RECOMBINATIONAL REPAIR OF GAPS IN DNA IS ASYMMETRIC IN USTILAGO-MAYDIS AND CAN BE EXPLAINED BY A MIGRATING D-LOOP MODEL

Recombinational repair of double-stranded DNA gaps was investigated in Ustilago maydis. The experimental system was designed for analysis of repair of an autonomously replicating plasmid containing a cloned gen e disabled by an internal deletion. It was discovered that crossing ov er rarely accompanied gap repair. The strong bias against crossing ove r was observed in three different genes regardless of gap size. These results indicate that gap repair in U. maydis is unlikely to proceed b y the mechanism envisioned in the double-stranded break repair model o f recombination, which was developed to account for recombination in S accharomyces cerevisiae. Experiments aimed at exploring processing of DNA ends were performed to gain understanding of the mechanism respons ible for the observed bias. A heterologous insert placed within a gap in the coding sequence of two different marker genes strongly inhibite d repair if the DNA was cleaved at the promoter-proximal junction join ing the insert and coding sequence but had little effect on repair if the DNA was cleaved at the promoter-distal junction. Gene conversion o f plasmid restriction fragment length polymorphism markers engineered in sequences flanking both sides of a gap accompanied repair but was d irectionally biased. These results are interpreted to mean that the DN A ends flanking a gap are subject to different types of processing. A model featuring a single migrating D-loop is proposed to explain the b ias in gap repair outcome based on the observed asymmetry in processin g the DNA ends.