6782 Nucleic Acids Research, 2012, Vol. 40, No. 14 afﬁnity 50-tail. We competed hDna2 pre-bound to the 15 nt gap-ﬂap structures with increasing concentrations of hFEN1 (Figure 5C). hDna2 pre-bound to the 5 and 2 nt single-stranded gap structures is shown in lanes 2 and 10, respectively. The titration of hFEN1 alone bound to the 5 and 2 nt single-stranded gap structures is shown in lanes 6–8 and 14–16, respectively. hFEN1 titrated into the reactions containing pre-bound hDna2 to the 5 and 2 nt single-stranded gap-ﬂap structures is shown in lanes 3–5 and 11–13, respectively. hDna2 was displaced similarly by hFEN1 from both gap-ﬂap struc- tures. The comparative displacement of hDna2 from the 5 and 2 nt gap-ﬂap structures relative to the initial amount bound is shown graphically in Figure 5D ‘15 nt 50 Flap 5 nt Gap’ and ‘15 nt 50 Flap 2 nt Gap’, respectively. These results suggest that hDna2 displacement by hFEN1 requires upstream DNA to stabilize hFEN1 binding and a shortened ﬂap to reduce hDna2-binding afﬁnity. DISCUSSION All homologs of eukaryotic FEN1 display high-afﬁnity binding and cleavage speciﬁcity for a double-ﬂap substrate with a 1 nt 30 ﬂap and a 50 ﬂap of variable length (15,20,37). During Okazaki fragment maturation, strand displacement synthesis by DNA polymerase d (pol d) creates a 50 ﬂap (38). A number of reports indicate that the steady-state structure during strand displacement syn- thesis is an annealed 30 terminus separated by a short gap from the 50-displaced ﬂap created by steric separation of the template and downstream complementary strand by the DNA polymerase (39,40). The manner in which the double ﬂap becomes available to FEN1 is currently not known. The efﬁciency and accuracy of short ﬂap process- ing suggest a simple mechanism in which the polymerase withdraws to expose the upstream synthesis strand and downstream 50 ﬂap for FEN1 binding and subsequent cleavage (41,42). Prior reports suggest a polymerase idling process during strand displacement, in which the polymerase backs up or dissociates, creating an opportun- ity for FEN1 cleavage and subsequent ligation at virtually every DNA nucleotide (42). For those ﬂaps that escape FEN1 cleavage and bind RPA and then Dna2, the mech- anism must have additional complexity involving a se- quential action of Dna2 and then FEN1 (8,43). To better understand how the two nucleases work together, we characterized the substrate speciﬁcities inﬂuencing hDna2 binding and cleavage functions and the sequential interaction of hFEN1 and hDna2 on Okazaki fragment intermediates. hDna2 bound and cleaved long 50 ﬂap structures with similar high afﬁnity and efﬁciency irrespective of the presence of an upstream segment creating a double-ﬂap structure. Binding afﬁnity and cleavage efﬁciency on substrates with 50 ﬂaps decreased progressively with ﬂap length to become negligible at lengths of 4–5 nt. Utilizing substrates with a single-stranded gap between the upstream primer and downstream 50 ﬂap, termed a gap-ﬂap, we acquired evidence that the upstream DNA improved hDna2 binding afﬁnity and cleavage efﬁciency on intermediate length 50 ﬂap structures. The decreased hDna2 binding for the intermediate length 50-tail substrates compared to the double ﬂap was unexpected. We previously reported that optimal scDna2 substrate recognition involves simul- taneous binding of the 50 ﬂap and downstream dsDNA (36). Either human and yeast Dna2 possess different binding speciﬁcities or hDna2 utilizes additional upstream primer-template contacts for intermediate length 50 ﬂap processing that were undetected previously with the long ﬂap substrates used to characterize scDna2. Evidence for these additional binding contacts suggests that hDna2 reorients to access the upstream DNA after partially processing long ﬂaps. During DNA replication, pol d initiates 50 ﬂap creation through strand displacement synthesis (38,41,44). Foot-printing data show that pol d protects 5–6 nt of ssDNA ahead of the most recently incorporated nucleo- tide (45). The combination of the displaced 50 ﬂap and single-stranded region protected by pol d comprises a gap-ﬂap structure. Current work and prior reports have shown that hFEN1 neither stably binds nor efﬁciently cleaves these gap-ﬂap structures requiring that the DNA be remodeled prior to hFEN1 cleavage (4,20,37). Pol d most likely releases from the strand being synthesized allowing a portion of the 50 ﬂap to re-anneal to the template ﬁlling the gap region. This re-annealing can generate the double ﬂap base structure preferred by hFEN1 for cleavage. During short ﬂap processing, this polymerase dissociation, 50 ﬂap re-annealing and ﬂap base acquisition by hFEN1 would need to be efﬁciently coordinated to process the estimated 20–50 million Okazaki fragments made prior to every mammalian cell division (38). The synchronized protein handoff is likely accomplished by the polymerase processivity factor, proliferating cell nuclear antigen (PCNA) (41). PCNA is a homo-trimeric protein known to promote pol d DNA substrate binding stability and synthesis processivity (46–48). Reports show that pol d, hFEN1 and DNA ligase I can concurrently bind individual subunits of PCNA improving enzymatic function through increased substrate binding afﬁnity, and presumably coordinating sequential action (49–51). The steps by which hDna2 hands off the substrate to hFEN1 during long ﬂap processing have not been clear. To improve our understanding, we analyzed hDna2 displacement by hFEN1 from substrates representing expected replication intermediates. Speciﬁcally, we measured hDna2 and hFEN1 binding to long 50 ﬂap sub- strates with the gap-ﬂap and FEN1-preferred double ﬂap conﬁgurations. Surprisingly, in contrast with the yeast system, hFEN1 was unable to displace hDna2 from any of the long ﬂap substrates. Additionally, hFEN1 and hDna2 concurrently bound the long double-ﬂap structure. This co-binding mode was visualized neither with the long gap-ﬂap nor the 50-tail structures. The inability of hFEN1 to displace hDna2 from long ﬂaps was surprising as we previously reported that scFEN1 displaces scDna2 from the ﬂap base prior to cleaving (17,25). The observed con- current binding suggests that hFEN1 and hDna2 are able to stably bind different locations of the substrate. hDna2 likely binds the upper portion of the 50 ﬂap and