<b>Spatial separation of replisome arrest sites influences homologous recombination quality at a Tus/</b><i><b>Ter</b></i><b>-mediated replication fork barrier</b>

The <i>Escherichia coli</i> replication fork arrest complex Tus/<i>Ter</i> mediates site-specific replication fork arrest and homologous recombination (HR) on a mammalian chromosome, inducing both conservative “short tract” gene conversion (STGC) and error-prone “long tract” gene conversion (LTGC) products. We showed previously that bidirectional fork arrest is required for the generation of STGC products at Tus/<i>Ter</i>-stalled replication forks and that the HR mediators BRCA1, BRCA2 and Rad51 mediate STGC but suppress LTGC at Tus/<i>Ter</i>-arrested forks. Here, we report the impact of <i>Ter</i> array length on Tus/<i>Ter</i>-induced HR, comparing HR reporters containing arrays of 6, 9, 15 or 21 <i>Ter</i> sites—each targeted to the <i>ROSA26</i> locus of mouse embryonic stem (ES) cells. Increasing <i>Ter</i> copy number within the array beyond 6 did not affect the magnitude of Tus/<i>Ter</i>-induced HR but biased HR in favor of LTGC. A “lock”-defective Tus mutant, F140A, known to exhibit higher affinity than wild type (wt)Tus for duplex <i>Ter</i>, reproduced these effects. In contrast, increasing <i>Ter</i> copy number within the array reduced HR induced by the I-SceI homing endonuclease, but produced no consistent bias towards LTGC. Thus, the mechanisms governing HR at Tus/<i>Ter</i>-arrested replication forks are distinct from those governing HR at an enzyme-induced chromosomal double strand break (DSB). We propose that increased spatial separation of the two arrested forks encountering an extended Tus/<i>Ter</i> barrier impairs the coordination of DNA ends generated by the processing of the stalled forks, thereby favoring aberrant LTGC over conservative STGC.