Numerous proteins have been implicated genetically and biochemically in targeting the D-loop intermediate for disruption as discussed in the introduction. In the budding yeast alone, the DNA helicases Srs2, Sgs1, and Mph1 (Fasching et al., 2015; Marini and Krejci, 2012; Prakash et al., 2009; Sebesta et al., 2011) (this study), the motor protein Rad54 (Bugreev et al., 2007a; Wright and Heyer, 2014), as well as Top3-Rmi1 (Fasching et al., 2015) were shown to disrupt D-loops in vitro. We highlight possible reasons for the involvement of multiple proteins in D-loop disruption. First, before extension by DNA polymerase, disruption of a nascent D-loop is an inherently anti-recombination activity, whereas disruption of a D-loop extended by DNA polymerases limits crossover and favors SDSA. What distinguished these D-loops is their potential length, although little is known about this in vivo, and the proteins bound to the intermediate. While the genetic signatures of anti-recombination and pro-SDSA are different, the biochemical reaction of disrupting nascent or extending D-loops is highly similar and protein interactions involving also posttranslational modifications between the various D-loop disrupting enzymes may only impart preference rather than absolute specificity. Second, D-loops are dynamic entities, likely with varying length and structure, where for example the 3’ end may be embedded in the heteroduplex or transiently extruded. As we show for Srs2 (Figure 4), such structural variations may influence the activity of the enzymes involved. Finally, D-loops occur not only in the context of DSB repair but also in the context of gap repair. The D-loop structure resulting from gap invasion differs, as there are no free ends available to allow true intertwining. We tried to model this reaction using invading DNA molecules with duplex heterology on both ends (Figure 2) and show that Srs2 can disrupt such pairing intermediate, albeit with lower efficiency than regular D-loops. It will be interesting to test the activity of other D-loop disrupting proteins on such substrates. We propose that multiple enzymes with individual preferences for specific D-loop substrates are acting in vivo in a partially overlapping fashion to control anti-recombination, crossover, and SDSA. This view is consistent with genetic data and the observations that mutations in SRS2, SGS1, and MPH1 engage in synthetic lethal/negative interactions in budding yeast (Gangloff et al., 2000; Ira et al., 2003; Mitchel et al., 2013; Prakash et al., 2009).