All homologs of eukaryotic FEN1 display high-affinity binding and cleavage specificity for a double-flap substrate with a 1
nt 3′ flap and a 5′ flap of variable length (15
). During Okazaki fragment maturation, strand displacement synthesis by DNA polymerase δ (pol δ) creates a 5′ flap (38
). A number of reports indicate that the steady-state structure during strand displacement synthesis is an annealed 3′ terminus separated by a short gap from the 5′-displaced flap created by steric separation of the template and downstream complementary strand by the DNA polymerase (39
). The manner in which the double flap becomes available to FEN1 is currently not known. The efficiency and accuracy of short flap processing suggest a simple mechanism in which the polymerase withdraws to expose the upstream synthesis strand and downstream 5′ flap for FEN1 binding and subsequent cleavage (41
). Prior reports suggest a polymerase idling process during strand displacement, in which the polymerase backs up or dissociates, creating an opportunity 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 mechanism must have additional complexity involving a sequential action of Dna2 and then FEN1 (8
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 5′ 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 5′ 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 5′ flap, termed a gap-flap, we acquired evidence that the upstream DNA improved hDna2 binding affinity and cleavage efficiency on intermediate length 5′ flap structures. The decreased hDna2 binding for the intermediate length 5′-tail substrates compared to the double flap was unexpected. We previously reported that optimal scDna2 substrate recognition involves simultaneous binding of the 5′ 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 5′ 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 δ initiates 5′ flap creation through strand displacement synthesis (38
). Foot-printing data show that pol δ protects 5–6
nt of ssDNA ahead of the most recently incorporated nucleotide (45
). The combination of the displaced 5′ flap and single-stranded region protected by pol δ 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
). Pol δ most likely releases from the strand being synthesized allowing a portion of the 5′ 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, 5′ 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 δ DNA substrate binding stability and synthesis processivity (46–48
). Reports show that pol δ, 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 5′ flap substrates 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 5′-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
). The observed concurrent binding suggests that hFEN1 and hDna2 are able to stably bind different locations of the substrate. hDna2 likely binds the upper portion of the 5′ flap and downstream DNA while hFEN1 binds the upstream dsDNA and the 3′ 1
nt flap (A). hFEN1 may also bind the lower portion of the 5′ flap and a portion of the downstream dsDNA.
Figure 6. Models of FEN1 and Dna2 Okazaki fragment substrate recognition and processing. (A) hDna2 and hFEN1 substrate recognition model. Pol δ synthesizes until encountering the downstream Okazaki fragment RNA/DNA primer. Pol δ then physically (more ...)
It is noteworthy that the human system has evolved to promote hDna2 binding to long flap structures independent 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 predominant 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 supporting this model show that scRPA displacement by scDna2 in vitro
is more efficient than hRPA displacement by hDna2 (33
, hDna2 likely processes these long 5′ flaps, to an intermediate length, at which point its substrate binding affinity is reduced. As noted earlier, the shortened 5′ flap may lead to the reorientation of hDna2 to bind both the shorter 5′ flap and the upstream DNA for both the 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 processing 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 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
). 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 5′ flap structures.
Based on current and prior results, we propose the following model for long flap processing (B). During synthesis, pol δ generates a gap-flap structure with a 5–6
nt single-stranded gap between the segment being synthesized and the downstream fragment. Recognizing the 5′ flap and downstream dsDNA, hDna2 binds the flap being extended by pol δ 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 δ. When this occurs, hDna2 will reorient to bind the shortened flap and upstream DNA, possibly aiding the re-annealing of the 5′ 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 5′ flap can compete for template annealing with the upstream newly synthesized strand (31
). 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 5′ 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 5′-tail structures. The possibility exists that hDna2 is not needed to process 5′-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 5′-tail substrates. Reports supporting this model demonstrate that multi-protein complexes, known to bind DSBs and necessary for telomere maintenance, recruit and stimulate hDna2. These reports also show that (i) the recruitment of RPA, a 3′–5′ helicase (such as BLM helicase), and Dna2 is necessary, (ii) the helicase activity of the 3′–5′ helicase and the 5′ nuclease activity of Dna2 are required and (iii) the helicase activity of Dna2 is dispensable for end resection in vitro
). During these processes, Dna2 is suggested to act on pseudo-Y structures cleaving only the 5′ flap. The abilities of Dna2 to bind and process long 5′ 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 3′–5′ 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 δ during long flap strand displacement synthesis. 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 currently 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 physiological DNA substrates, our results in vitro define a model in which hDna2 plays an important role to promote genomic stability during DNA replication.