Reconstituted Okazaki Fragment Processing Indicates Two Pathways of Primer Removal

Abstract

Eukaryotic DNA replication proceeds discontinuously via Okazaki fragment synthesis and maturation on the lagging strand. DNA polymerase ␣ primes the lagging strand, synthesizing about 10 nucleotides (nt) 2 of RNA followed by an additional 10 -20 nt of DNA. The primer is extended by a complex of DNA polymerase ␦ (pol ␦), the sliding clamp proliferating cell nuclear antigen (PCNA), and the clamp loader replication factor C (RFC). Pol ␦ continues extension of the upstream fragment until it runs into the downstream fragment, displacing its 5Ј-end region into a flap. The flap can be cleaved by a nuclease to form a nick, which will be sealed by DNA ligase I to generate the continuous double-stranded DNA (1, 2).Cleavage of the flap can be completed by flap endonuclease 1 (FEN1), a 5Ј structure-specific endonuclease and the product of the RAD27 gene in Saccharomyces cerevisiae (1). One proposed model suggests FEN1 cleavage of primarily short flaps displaced by pol ␦ (3-5). As pol ␦ displaces the downstream primer into a flap, FEN1 can track from the 5Ј-end (6, 7) to the base of the flap for cleavage (6,8,9) that generates a nick for ligation. Through Okazaki fragment processing reconstitution studies in vitro, Ayyagari et al. (3) showed that short flaps, 10 nt in length, are processed efficiently by FEN1 to generate ligatable replication intermediates. More recent experimentation revealed that in the presence of pol ␦ and FEN1, strand-displacement and cleavage generated mainly mononucleotide products (5). In contrast, strand-displacement by mutant forms of pol ␦, deficient in its proofreading 3Ј-5Ј exonuclease activity, and cleavage by FEN1 generated mono-through hexanucleotide products (5). It was proposed that the 3Ј-5Ј exonuclease activity of pol ␦, coordinated with FEN1, prevents extensive strand-displacement because pol ␦ 3Ј-5Ј exonuclease mutants exhibit greater stranddisplacement activity than wild-type pol ␦ (4, 5). This idea is supported by the synthetic lethal interaction between 3Ј-5Ј exonuclease mutants of pol ␦ (pol3-exo) and either rad27 deletion mutants or those that are compromised for interaction with its cofactor PCNA (rad27-p) (10 -12). In addition, the rare pol3-exo rad27-p double mutants that are not lethal require intact recombination and double-strand break repair machinery (12). Altogether, these results suggest that with wild-type proteins, the system is generally designed to strand-displace and cleave short flaps, while failure to cleave at the short stage, either because strand-displacement is enhanced or nuclease cleavage inhibited, leads to the possibility that long flaps occasionally form (3-5).If a flap escapes cleavage by FEN1, it can become long and follow a secondary pathway. A model put forward by Bae et al. (13) asserts that flaps greater than 27 nt in length are coated by the single-stranded-binding protein replication protein A (RPA). RPA inhibits cleavage by FEN1 but stimulates cleavage by Dna2 nuclease/helicase (13,14). Dna2, like FEN1, must track from the 5Ј-end of the flap to...

The N-terminal 45-kDa Domain of Dna2 Endonuclease/Helicase Targets the Enzyme to Secondary Structure DNA

“…In keeping with this, it was shown that calf thymus pol ␦ displaces downstream duplex DNAs longer than 200 bp in vitro, revealing its intrinsic ability to form extensively long flaps (31,32). In vitro reconstitution experiments using yeast enzymes also showed that a portion of flaps grows long, up to 20 -30 nt, although the flaps formed in vitro are primarily short, up to 8 nt in length (33). The observation that overexpression of RPA alleviates the requirement of Dna2 helicase activity (13) is also consistent with formation of long flaps in vivo.…”

Sequential switching of binding partners on PCNA during in vitro Okazaki fragment maturation

“…Having observed the activities of Pol δ and FEN1 separately, we next examined whether the cooperation between these two enzymes during Okazaki fragment maturation requires multiple binding sites on the PCNA trimer, as posited by the toolbelt model. Nick translation, the result of iterative, sequential strand displacement synthesis by Pol δ and flap cleavage by FEN1, requires rapid and efficient cooperation between the enzymes in the presence of PCNA (7,10,11). We examined the kinetics of nick translation on an oligonucleotide-based model substrate in the presence of different PCNA trimers.…”

“…In particular, removal of the initiator RNA requires several rapid iterative switches between Pol δ and FEN1 (7). This PCNA-dependent cooperation is particularly important to ensure that flaps will not become too long for processing by this short-flap pathway (7,10,11).…”

Pif1 Helicase Lengthens Some Okazaki Fragment Flaps Necessitating Dna2 Nuclease/Helicase Action in the Two-nuclease Processing Pathway

“…In reconstitution of Okazaki fragment processing with purified proteins, even though some flaps became long enough to bind RPA, FEN1 was very effective at cleaving essentially all of the generated flaps (13,14). Evidently, FEN1 could engage the flaps before binding of RPA.…”

“…In addition, the tightly coordinated action of pol ␦ and FEN1 also tends to keep flaps short. However, biochemical reconstitution studies demonstrate that some flaps can become long (13,14). Once these flaps reach ϳ30 nt, they can be bound by the eukaryotic single strand binding protein replication protein A (RPA) (15).…”