6784 Nucleic Acids Research, 2012, Vol. 40, No. 14 substrate binding pending acquisition and activity of hFEN1 with lower binding afﬁnity. The hFEN1–hDna2 protein–protein interaction has been shown to result in stimulation of both nuclease activities (24,52). The ability of hFEN1 to displace hDna2 from intermediate double-ﬂap structures taken together with the improved hDna2 upstream DNA binding for intermediate length supports a mechanism in which the nucleases have evolved to sequentially and cooperatively process elongated 50 ﬂap structures. Based on current and prior results, we propose the fol- lowing model for long ﬂap processing (Figure 6B). During synthesis, pol d generates a gap-ﬂap structure with a 5–6 nt single-stranded gap between the segment being synthesized and the downstream fragment. Recognizing the 50 ﬂap and downstream dsDNA, hDna2 binds the ﬂap being extended by pol d strand displacement. hDna2 cleaves generating an intermediate length ﬂap, disrupting stable RPA binding. hFEN1 is unable to bind or cleave this gap-ﬂap conﬁguration. Upstream DNA must be exposed by displacement or idling of pol d. When this occurs, hDna2 will reorient to bind the shortened ﬂap and upstream DNA, possibly aiding the re-annealing of the 50 ﬂap to the template forming a double-ﬂap structure. In line with this idea, prior reports show that hDna2 possesses a DNA strand annealing activity (55) and promotes hFEN1 nick generation in the presence of equilibrating ﬂaps where the long 50 ﬂap can compete for template annealing with the upstream newly synthesized strand (31,34). The combination of the double-ﬂap conﬁguration and hDna2 movement in the upstream DNA direction would enable hFEN1 to bind hDna2 and provide access to the base of the correctly formed double-ﬂap substrate. hFEN1 bound to the ﬂap base and the protein–protein interaction would enable hDna2 displacement from the substrate and stimulate hFEN1 50 ﬂap cleavage to efﬁciently create the nicked structure for ligation. While the ability of hDna2 to bind upstream DNA during replication may be beneﬁcial, this binding site structure may not be available during DSB repair or telomere maintenance. The loss of the upstream binding contact might lead to the reduction in cleavage activity as visualized for the intermediate 50-tail structures. The pos- sibility exists that hDna2 is not needed to process 50-tail substrates in these pathways until they are lengthened. Furthermore, protein cofactors may be required to generate long ﬂaps and recruit hDna2 to these long 50-tail substrates. Reports supporting this model demon- strate that multi-protein complexes, known to bind DSBs and necessary for telomere maintenance, recruit and stimulate hDna2. These reports also show that (i) the re- cruitment of RPA, a 30–50 helicase (such as BLM helicase), and Dna2 is necessary, (ii) the helicase activity of the 30–50 helicase and the 50 nuclease activity of Dna2 are required and (iii) the helicase activity of Dna2 is dispensable for end resection in vitro (29,30). During these processes, Dna2 is suggested to act on pseudo-Y structures cleaving only the 50 ﬂap. The abilities of Dna2 to bind and process long 50 ﬂaps independent of the upstream DNA properties support correct DNA product formation during DSB and telomere maintenance. Our binding and cleavage data taken together with these prior results support the model that protein interactions and ﬂap elongation by a 30–50 helicase are likely necessary to activate Dna2 during DSB repair and telomere maintenance. In summary, we have shown that hDna2 binds ﬂap structures with higher afﬁnity as the ﬂap length increases. In the human system, this increased long ﬂap binding afﬁnity prevents displacement of hDna2 by hFEN1. As hDna2 cleaves the ﬂap to an intermediate length, the nuclease is able to bind upstream DNA improving binding afﬁnity and cleavage efﬁciency. We suggest a model in which this additional hDna2 binding aids in generating the double-ﬂap structure from the gap-ﬂap created by pol d during long ﬂap strand displacement syn- thesis. hDna2 can remain bound to the ﬂap base during acquisition of hFEN1. It is likely that the two proteins transiently bind each other and the substrate and then, as the ﬂap is further shortened, hDna2 dissociates and hFEN1 cleaves at the base. While further analysis is needed to validate the model in a cellular system, the dynamic characteristics of the replication fork do not cur- rently allow for measurement of these properties in vivo. Additionally, lack of a crystal structure for hDna2 limits designing experiments in vivo that would speciﬁcally deﬁne the sequential action of the proteins on the replication fork. While structural analysis of hDna2 is needed to validate the speciﬁc binding contacts and protein– protein interactions in the presence of various physio- logical DNA substrates, our results in vitro deﬁne a model in which hDna2 plays an important role to promote genomic stability during DNA replication. SUPPLEMENTARY DATA Supplementary Data are available at NAR Supplementary Figure 1. Online: ACKNOWLEDGEMENTS We would like to thank the Bambara and Campbell research groups for critical discussion during the course of this work and comments on the manuscript. FUNDING The National Institutes of Health [GM024441 to R.A.B., GM068411 to J.W.G., GM098328-01A1 to L.B., GM078666 to J.L.C.]. Funding for open access charge: The National Institutes of Health [GM024441]. Conﬂict of interest statement. None declared. 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