Mismatch Repair Regulates Homologous Recombination, but Has Little Influence on Antigenic Variation, in Trypanosoma brucei
2003; Elsevier BV; Volume: 278; Issue: 46 Linguagem: Inglês
10.1074/jbc.m308123200
ISSN1083-351X
AutoresJ. S. Bell, Richard McCulloch,
Tópico(s)Parasites and Host Interactions
ResumoAntigenic variation is critical in the life of the African trypanosome, as it allows the parasite to survive in the face of host immunity and enhance its transmission to other hosts. Much of trypanosome antigenic variation uses homologous recombination of variant surface glycoprotein (VSG)-encoding genes into specialized transcription sites, but little is known about the processes that regulate it. Here we describe the effects on VSG switching when two central mismatch repair genes, MSH2 and MLH1, are mutated. We show that disruption of the parasite mismatch repair system causes an increased frequency of homologous recombination, both between perfectly matched DNA molecules and between DNA molecules with divergent sequences. Mismatch repair therefore provides an important regulatory role in homologous recombination in this ancient eukaryote. Despite this, the mismatch repair system has no detectable role in regulating antigenic variation, meaning that VSG switching is either immune to mismatch selection or that mismatch repair acts in a subtle manner, undetectable by current assays. Antigenic variation is critical in the life of the African trypanosome, as it allows the parasite to survive in the face of host immunity and enhance its transmission to other hosts. Much of trypanosome antigenic variation uses homologous recombination of variant surface glycoprotein (VSG)-encoding genes into specialized transcription sites, but little is known about the processes that regulate it. Here we describe the effects on VSG switching when two central mismatch repair genes, MSH2 and MLH1, are mutated. We show that disruption of the parasite mismatch repair system causes an increased frequency of homologous recombination, both between perfectly matched DNA molecules and between DNA molecules with divergent sequences. Mismatch repair therefore provides an important regulatory role in homologous recombination in this ancient eukaryote. Despite this, the mismatch repair system has no detectable role in regulating antigenic variation, meaning that VSG switching is either immune to mismatch selection or that mismatch repair acts in a subtle manner, undetectable by current assays. African trypanosomes are protistan parasites that infect mammals and are considered to have diverged early in eukaryotic evolution (1Sogin M.L. Elwood H.J. Gunderson J.H. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 1383-1387Crossref PubMed Scopus (367) Google Scholar), prior to the radiation of animals, plants, and fungi. In the mammal Trypanosoma brucei resides in the blood-stream and tissue fluids, where it is subject to immune attack. To evade immune killing, it undergoes antigenic variation, a strategy for changing surface coats found in a diverse range of microbes (2Barry J.D. McCulloch R. Adv. Parasitol. 2001; 49: 1-70Crossref PubMed Google Scholar, 3Donelson J.E. Acta Trop. 2003; 85: 391-404Crossref PubMed Scopus (137) Google Scholar). The surface coat of T. brucei is composed of variant surface glycoprotein (VSG) 1The abbreviations used are: VSG, variant surface glycoprotein; ES, expression sites; MMR, mismatch repair; MEPS, minimal efficient processing segments.1The abbreviations used are: VSG, variant surface glycoprotein; ES, expression sites; MMR, mismatch repair; MEPS, minimal efficient processing segments. (4Cross G.A. Parasitology. 1975; 71: 393-417Crossref PubMed Scopus (636) Google Scholar). VSG genes are expressed from telomeric transcription units, called expression sites (ES), of which ∼20 can be used while the parasite is present in the mammalian host. Only one ES is expressed at a given time from a specific sub-nuclear domain (5Navarro M. Gull K. Nature. 2001; 414: 759-763Crossref PubMed Scopus (250) Google Scholar), but coordinated transcriptional switches (termed in situ switches) can activate a silent ES and inactivate the transcribed site (6Cross G.A. Wirtz L.E. Navarro M. Mol. Biochem. Parasitol. 1998; 91: 77-91Crossref PubMed Scopus (79) Google Scholar, 7Navarro M. Cross G.A. Wirtz E. EMBO J. 1999; 18: 2265-2272Crossref PubMed Scopus (73) Google Scholar, 8Vanhamme L. Pays E. McCulloch R. Barry J.D. Trends Parasitol. 2001; 17: 338-343Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar, 9Vanhamme L. Lecordier L. Pays E. Int. J. Parasitol. 2001; 31: 523-531Crossref PubMed Scopus (28) Google Scholar, 10Vanhamme L. Poelvoorde P. Pays A. Tebabi P. Van Xong H. Pays E. Mol. Microbiol. 2000; 36: 328-340Crossref PubMed Scopus (106) Google Scholar, 11Chaves I. Rudenko G. Dirks-Mulder A. Cross M. Borst P. EMBO J. 1999; 18: 4846-4855Crossref PubMed Scopus (91) Google Scholar, 12Borst P. Chaves I. Trends Genet. 1999; 15: 95-96Abstract Full Text Full Text PDF PubMed Scopus (6) Google Scholar). T. brucei also contains hundreds of silent VSG genes, both in multigene arrays in the megabase chromosomes and at the telomeres of minichromosomes. This silent reservoir is activated by recombination reactions that move the genes into the ES, normally by a gene conversion process (13Borst P. Cross G.A. Cell. 1982; 29: 291-303Abstract Full Text PDF PubMed Scopus (259) Google Scholar, 14Pays E. Prog. Nucleic Acids Res. Mol. Biol. 1985; 32: 1-26Crossref PubMed Scopus (34) Google Scholar, 15Robinson N.P. Burman N. Melville S.E. Barry J.D. Mol. Cell. Biol. 1999; 19: 5839-5846Crossref PubMed Scopus (111) Google Scholar). The available evidence suggests that recombinational VSG switching occurs by homologous recombination. No specific sequence has been shown to be essential in activating VSG gene conversion, but instead the reactions use variable amounts of flanking sequence homologies (2Barry J.D. McCulloch R. Adv. Parasitol. 2001; 49: 1-70Crossref PubMed Google Scholar). In addition, disruption of a gene encoding a major enzyme of eukaryotic homologous recombination, RAD51, impairs VSG switching (16McCulloch R. Barry J.D. Genes Dev. 1999; 13: 2875-2888Crossref PubMed Scopus (120) Google Scholar). Consistent with this genetic analysis, inactivation of KU70 or KU80, which catalyze a non-homologous end-joining pathway of DNA repair in other organisms (17Featherstone C. Jackson S.P. Curr. Biol. 1999; 9: R759-R761Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar, 18Barnes D.E. Curr. Biol. 2001; 11: R455-R457Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar), does not affect VSG switching (19Conway C. McCulloch R. Ginger M.L. Robinson N.P. Browitt A. Barry J.D. J. Biol. Chem. 2002; 277: 21269-21277Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar). Surprisingly, mutation of MRE11 also does not affect VSG switching (20Robinson N.P. McCulloch R. Conway C. Browitt A. Barry J.D. J. Biol. Chem. 2002; 277: 26185-26193Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar), despite this gene encoding an enzyme that has been proposed to have many roles in homologous and non-homologous recombination (21D'Amours D. Jackson S.P. Nat. Rev. Mol. Cell. Biol. 2002; 3: 317-327Crossref PubMed Scopus (712) Google Scholar). Mismatch repair (MMR) plays a critical role in maintaining genetic stability, in part by correcting base mismatches that can arise through replication errors or chemical damage (reviewed in Refs. 22Modrich P. Lahue R. Annu. Rev. Biochem. 1996; 65: 101-133Crossref PubMed Scopus (1322) Google Scholar, 23Yang W. Mutat. Res. 2000; 460: 245-256Crossref PubMed Scopus (90) Google Scholar, 24De Wind N. Hays J.B. Curr. Biol. 2001; 11: R545-R548Abstract Full Text Full Text PDF PubMed Scopus (9) Google Scholar, 25Hsieh P. Mutat. Res. 2001; 486: 71-87Crossref PubMed Scopus (149) Google Scholar). In most bacteria, a homodimer of MutS binds mismatched DNA and is then recognized by a homodimer of MutL, which is thought to recruit downstream factors that catalyze the repair reaction. Eukaryotic MMR is catalyzed by MutS and MutL homologues, but here the proteins act as heterodimers. In Saccharomyces cerevisiae there are two MutS-related heterodimers. One is composed of MSH2 and MSH6 and recognizes base-base mismatches and small insertion/deletion loops, whereas the second is composed of MSH2 and MSH3 and binds a larger range of insertion/deletion loops. Two MutL heterodimers, composed of MLH1 and either PMS1 or MLH3, have been well characterized in S. cerevisiae, and a third (MLH1–MLH2) has recently come to light (26Durant S.T. Morris M.M. Illand M. McKay H.J. McCormick C. Hirst G.L. Borts R.H. Brown R. 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An MSH2-related gene has also been characterized in Trypanosoma cruzi (31Augusto-Pinto L. Bartholomeu D.C. Teixeira S.M. Pena S.D. Machado C.R. Gene (Amst.). 2001; 272: 323-333Crossref PubMed Scopus (25) Google Scholar, 32Augusto-Pinto L. Teixeira S.M. Pena S.D. Machado C.R. Genetics. 2003; 164: 117-126PubMed Google Scholar). Mismatches can also arise during the DNA strand exchange step of recombination between non-identical DNA substrates. These are recognized by the MMR machinery, which either triggers mismatch correction, resulting in gene conversion, or recombination abortion (reviewed in Refs. 33Harfe B.D. Jinks-Robertson S. Annu. Rev. Genet. 2000; 34: 359-399Crossref PubMed Scopus (498) Google Scholar and 34Evans E. Alani E. Mol. Cell. Biol. 2000; 20: 7839-7844Crossref PubMed Scopus (93) Google Scholar). MMR anti-recombination activity has been described in bacteria, yeast, and mammals (35Rayssiguier C. Thaler D.S. Radman M. Nature. 1989; 342: 396-401Crossref PubMed Scopus (585) Google Scholar, 36Alani E. Reenan R.A. Kolodner R.D. Genetics. 1994; 137: 19-39Crossref PubMed Google Scholar, 37De Wind N. Dekker M. Berns A. Radman M. te Riele R.H. Cell. 1995; 82: 321-330Abstract Full Text PDF PubMed Scopus (724) Google Scholar) and plays roles in speciation (35Rayssiguier C. Thaler D.S. Radman M. Nature. 1989; 342: 396-401Crossref PubMed Scopus (585) Google Scholar, 38Radman M. Genome. 1989; 31: 68-73Crossref PubMed Scopus (50) Google Scholar, 39Hunter N. Chambers S.R. Louis E.J. Borts R.H. EMBO J. 1996; 15: 1726-1733Crossref PubMed Scopus (246) Google Scholar) and in maintaining genome stability (40Petit M.A. Dimpfl J. Radman M. Echols H. Genetics. 1991; 129: 327-332Crossref PubMed Google Scholar, 41Reitmair A.H. Schmits R. Ewel A. Bapat B. Redston M. Mitri A. Waterhouse P. Mittrucker H.W. Wakeham A. Liu B. Thomason A. Griesser H. Gallinger S. Ballhausen W.G. Fishel R. Mak T.W. Nat. Genet. 1995; 11: 64-70Crossref PubMed Scopus (348) Google Scholar, 42Lin C.T. Lyu Y.L. Xiao H. Lin W.H. Whang-Peng J. Nucleic Acids Res. 2001; 29: 3304-3310Crossref PubMed Scopus (34) Google Scholar). The mechanism(s) by which the MMR machinery aborts homologous recombination is not yet clear, but destruction of DNA strand exchange heteroduplexes by MMR-catalyzed nicking and non-destructive reversal or rejection of the heteroduplexes has been suggested (33Harfe B.D. Jinks-Robertson S. Annu. Rev. Genet. 2000; 34: 359-399Crossref PubMed Scopus (498) Google Scholar, 34Evans E. Alani E. Mol. Cell. Biol. 2000; 20: 7839-7844Crossref PubMed Scopus (93) Google Scholar, 35Rayssiguier C. Thaler D.S. Radman M. Nature. 1989; 342: 396-401Crossref PubMed Scopus (585) Google Scholar). In T. brucei, transformed DNA appears to integrate into the genome almost exclusively by homologous recombination (43Conway C. Proudfoot C. Burton P. Barry J.D. McCulloch R. Mol. Microbiol. 2002; 45: 1687-1700Crossref PubMed Scopus (60) Google Scholar), suggesting that, as in yeast, homologous recombination is the major DNA double strand break repair pathway. Indirect evidence from such transformation experiments has suggested that MMR influences homologous recombination in both T. brucei (44Blundell P.A. Rudenko G. Borst P. Mol. Biochem. Parasitol. 1996; 76: 215-229Crossref PubMed Scopus (41) Google Scholar) and the related parasite Leishmania (45Papadopoulou B. Dumas C. Nucleic Acids Res. 1997; 25: 4278-4286Crossref PubMed Scopus (49) Google Scholar). Given that T. brucei antigenic variation is heavily dependent on homologous recombination, mismatch repair might be envisaged to be an important regulatory component of the immune evasion process. Here we test the contribution that MMR makes to antigenic variation, and we quantify the constraint that MMR places upon homologous recombination in this parasite. T. brucei Strains and Growth—T. brucei Lister 427 bloodstream cells were used and grown at 37 °C in HMI-9 medium (46Hirumi H. Hirumi K. J. Parasitol. 1989; 75: 985-989Crossref PubMed Scopus (765) Google Scholar). MSH2 and MLH1 mutants for assaying VSG switching were made in strain 3174,2 a derivative of MITat1.2a (47McCulloch R. Rudenko G. Borst P. Mol. Cell. Biol. 1997; 17: 833-843Crossref PubMed Scopus (81) Google Scholar). Determination of VSG switching frequency and pattern has been described previously (16McCulloch R. Barry J.D. Genes Dev. 1999; 13: 2875-2888Crossref PubMed Scopus (120) Google Scholar, 19Conway C. McCulloch R. Ginger M.L. Robinson N.P. Browitt A. Barry J.D. J. Biol. Chem. 2002; 277: 21269-21277Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar, 20Robinson N.P. McCulloch R. Conway C. Browitt A. Barry J.D. J. Biol. Chem. 2002; 277: 26185-26193Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar). To assay the effect of base mismatches on recombination, MITat1.2a cells were transformed with the construct pTHT (43Conway C. Proudfoot C. Burton P. Barry J.D. McCulloch R. Mol. Microbiol. 2002; 45: 1687-1700Crossref PubMed Scopus (60) Google Scholar, 44Blundell P.A. Rudenko G. Borst P. Mol. Biochem. Parasitol. 1996; 76: 215-229Crossref PubMed Scopus (41) Google Scholar); targeting of the HYG gene (see text) to the tubulin array was confirmed by Southern mapping with KpnI- or EcoRI-digested genomic DNA (data not shown). The constructs used to create MSH2 and MLH1 mutants will be described elsewhere, as will the approaches to confirm the mutations.2 Generation of HYG Targeting Constructs—Constructs to target HYG in the HTUB cell line were made by PCR on 2 ng of plasmid pTHT (43Conway C. Proudfoot C. Burton P. Barry J.D. McCulloch R. Mol. Microbiol. 2002; 45: 1687-1700Crossref PubMed Scopus (60) Google Scholar, 44Blundell P.A. Rudenko G. Borst P. Mol. Biochem. Parasitol. 1996; 76: 215-229Crossref PubMed Scopus (41) Google Scholar) with primers HYGFor (5′-AAGGCGCGCCAGCCTGAACTCACCGCGACG-3′; AscI site underlined) and HYGRev (5′-ATGGCGCGCCCTCCGGATCGGACGATTGCG-3′; AscI underlined). Reactions were performed at 94 °C for 10 min, 30 cycles of 94 °C for 1 min, 65 °C for 1 min, 72 °C for 1 min, and 72 °C for 10 min; the 914-bp products were cloned into pCR2.1-TOPO (Invitrogen). Herculase polymerase (Stratagene) was used to make flanks homologous to HYG; sequencing revealed no base changes relative to HYG in pTHT. Two rounds of PCR with Taq polymerase (Advanced Biotechnologies) were used to make HYG mismatched flanks. The first round reaction contained, in addition to standard dNTPs, dPTP and 8-oxo-dGTP (Amersham Biosciences; 100 μm). After cycles 5, 10, 15, 20, 25, and 30, a 1-μl sample was removed for template in a second TaqPCR with a standard set of nucleotides. A number of products from the 6-second round amplifications were sequenced, and clones were chosen for their percent sequence divergence (to the nearest integer) relative to HYG in pTHT. All mutations were base changes and the majority were transitions. In all but the 1% divergent clone, the mismatches were spread throughout the PCR product (Fig. 3B). To generate targeting constructs from each recombination flank, a bleomycin resistance cassette (BLE) was cloned into a central NdeI site, generating the plasmids HYGwt::BLE, HYG01::BLE, HYG02::BLE, etc. BLE contains a bleomycin resistance protein open reading frame flanked upstream and downstream by intergenic sequence from the actin and calmodulin loci, respectively (47McCulloch R. Rudenko G. Borst P. Mol. Cell. Biol. 1997; 17: 833-843Crossref PubMed Scopus (81) Google Scholar); these provide processing signals to make mature mRNA and are unlinked in the T. brucei genome, so homologous recombination can only occur via the HYG targeting flanks. Determination of Transformation Frequency—Transformation of the HYG targeting constructs was performed as described previously (43Conway C. Proudfoot C. Burton P. Barry J.D. McCulloch R. Mol. Microbiol. 2002; 45: 1687-1700Crossref PubMed Scopus (60) Google Scholar), but with modifications to provide reproducibility. T. brucei were grown maximally to 3 × 106 cells·ml–1 and minimally to 1.5 × 106 cells·ml–1, prior to transformation. 2.5 × 107 cells were used in each transformation, harvested by centrifugation at 600 × g for 10 min at room temperature, resuspended in 0.5 ml of ZMG buffer (43Conway C. Proudfoot C. Burton P. Barry J.D. McCulloch R. Mol. Microbiol. 2002; 45: 1687-1700Crossref PubMed Scopus (60) Google Scholar) pre-warmed to 37 °C, and electroporated with 3 μg of linearized DNA with a single pulse of 1.4 kV, 25 microfarads using a Bio-Rad Gene Pulser II. The cells were recovered for 18 h in 10 ml of HMI-9, harvested as before, then resuspended in HMI-9 containing 2.5 μg·ml–1 phleomycin (Cayla), and spread in 1.0-ml aliquots over a 24-well dish. To ensure clonal growth as far as possible, between 1 × 106 and 7.5 × 106 cells were plated out in this way. At least three selection plates were used in each transformation, and each construct was transformed into each cell line at least three times. Transformation efficiency was measured as the number of wells containing phleomycin-resistant cells after 8 days of growth. DNA constructs were AscI-digested prior to transformation and purified as before (43Conway C. Proudfoot C. Burton P. Barry J.D. McCulloch R. Mol. Microbiol. 2002; 45: 1687-1700Crossref PubMed Scopus (60) Google Scholar). The DNA concentration of each linear construct was determined by electrophoretic separation of serial dilutions relative to a 1.0-kbp size ladder (Invitrogen), and adjusted to 1 μg·μl–1. The length of the minimal efficient processing segment (MEPS) in MMR+ and MMR– cells was calculated using the formula described by Datta et al. (48Datta A. Hendrix M. Lipsitch M. Jinks-Robertson S. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 9757-9762Crossref PubMed Scopus (184) Google Scholar): L(1 – ea). In this equation, MEPS are calculated according to the slope a of a regression plot of ln (recombination frequency) versus the number of mismatches, where L is the length of the substrate. For L, we have assumed that each linear end of the constructs is used as a separate substrate, in that each end will search for homology independently, and therefore L is 450 bp, the size of each HYG recombination flank. Mutation of T. brucei MSH2 Does Not Affect VSG Switching—To examine the role of MMR in VSG switching, we first made mutants of MSH2 in the T. brucei strain 3174. This strain has been described before (47McCulloch R. Rudenko G. Borst P. Mol. Cell. Biol. 1997; 17: 833-843Crossref PubMed Scopus (81) Google Scholar) and contains copies of genes encoding resistance to the antibiotics hygromycin B and G418 in the actively transcribed ES (see Fig. 2A), allowing the frequency of VSG switching to be calculated and the types of switching events that occur to be assessed. Three independent MSH2 heterozygotic (+/–) and homozygotic (–/–) mutants were created by sequential transformation of the constructs ΔMSH2::BSD and ΔMSH2::PUR.2 In addition, an intact copy of MSH2 was re-integrated into the disrupted allele in one MSH2–/– cell line, re-expressing the gene (MSH2–/–/+). Southern analysis confirmed that the MSH2 open reading frame had been precisely deleted in the mutants, and reverse transcriptase-PCR showed that no intact MSH2 transcript was generated in the –/– cell lines (data not shown). Mutation of MSH2 had no discernible effect on the growth of T. brucei in vitro, but the –/– mutants displayed increased tolerance to the alkylating agent N-methyl-N′-nitro-N-nitrosoguanidine and increased frequencies of size changes at a number of microsatellite repeat loci,2 both phenotypes consistent with impaired MMR. The estimated VSG switching frequencies of the 3 MSH2+/– and MSH2–/– cells lines are shown in Fig. 1, compared with the wild type cells and the homozygotic mutant re-expressing MSH2. There is considerable variation in the VSG switching frequencies measured by this assay (16McCulloch R. Barry J.D. Genes Dev. 1999; 13: 2875-2888Crossref PubMed Scopus (120) Google Scholar, 47McCulloch R. Rudenko G. Borst P. Mol. Cell. Biol. 1997; 17: 833-843Crossref PubMed Scopus (81) Google Scholar), due almost certainly to fluctuations in the timing of individual VSG switching events during population growth, reflecting the randomness of this process in T. brucei. Nevertheless, statistical examination of the data revealed no significant difference in VSG switching frequency in any of the MSH2 cell lines, indicating that MSH2 mutation does not detectably influence the overall frequency. VSG switching is a combination of in situ transcriptional switching events, which are not believed to involve recombination, and recombination events. Because it is possible that a high frequency of in situ switching might obscure an alteration in a relatively lower frequency of VSG recombinational switching resulting from MSH2 deletion, we characterized the switching events in a number of clonal switched variants. By using the antibiotic markers in the active ES, we can distinguish three types of switching reaction: in situ switches; gene conversions that remove the VSG gene and both upstream antibiotic markers (we term this ES gene conversion); and more localized gene conversions that remove just the VSG gene and proximal G418 resistance marker (VSG gene conversion). Again, we found considerable fluctuation in these data (Fig. 2). Comparing the wild type, MSH2+/–, and MSH2 re-expressing cell lines showed that, in this T. brucei strain, VSG gene conversion is the rarest event (0–37%) and in situ switching and ES gene conversion are more frequent (5–50% and 35–78% respectively), with the latter slightly predominating. The same general pattern was true in the MSH2–/– cell lines. Inactivation of MMR would most likely be expected to increase recombination rates, but we found that neither recombinational VSG switching event became predominant or, alternatively, was reduced. MSH2 mutation does not appear to detectably alter the profile of VSG switching. Mutation of T. brucei MLH1 Does Not Affect VSG Switching—Although MSH2 acts to suppress recombination, in S. cerevisiae it also functions, paradoxically, to promote homologous recombination in some circumstances. MSH2 and MSH3 cooperate with the nucleotide excision repair proteins RAD1 and RAD10 (49Fishman-Lobell J. Haber J.E. Science. 1992; 258: 480-484Crossref PubMed Scopus (307) Google Scholar, 50Saparbaev M. Prakash L. Prakash S. Genetics. 1996; 142: 727-736Crossref PubMed Google Scholar, 51Sugawara N. Paques F. Colaiacovo M. Haber J.E. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 9214-9219Crossref PubMed Scopus (270) Google Scholar, 52Evans E. Sugawara N. Haber J.E. Alani E. Mol. Cell. 2000; 5: 789-799Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar) to remove non-homologous 3′ tails during the strand invasion step of homologous recombination and in another homology-dependent DNA repair pathway termed single strand annealing (reviewed in Ref. 53Paques F. Haber J.E. Microbiol. Mol. Biol. Rev. 1999; 63: 349-404Crossref PubMed Google Scholar). No other yeast MMR proteins are involved in this tail removal (51Sugawara N. Paques F. Colaiacovo M. Haber J.E. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 9214-9219Crossref PubMed Scopus (270) Google Scholar), but all appear to contribute to the suppression of recombination between non-identical DNA substrates (54Nicholson A. Hendrix M. Jinks-Robertson S. Crouse G.F. Genetics. 2000; 154: 133-146Crossref PubMed Google Scholar). We do not yet know the detailed molecular mechanisms of VSG switching, and therefore one potential explanation for the lack of altered VSG switching in the MSH2 mutants is that the enzyme both promotes the process, by processing 3′ tails, and suppresses it by selecting against insufficiently matched recombining sequences. To address this, we examined the influence of MLH1 on VSG switching. Although MLH1 promotes meiotic recombination in at least some organisms (55Wang T.F. Kleckner N. Hunter N. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 13914-13919Crossref PubMed Scopus (238) Google Scholar, 56Lipkin S.M. Moens P.B. Wang V. Lenzi M. Shanmugarajah D. Gilgeous A. Thomas J. Cheng J. Touchman J.W. Green E.D. Schwartzberg P. Collins F.S. Cohen P.E. Nat. Genet. 2002; 31: 385-390Crossref PubMed Scopus (289) Google Scholar), it appears only to suppress mitotic recombination events, and T. brucei VSG switching occurs in dividing bloodstream stage cells that do not undergo meiosis. MLH1 mutants were created in T. brucei strain 3174, again precisely deleting the open reading frame by two rounds of transformation (with the constructs ΔMLH1::BSD and ΔMLH1::PUR).2 Two independent MLH1+/– and MLH1–/– mutants were confirmed by Southern and reverse transcriptase-PCR analysis and, as for MSH2, caused no detectable growth alteration in vitro, and led to the same MMR-associated phenotypes described above. Deletion of MLH1 had no statistically significant effect on the calculated VSG switching frequencies (Fig. 1) and caused no discernible alteration to the profile of VSG switching reactions used to generate switched variants (Fig. 2). This suggests that MMR in general, rather than MSH2 alone, plays little or no role in regulating this process. Mismatch Repair Regulates Homologous Recombination in T. brucei—Although previous work has suggested indirectly that MMR influences recombination of transformed DNA constructs in T. brucei (44Blundell P.A. Rudenko G. Borst P. Mol. Biochem. Parasitol. 1996; 76: 215-229Crossref PubMed Scopus (41) Google Scholar), the apparent lack of involvement in VSG switching made it important to test this genetically. To do this, we integrated a copy of the hygromycin phosphotransferase gene (HYG) into the tubulin array of the T. brucei genome (Fig. 3A), creating a unique sequence for recombination of transformed DNA (see below). Two independent MSH2 mutants were created in the HYG transformant strain, each by two rounds of transformation with the constructs ΔMSH2:: BSD and ΔMSH2::PUR. Southern analysis confirmed that HYG was retained in the +/– and –/– cells lines and that the MSH2 open reading frames had been deleted as expected; in addition, reverse transcriptase-PCR demonstrated that intact MSH2 transcript was no longer expressed in the –/– cells (data not shown). To assess the impact of MMR on homologous recombination, we created a series of DNA constructs designed to integrate into the HYG marker following transformation of wild type, +/–, and –/– cells (Fig. 3, A and B). All the constructs contained a bleomycin resistance cassette (BLE) to provide selection for recombination into the T. brucei genome. The resistance cassette was flanked upstream and downstream, respectively, by 445 and 449 bp of sequence derived from HYG, providing substrates for homologous recombination. The constructs in the series differed from each other in that the overall sequence identity between the HYG targeting flanks and the genomic HYG marker reduced progressively from 100 to 89% (Fig. 3B). The consequences of increasing sequence divergence for homologous recombination was compared in MSH2wt and mutant cells by assaying the efficiency with which stable transformants arose following transformation of a fixed amount of each linear DNA construct. This is a valid measurement of homologous recombination efficiency because in RAD51wt T. brucei essentially all stable transformants integrate linear DNA by homologous recombination rather than by non-homologous reactions (43Conway C. Proudfoot C. Burton P. Barry J.D. McCulloch R. Mol. Microbiol. 2002; 45: 1687-1700Crossref PubMed Scopus (60) Google Scholar), and the formation of extrachromosomal replicons to yield antibiotic resistant T. brucei following the transformation of small, linear constructs has never been described (57Alsford N.S. Navarro M. Jamnadass H.R. Dunbar H. Ackroyd M. Murphy N.B. Gull K. Ersfeld K. Mol. Microbiol. 2003; 47: 277-289Crossref PubMed Scopus (14) Google Scholar). The results of this analysis are graphed in Fig. 4 and summarized in Table I.Table ISummary of recombination frequenciesTable ISummary of recombination frequencies In the wild type and MSH2+/– (MMR+) cells there was a 2.8-fold decrease in transformation efficiency with construct HYG01 (1% sequence divergence) compared with HYGwt (100% sequence identity), showing that even a small number of mismatches affect homologous recombination. This was true also in the MSH2–/– cells, where a small
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