Deletion of the Tetrahymena thermophila rDNA replication fork barrier region disrupts macronuclear rDNA excision and creates a fragile site in the micronuclear genome

ABSTRACTDuring macronuclear development the Tetrahymenathermophila ribosomal RNA gene is excised frommicronuclear chromosome 1 by site-specific cleav-age at chromosome breakage sequence (Cbs)elements, rearranged into a ‘palindromic’ 21 kb mini-chromosome and extensively amplified. Geneamplification initiates from origins in the 5 0 non-transcribed spacer, and forks moving toward thecenter of the palindrome arrest at a developmentallyregulated replication fork barrier (RFB). The RFB isinactive during vegetative cell divisions, suggestinga role in the formation or amplification of macro-nuclear rDNA. Using micronuclear (germline) trans-formation, we show that the RFB region facilitatesCbs-mediated excision. Deletion of the RFB inhi-bits chromosome breakage in a sub-population ofdeveloping macronuclei and promotes alternativeprocessing by a Cbs-independent mechanism.Remarkably, the RFB region prevents spontaneousbreakage of chromosome 1 in the diploid micronu-cleus. Strains heterozygous for DRFB and wild-typerDNA lose the DRFB allele and distal left arm ofchromosome 1 during vegetative propagation. Thewild-type chromosome is subsequently fragmentednear the rDNA locus, and both homologs are pro-gressively eroded, suggesting that broken micronu-clear chromosomes are not ‘healed’ by telomerase.Deletion of this 363 bp segment effectively createsa fragile site in the micronuclear genome, provid-ing the first evidence for a non-telomere cis-actingdeterminant that functions to maintain the structuralintegrity of a mitotic eukaryotic chromosome.INTRODUCTIONThe initiation of DNA replication is precisely regulated toassure that chromosomes are duplicated once and only onceper cell division. Eukaryotic genomes contain hundreds tothousands of initiation sites that are relatively evenly spacedthroughout chromosomes (1). The convergence of neighboringreplication forks allows for the complete replication of chro-mosomes. Elongating replication forks can stall when theyencounter DNA damage at random sites in the genome. Stalledforks are recognized by the ATR (Ataxia-Telangectasia andRad3-related) kinase, which activates a checkpoint pathwaythat prevents the fork from collapsing and initiates the DNArepair response [reviewed in (2)]. Replication forks can alsoarrest at speciﬁed sites in the genome. Transient pausing ofreplication forks frequently occurs near coding regions andmay help coordinate replication and transcription. Similarto sites of DNA damage, the paused replication machineryresumes DNA synthesis once the impediment has beenremoved. More pronounced replication fork barriers (RFBs)irreversibly block the movement of the replication fork, andrequire the activity of a converging fork to replicate sequencesdownstream of the RFB [reviewed in (3)].Natural RFBs occur in both prokaryotes and eukaryotes,and have been shown to coordinate certain events withDNA replication. For example, unidirectional RFBs allowconverging forks to enter, but not exit the replication terminusof circular bacterial chromosomes (3,4). RFBs in the inter-genic regions of tandemly arrayed ribosomal RNA genesprevent head on collisions between the replication and tran-scription machinery (5–7). The contributions of RFBs tochromosome function are offset by the fact that they createhot spots for programmed or spontaneous genome rearrange-ment. For example, mating type switching in the ﬁssion yeastSchizosaccharomyces pombe requires that the mat locus bereplicated from forks progressing in one direction (2,8). TheSaccharomyces cerevisiae ribosomal DNA RFB promotes the