downstream DNA while hFEN1 binds the upstream dsDNA and the 30 1 nt ﬂap (Figure 6A). hFEN1 may also bind the lower portion of the 50 ﬂap and a portion of the downstream dsDNA. It is noteworthy that the human system has evolved to promote hDna2 binding to long ﬂap structures independ- ent of the ﬂap base conﬁguration even in the presence of hFEN1. Original genetic analyses of the yeast homologs suggested that long ﬂap creation and removal is the pre- dominant pathway of Okazaki fragment processing (8). Later, biochemical analyses demonstrated that most ﬂaps are processed while short and that the long ﬂap pathway is possibly used as a backup (43). While the yeast system may use scRPA in combination with scDna2 to process long ﬂaps, the human system may have evolved to more efﬁciently use hDna2 to cleave elongating ﬂaps prior to stably binding hRPA. Data sup- porting this model show that scRPA displacement by scDna2 in vitro is more efﬁcient than hRPA displacement by hDna2 (33,34). In vivo, hDna2 likely processes these long 50 ﬂaps, to an intermediate length, at which point its substrate binding afﬁnity is reduced. As noted earlier, the shortened 50 ﬂap may lead to the reorientation of hDna2 to bind both the shorter 50 ﬂap and the upstream DNA for both the Nucleic Acids Research, 2012, Vol. 40, No. 14 6783 gap-ﬂap and double-ﬂap structures. Our competition data show that hFEN1 effectively displaces hDna2 from these intermediate length double-ﬂap structures. Considering this displacement and the need for hFEN1 to bind the upstream DNA prior to cleaving, it was counterintuitive that hDna2 bound the upstream DNA of intermediate length ﬂaps. We envision two scenarios in which this additional binding would be advantageous. First, binding the upstream DNA may allow hDna2 to interact with proteins located upstream of the ﬂap base. Second, binding the upstream DNA may prepare hDna2 for cleavage activity needed in Okazaki fragment process- ing but not DSB repair. Consistent with the ﬁrst scenario, hDna2 has been shown to directly interact with hFEN1 (52). The additional upstream binding contact may bring hDna2 in contact with hFEN1 across a gap-ﬂap structure. This contact may be part of the mechanism by which hFEN1 displaces hDna2. In line with the second scenario, FEN1 substrate localization and cleavage activity are diminished by certain FEN1 post-translational modiﬁcations. Speciﬁcally, phosphorylation leads to reduced FEN1 substrate localization (53) and acetylation diminishes substrate afﬁnity and cleavage activity (54). In both binding scenarios, the increased binding afﬁnity imparted by the upstream DNA would stabilize hDna2 Figure 6. Models of FEN1 and Dna2 Okazaki fragment substrate recognition and processing. (A) hDna2 and hFEN1 substrate recognition model. Pol d synthesizes until encountering the downstream Okazaki fragment RNA/DNA primer. Pol d then physically separates the two strands of the duplex DNA to form a 50 ﬂap. The active site of the polymerase protects a 5–6 nt single-stranded region on the template creating a structure with a 50-displaced ﬂap and a single-stranded gap between the upstream and downstream primers, termed a gap-ﬂap. Left arrow: short ﬂap pathway. During short ﬂap processing, the polymerase idles back or dissociates from the synthesis fragment allowing the 50 ﬂap to re-anneal with the template generating a short double-ﬂap structure. hFEN1 binds the double-ﬂap substrate using three contact locations, indicated by ‘F’, (i) upstream dsDNA, (ii) downstream dsDNA and (iii) the 30 1 nt ﬂap. Right arrow: long ﬂap pathway. During long ﬂap processing, ﬂaps escape hFEN1 cleavage. hDna2 binds the long gap-ﬂap using two contact locations, indicated by ‘D’, the downstream dsDNA and the long 50 ﬂap. Dna2 threads the long ﬂap and begins cleaving. The polymerase idles back or dissociates from the synthesis fragment allowing Dna2 to continue to cleave the long ﬂap. hDna2 binding afﬁnity for the 50 ﬂap is reduced and it reorients to bind the upstream DNA. This hDna2 reorientation promotes the re-annealing of the 50 ﬂap with the template, ﬁlling the single-stranded gap. The combination of hDna2 upstream DNA binding and the formation of the double ﬂap promote hFEN1 ﬂap base localization. hDna2 releases from the upstream DNA and the 50 ﬂap allowing hFEN1 binding contacts with the upstream dsDNA and the 1 nt 30 ﬂap. The 50 ﬂap transitions to hFEN1 and hFEN1 binds the downstream DNA. hFEN1 is now poised to thread and cleave the substrate and generate a nicked product. (B) Long Flap Pathway Model. As in (A), pol d displaces the 50 ﬂap into a 5–6 nt gap-ﬂap structure. Flaps escaping hFEN1 cleavage become long and are bound by hRPA. hDna2 binds the ﬂap displacing hRPA. hDna2 cleaves the long ﬂap as pol d idles back or dissociates from the synthesis fragment. hDna2 reorients promoting 50 ﬂap re-annealing with the template, as shown in (A). The double-ﬂap structure formation and hDna2–hFEN1 protein interactions allow hFEN1 to bind the ﬂap base. The reduced hDna2-binding afﬁnity for the shortened 50 ﬂap permits hFEN1 acquisition of the ﬂap for threading. hDna2 stimulates the cleavage activity of hFEN1 to create a nicked product.