restart proteins as attractive candidates for playing a role in the late steps of retrohoming .', 'We therefore carried out systematic Taqman qPCR assays of replication restart mutants and found significant reductions in retrohoming in Keio deletions of priA , priC , and dnaT , and in a temperature-sensitive mutant of dnaB ( Table 1 ) .', 'PriA and PriC are key proteins that independently recognize stalled or collapsed replication forks in the three major E . coli replication restart pathways ( denoted the PriA-PriB , PriA-PriC , and PriC-Rep pathways; [47] , [52] ) , while DnaT interacts with PriA and PriC to load the replicative DNA helicase DnaB [53] , [54] .', 'We also found decreased retrohoming efficiencies in temperature-sensitive mutants of several essential genes that function in replication restart , including those encoding DnaC , which interacts with DnaB prior to loading [55]; DnaG , the DNA primase [56]; and the single-stranded DNA binding protein Ssb , which has been shown to promote the formation of the primosome at the chromosomal replication origin ( oriC ) and interacts with PriA to stimulate the loading of DnaB during replication restart [57] , [58] .', 'However , deletion of the genes encoding PriB , an auxiliary component of the PriA-dependent pathway , and Rep , which functions in conjunction with PriC [59] , showed only small ( 1–21% ) reductions in retrohoming efficiency .', 'We also carried out Taqman qPCR assays of retrohoming in a different set of replication restart mutants in the genetic background of E . coli SS996 , a recA+ strain containing a GFP reporter for SOS induction .', 'The results indicated that the decreased retrohoming efficiencies in the affected replication restart mutants are not allele specific and are larger than can be accounted for by the proportion of cells undergoing the SOS response ( Figure S5; different alleles tested for all genes except dnaE and dnaB ) .', 'Additionally , the mutant strain SS4610 ( lexA51::Tn5 ) , which has a lexA null allele and is constitutively induced for the SOS response in nearly 100% of cells [60] , showed only minimally decreased retrohoming frequencies in the Taqman qPCR assay ( 81–87% wild type; Figure S5 and Table 1 ) .', 'Collectively , the above findings indicate that the decreased retrohoming efficiency in the replication restart mutants is not a secondary effect of cell cycle arrest during SOS induction and indicate a requirement for replication restart proteins in group II intron retrohoming .', 'To further test the function of individual host proteins , we developed a biochemical assay in which host factors function together with group II intron RNPs to reconstitute the complete retrohoming reaction in vitro .', 'This assay uses an E . coli S12 extract similar to those used for in vitro transcription and translation [61] , [62] .', 'Figure 3 shows experiments in which 5′ top- or bottom-strand labeled , 73-bp DNA oligonucleotide substrates containing the Ll . LtrB target site were incubated with group II intron RNPs in the presence of the extract , dNTPs ( dATP , dCTP , dGTP , and dTTP ) , ATP , and an ATP-regenerating system ( phosphoenolpyruvate+pyruvate kinase ) at 37°C .', 'The products were then analyzed in a denaturing polyacrylamide gel before and after digestion with RNases A+H to degrade the reverse spliced intron RNA leaving only 5′-labeled DNA products .', 'Time courses with the 5′-labeled bottom-strand substrate , which monitors cDNA synthesis , showed that an RNase-resistant band of the size expected for full-length bottom strand ( 988 nt ) appeared after 5 min and accumulated for up to 30 min , along with a series of smaller bands ( Figure 3A ) .', 'These smaller bands are likely incomplete cDNAs rather than degradation products , as controls showed that exogenous 32P-labeled ssDNA and dsDNA corresponding to the retrohoming products were not degraded when incubated in the extracts under the same conditions ( Figure S6 ) .', 'Time courses with the 5′-labeled top-strand substrate , which monitors second-strand DNA synthesis , showed that an RNase-resistant band of the size expected for full-length top strand appeared later ( 10 min ) and continued to accumulate during the reaction ( Figure 3A , bottom right panel ) .', 'We confirmed by primer extension that both the 5′- and 3′-junctions in the newly synthesized top strand DNA are continuous ( Figure 3B ) .', 'Figure 3C shows that the appearance of labeled top- and bottom-strand products is dependent upon the addition of RNPs ( lanes 4 and 8 ) and that top-strand DNA synthesis is completely dependent upon the presence of extract ( lane 6 ) .', 'ATP increased the levels of reverse splicing and cDNA synthesis and was required for top-strand DNA synthesis in the extracts ( cf . , lane 1 and 5 with lanes 3 and 7 ) , indicating that energy-dependent processes , perhaps involving DNA or RNA helicases , are involved at both stages of the reaction .', 'Further , RNPs containing a mutant LtrA protein that lacks RT activity ( RT−; YADD→YAAA ) carried out reverse splicing , but showed no detectable cDNA synthesis , indicating that bottom-strand synthesis is dependent upon the group II intron RT activity and is not done by a host polymerase in the extracts ( Figure 3D ) .', 'Finally , the E . coli extract assay enabled us to directly analyze bottom- and top-strand DNA synthesis in retrohoming-deficient mutants identified in genetic screens .', 'In these experiments , extracts from Keio deletion or temperature-sensitive mutants were compared with those from their parental wild-type strains .', 'In most cases , extracts were prepared from cells grown continuously at 37°C , which is a semi-permissive temperature for most of the temperature-sensitive mutants .', 'The priA deletion strain was grown at 30°C to avoid accumulation of suppressor mutations , and five temperature-sensitive mutants ( dnaEts , gyrBts , ligAts , rpoHts , and ssbts ) that could not grow at 37°C along with their parental wild-type strains were grown at 30°C and then shifted to 37°C for 2 h before preparing the extracts .', 'For all strains , the extract assays were done at 37°C for relatively short times ( 15 min ) to remain within the linear range and minimize dephosphorylation of the labeled DNA substrate for several mutants whose extracts appear to have elevated phosphatase activity ( DnaQ , DnaT , PriA , and DnaB ) .', 'Table 2 and Table S6 summarize quantitation of the assays , and representative assays for notable mutants are shown in Figure 4 and for the remaining mutants in Figure S7 .', 'All values shown in Table 2 and Table S6 were reproducible to within <30% in replicate experiments .', 'First , the biochemical assays confirmed the function of several candidate retrohoming factors that were expected to be required for top-strand DNA synthesis , including RNase H1 ( rnhA ) , the 5′→3′ exonuclease activity of Pol I ( polAexts ) , and the replicative polymerase Pol III ( dnaQ , dnaEts ) .', 'Extracts from Keio deletions or mutants having temperature-sensitive defects in these activities showed high levels of reverse splicing and cDNA synthesis , but strongly decreased top-strand DNA synthesis ( rnhA and dnaQ , 2% wild type; polAexts , 34% wild type; and dnaEts , 10% wild type; Figure 4A , Figure 4C , and Table 2 ) .', 'The ligAts mutant also showed a substantial decrease in top-strand DNA synthesis in the extract assays ( 45% wild type; Figure 4C ) , consistent with a role for DNA ligase A in sealing nicks .', 'The incomplete inhibition of top-strand DNA synthesis in the temperature-sensitive mutants may reflect residual activity at the semi-permissive temperature used in the experiment .', 'The Keio deletion of the second E . coli RNase H gene , rnhB , which had no effect on retrohoming in vivo , did not inhibit top-strand synthesis in the in vitro assays ( 153% wild type; Figure 4A ) .', 'However , extracts from both the rnhA and rnhB deletions showed elevated levels of full-length intron cDNAs ( 239 and 218% wild type , respectively ) , possibly reflecting that RNase H2 makes some contribution to degrading the intron RNA template in vitro .', 'Among the replication restart mutants , we found substantial decreases in top-strand synthesis in extracts from Keio deletions priA ( 1% wild type ) , priC ( 60% wild type ) , and dnaT ( 50% wild type ) , as well as the temperature-sensitive mutants of dnaB , which encodes the replicative DNA helicase ( 0% ) , and dnaC , which interacts with DnaB prior to loading ( 18% wild type ) ( Figure 4A , Table 2 ) .', 'The greater effect of the Keio priA deletion in vitro than in vivo may reflect that the short time of the in vitro assays ( 15 min ) favors intermediates with short gaps that are recognized by PriA , while the longer time of the in vivo assays ( 1', 'h ) favors intermediates with longer gaps that are recognized by PriC [47] .', 'In agreement with Taqman qPCR assays , we found no effect for the deletion of the genes encoding PriB , an accessory protein that facilitates PriA-DnaT complex formation ( 116% wild type; Figure 4B ) [63] , nor the DNA helicase Rep ( 96% wild type; Figure 4A ) , which ordinarily functions in conjunction with PriC [59] .', 'We also found severe defects in top-strand DNA synthesis in the extracts from temperature-sensitive mutants of two essential proteins that function in replication restart pathways , the single-stranded DNA-binding protein Ssb ( ssbts; 8% wild type ) [57] , [58] and the DNA primase ( dnaGts , 0%; [56] ) ( Figure 4C ) .', 'Surprisingly , the DNA primase mutant ( dnaGts ) was also strongly inhibited in bottom-strand cDNA synthesis ( <1% wild type; Table 2; see Discussion ) .', 'Importantly , the LexA SS4610 ( lexA51::Tn5 ) mutant , which has a constitutively induced SOS response [60] , showed no significant decrease in top- or bottom-strand DNA synthesis in the extract assays ( 92 and 121% wild type , respectively; Table 2 , Figure S7 ) , consistent with the minimal effect of this mutation on retrohoming in vivo ( Table 1 and Figure S5 ) .', 'Only four other protein mutants showed significantly decreased retrohoming in the extract assays ( <70% wild-type top-strand synthesis ) : the Keio deletions of dinB ( Pol IV; 63% wild type ) , seqA ( 51% wild type ) , and stpA ( 42% wild type; Figure 4A ) ; and a pnp mutant that retains <10% of the wild-type PNPase activity ( 
['Introduction', 'Results', 'Discussion', 'Materials and Methods']
['Mobile group II introns retrohome by an RNP-based mechanism in which the intron RNA reverse splices into a DNA site and is reverse transcribed by the associated intron-encoded protein .', 'The resulting intron cDNA is then integrated into the genome by cellular mechanisms that have remained unclear .', 'Here , we used an Escherichia coli genetic screen and Taqman qPCR assay that mitigate indirect effects to identify host factors that function in retrohoming .', 'We then analyzed mutants identified in these and previous genetic screens by using a new biochemical assay that combines group II intron RNPs with cellular extracts to reconstitute the complete retrohoming reaction in vitro .', 'The genetic and biochemical analyses indicate a retrohoming pathway involving degradation of the intron RNA template by a host RNase H and second-strand DNA synthesis by the host replicative DNA polymerase .', 'Our results reveal ATP-dependent steps in both cDNA and second-strand synthesis and a surprising role for replication restart proteins in initiating second-strand synthesis in the absence of DNA replication .', 'We also find an unsuspected requirement for host factors in initiating reverse transcription and a new RNA degradation pathway that suppresses retrohoming .', 'Key features of the retrohoming mechanism may be used by human LINEs and other non-LTR-retrotransposons , which are related evolutionarily to mobile group II introns .', 'Our findings highlight a new role for replication restart proteins , which function not only to repair DNA damage caused by mobile element insertion , but have also been co-opted to become an integral part of the group II intron retrohoming mechanism .']
['Mobile group II introns are bacterial retrotransposons that are evolutionarily related to introns and retroelements in higher organisms .', 'They spread within and between genomes by a mechanism termed “retrohoming” in which the intron RNA inserts directly into a DNA site and is reverse transcribed by an intron-encoded reverse transcriptase .', 'The resulting intron cDNA is integrated into the genome by host factors , but how it occurs has remained unclear .', 'Here , we investigated the function of host factors in retrohoming by genetic and biochemical approaches , including a new biochemical assay that reconstitutes the complete retrohoming reaction in vitro .', 'Our results lead to a comprehensive model for retrohoming , which includes a surprising role for replication restart proteins in recruiting the host replicative DNA polymerase to copy the intron cDNA into the genome in the absence of DNA replication .', 'We also find an unexpected contribution of host factors to initiating reverse transcription and a new RNA degradation pathway that suppresses retrohoming .', 'We suggest that key features of the group II intron retrohoming mechanism may be used by human LINE elements and other non-LTR-retrotransposons .', 'Additionally , our results provide new insights into the function of replication restart proteins , which are critical for surviving DNA damage in all organisms .']
['biochemistry', 'rna', 'transposons', 'retrotransposons', 'dna replication', 'nucleic acids', 'genetics', 'molecular genetics', 'dna', 'biology', 'molecular cell biology', 'rna stability']