A yeast-endonuclease-generated DNA break induces antigenic switching in Trypanosoma brucei.
Trypanosoma brucei is the causative agent of African sleeping sickness in humans and one of the causes of nagana in cattle. This protozoan parasite evades the host immune system by antigenic variation, a periodic switching of its variant surface glycoprotein (VSG) coat. VSG switching is spontaneous and occurs at a rate of about 10(-2)-10(-3) per population doubling in recent isolates from nature, but at a markedly reduced rate (10(-5)-10(-6)) in laboratory-adapted strains. VSG switching is thought to occur predominantly through gene conversion, a form of homologous recombination initiated by a DNA lesion that is used by other pathogens (for example, Candida albicans, Borrelia sp. and Neisseria gonorrhoeae) to generate surface protein diversity, and by B lymphocytes of the vertebrate immune system to generate antibody diversity. Very little is known about the molecular mechanism of VSG switching in T. brucei. Here we demonstrate that the introduction of a DNA double-stranded break (DSB) adjacent to the approximately 70-base-pair (bp) repeats upstream of the transcribed VSG gene increases switching in vitro approximately 250-fold, producing switched clones with a frequency and features similar to those generated early in an infection. We were also able to detect spontaneous DSBs within the 70-bp repeats upstream of the actively transcribed VSG gene, indicating that a DSB is a natural intermediate of VSG gene conversion and that VSG switching is the result of the resolution of this DSB by break-induced replication.
Laboratory of Lymphocyte Biology, The Rockefeller University, New York, New York 10065, USA.
Catharine E
CE
Boothroyd
Oliver
O
Dreesen
Tatyana
T
Leonova
K Ina
KI
Ly
Luisa M
LM
Figueiredo
George A M
GA
Cross
F Nina
FN
Papavasiliou
D000818:Animals; D000940:Antigenic Variation / Q000235:genetics*; D053903:DNA Breaks, Double-Stranded; D004260:DNA Repair / Q000235:genetics; D004261:DNA Replication; D015252:Deoxyribonucleases, Type II Site-Specific / Q000235:genetics / Q000378:metabolism*; D005785:Gene Conversion / Q000235:genetics; D008957:Models, Genetic; D016133:Polymerase Chain Reaction; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism*; D017421:Sequence Analysis; D014346:Trypanosoma brucei brucei / Q000235:genetics* / Q000276:immunology*; D014643:Variant Surface Glycoproteins, Trypanosoma / Q000235:genetics / Q000276:immunology
D029701:Saccharomyces cerevisiae Proteins; D014643:Variant Surface Glycoproteins, Trypanosoma; C037725:SCEI protein, S cerevisiae; D015252:Deoxyribonucleases, Type II Site-Specific