cerevisiae, neither RAD27 nor DNA2 is strictly essential for replication or viability because the lethality of dna2 null mutants can be suppressed by deletion of RAD9 ( 14, 15). Genetic data suggest additional redundancy in lagging-strand processing, and point to the likely involvement of Exo1 as a third Okazaki nuclease. However, recent reports indicate that Dna2 activity is sufficient to process 5′ flaps into ligatable nicks in vitro ( 13). Dna2 cleaves to leave a short flap such that RPA dissociates and Rad27 can cleave ( 11, 12). Long, RPA-coated flaps are optimal substrates for Dna2 in vitro. Rad27 readily cleaves short flaps longer flaps are competent to bind RPA and thereby become refractory to Rad27 cleavage ( 10). Rad27 and Dna2 show distinct substrate requirements in vitro. If Pol δ extension outpaces Rad27 cleavage, Dna2 is required to process the resulting long flap. According to this model, ( 9) iterative extension by Pol δ is followed by immediate cleavage of short DNA flaps by Rad27. Genetic and biochemical work has given rise to a ‘two-nuclease’ model ( 1, 8). The nucleases Rad27 and Dna2 have been proposed to cleave the majority of flaps during Okazaki fragment maturation. cerevisiae and millions of times per human cell division. Nuclease cleavage during Okazaki fragment biogenesis represents an extremely abundant DNA transaction – one that must occur tens of thousands of times during each S-phase in S. Iterative rounds of extension, followed by flap cleavage or nick regeneration by the 3′-5′ exonuclease activity of Pol δ ( 5), maintain a ligatable nick that persists until it is sealed by DNA ligase I, encoded by the CDC9 gene in Saccharomyces cerevisiae ( 7). The displaced 5′ flap serves as a substrate for nucleases ( 6), which cleave to generate a nick between Okazaki fragment termini. Pol δ synthesizes DNA to the 5′ end of the preceding fragment and continues beyond this point ( 5), generating a 5′ flap structure. The high-fidelity, PCNA-associated polymerase δ (Pol δ) is subsequently loaded onto the 3′ terminus of the initiating fragment in a reaction catalyzed by the RFC clamp loader ( 4). The primase component of the Pol α-primase complex synthesizes a short RNA primer, which is further extended by the error-prone DNA polymerase α (Pol α) ( 2, 3). The synthesis of each Okazaki fragment requires several distinct enzymatic activities ( 1). Further, using cell cycle-restricted constructs, we demonstrate that both the nucleolytic processing and the ligation of Okazaki fragments can be uncoupled from DNA replication and delayed until after synthesis of the majority of the genome is complete. When nuclease cleavage is impaired, we observe a reduction in strand-displacement synthesis as opposed to the widespread generation of long Okazaki fragment 5′ flaps, as predicted by some models. We find that Rad27 processes the majority of lagging-strand flaps, with a significant additional contribution from Exo1 but not from Dna2. By conditionally depleting lagging-strand nucleases and directly analyzing Okazaki fragments synthesized in vivo in Saccharomyces cerevisiae, we conduct a systematic evaluation of the impact of Rad27, Dna2 and Exo1 on lagging-strand synthesis. However, neither the contributions of individual nucleases to lagging-strand synthesis nor the structure of the DNA intermediates formed in their absence have been fully defined in vivo. At least three DNA nucleases: Rad27 (Fen1), Dna2 and Exo1, have been implicated in processing Okazaki fragment flaps. Nuclease cleavage takes place in the context of 5′ flap structures generated via strand-displacement synthesis by DNA polymerase delta. Prior to ligation, each Okazaki fragment synthesized on the lagging strand in eukaryotes must be nucleolytically processed.
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