Eukaryotic Okazaki fragment maturation requires complete removal of the initiating RNA primer before ligation occurs. Polymerase ␦ (Pol ␦) extends the upstream Okazaki fragment and displaces the 5-end of the downstream primer into a single nucleotide flap, which is removed by FEN1 nuclease cleavage. This process is repeated until all RNA is removed. However, a small fraction of flaps escapes cleavage and grows long enough to be coated with RPA and requires the consecutive action of the Dna2 and FEN1 nucleases for processing. Here we tested whether RPA inhibits FEN1 cleavage of long flaps as proposed. Surprisingly, we determined that RPA binding to long flaps made dynamically by polymerase ␦ only slightly inhibited FEN1 cleavage, apparently obviating the need for Dna2. Therefore, we asked whether other relevant proteins promote long flap cleavage via the Dna2 pathway. The Pif1 helicase, implicated in Okazaki maturation from genetic studies, improved flap displacement and increased RPA inhibition of long flap cleavage by FEN1. These results suggest that Pif1 accelerates long flap growth, allowing RPA to bind before FEN1 can act, thereby inhibiting FEN1 cleavage. Therefore, Pif1 directs long flaps toward the two-nuclease pathway, requiring Dna2 cleavage for primer removal.During eukaryotic DNA replication, the lagging strand is replicated via synthesis and maturation of Okazaki fragments. These fragments are short stretches of DNA that are joined to generate a continuous strand (1). Each fragment is initiated when DNA polymerase ␣/primase (Pol ␣) 3 makes an RNA/ DNA primer, synthesizing ϳ10 nucleotides (nt) of RNA followed by 10 -20 nt of DNA (2). The primer is then extended by a complex of DNA polymerase ␦ (Pol ␦), the sliding clamp, proliferating cell nuclear antigen (PCNA), and the clamp loader, replication factor C (RFC). When Pol ␦ encounters the 5Ј-end of the downstream Okazaki fragment, it displaces it into a flap. Cleavage of the flap by nucleases generates a nick, which is subsequently sealed by DNA ligase I to form continuous double-stranded DNA (3, 4).One pathway for cleavage of the flap employs flap endonuclease 1 (FEN1). FEN1 is a single-strand, structure-specific endonuclease that enters the 5Ј-end of the flap and tracks to the base for cleavage (3,5,6). Following displacement of a short flap, less than about 12 nt, by Pol ␦, FEN1 cleaves leaving a nick, the substrate for DNA ligase I (7-9). Because the RNA initiating the Okazaki fragments is ϳ10 nt in length, short flaps composed entirely of RNA are first displaced by Pol ␦. This does not obstruct FEN1, which is active on RNA (10, 11). In addition, displacement and cleavage occurs mostly within the first 25 nt of the downstream fragment, sufficient to remove the entire RNA/DNA primer, which is ϳ20 -30 nt in length (11). Previous biochemical studies demonstrated that primarily short flaps are cleaved by FEN1. It is likely that in vivo a series of short successive displacements by Pol ␦ and cleavages by FEN1 are effective for removal of the entire init...

We have developed a system to reconstitute all of the proposed steps of Okazaki fragment processing using purified yeast proteins and model substrates. DNA polymerase ␦ was shown to extend an upstream fragment to displace a downstream fragment into a flap. In most cases, the flap was removed by flap endonuclease 1 (FEN1), in a reaction required to remove initiator RNA in vivo. The nick left after flap removal could be sealed by DNA ligase I to complete fragment joining. An alternative pathway involving FEN1 and the nuclease/helicase Dna2 has been proposed for flaps that become long enough to bind replication protein A (RPA). RPA binding can inhibit FEN1, but Dna2 can shorten RPA-bound flaps so that RPA dissociates. Recent reconstitution results indicated that Pif1 helicase, a known component of fragment processing, accelerated flap displacement, allowing the inhibitory action of RPA. In results presented here, Pif1 promoted DNA polymerase ␦ to displace strands that achieve a length to bind RPA, but also to be Dna2 substrates. Significantly, RPA binding to long flaps inhibited the formation of the final ligation products in the reconstituted system without Dna2. However, Dna2 reversed that inhibition to restore efficient ligation. These results suggest that the two-nuclease pathway is employed in cells to process long flap intermediates promoted by Pif1.Eukaryotic cellular DNA is replicated semi-conservatively in the 5Ј to 3Ј direction. A leading strand is synthesized by DNA polymerase ⑀ in a continuous manner in the direction of opening of the replication fork (1, 2). A lagging strand is synthesized by DNA polymerase ␦ (pol ␦) 3 in the opposite direction in a discontinuous manner, producing segments called Okazaki fragments (3). These stretches of ϳ150 nucleotides (nt) must be joined together to create the continuous daughter strand. DNA polymerase ␣/primase (pol ␣) initiates each fragment by synthesizing an RNA/DNA primer consisting of ϳ1-nt of RNA and ϳ10 -20 nt of DNA (4). The sliding clamp proliferating cell nuclear antigen (PCNA) is loaded on the DNA by replication factor C (RFC). pol ␦ then complexes with PCNA and extends the primer. When pol ␦ reaches the 5Ј-end of the downstream Okazaki fragment, it displaces the end into a flap while continuing synthesis, a process known as strand displacement (5, 6). These flap intermediates are cleaved by nucleases to produce a nick for DNA ligase I (LigI) to seal, completing the DNA strand.In one proposed mechanism for flap processing, the only required nuclease is flap endonuclease 1 (FEN1). pol ␦ displaces relatively short flaps, which are cleaved by FEN1 as they are created, leaving a nick for LigI (7-9). FEN1 binds at the 5Ј-end of the flap and tracks down the flap cleaving only at the base (5, 10, 11). Because pol ␦ favors the displacement of RNA-DNA hybrids over DNA-DNA hybrids, strand displacement generally is limited to that of the initiator RNA of an Okazaki fragment (12). In addition, the tightly coordinated action of pol ␦ and FEN1 also tends to keep flaps...

During eukaryotic DNA replication, the lagging strand is synthesized in a series of segments, each ϳ150 nucleotides (nt) 2 long, called Okazaki fragments (1). An Okazaki fragment is initiated by DNA polymerase ␣/primase (pol ␣), which synthesizes a primer beginning with 10 -12 nt of RNA followed by ϳ20 nt of DNA (2). After primer synthesis, the sliding clamp proliferating cell nuclear antigen (PCNA) is loaded on the primer-template DNA by replication factor C (RFC).DNA polymerase ␦ (pol ␦) then conjugates with PCNA and continues rapid and efficient extension of the primer. Upon reaching the downstream Okazaki fragment, pol ␦ displaces its 5Ј end into a single-stranded flap that must be removed by nucleases (3, 4). Cleavage of the flap produces a nick that DNA ligase I (LigI) will seal to complete the continuous DNA strand.Two pathways are proposed to process Okazaki flaps. In the first pathway, only one nuclease, flap endonuclease I (FEN1), is employed. In reconstitution studies, pol ␦ displaces short flaps, ϳ1-5 nt long, that are efficiently cleaved by FEN1 to produce a nicked intermediate (5-7). FEN1 binds the 5Ј end of the flap, tracks down the flap, and cleaves once at the base (8, 9). PCNA binds and stimulates both pol ␦ and FEN1, allowing for tight coordination between flap displacement and cleavage (10). This cooperation keeps flaps short, and the FEN1-only pathway has the potential to process virtually all flaps. However, reconstitutions have shown that some flaps can escape immediate cleavage and become long (11-13). When flaps become ϳ25-30 nt long, the eukaryotic single strand-binding protein replication protein A (RPA) can bind the flap stably (14). RPA binding inhibits FEN1 cleavage (15), necessitating the second pathway.This second, or two-nuclease, pathway was proposed to utilize Dna2 in addition to FEN1 to process long RPA-bound flaps (15). Dna2 displays both 5Ј-3Ј helicase and endonuclease activities (16 -18). Dna2, like FEN1, cleaves a 5Ј flap structure by binding the 5Ј end and tracking toward the base (19). However, Dna2 cleaves multiple times before approaching the base, finally leaving a short flap of ϳ5-10 nt (20). Dna2 is capable of cleaving an RPA-bound flap by displacing the RPA as it tracks (15, 21). Dna2 cleavage ultimately produces a short flap that RPA can no longer bind. FEN1 will then complete cleavage of the short flap, leaving a nick to be sealed by LigI. The importance of Dna2 cleavage is highlighted by the observation that Dna2 nuclease is essential in Saccharomyces cerevisiae (17,22). In the absence of Dna2, it is likely that long RPA-bound flaps cannot be properly processed, leading to genomic instability and cell death.Genetic evidence suggests that Pif1 helicase influences the pathway chosen for flap processing by lengthening displaced flaps. Deletion of PIF1 rescues the lethality of dna2⌬ in S. cerevisiae (23, 24), suggesting that in the absence of Pif1, flaps do not become long enough to require cleavage by Dna2. Our biochemical studies support this conclusion (11,13). U...

Bloom syndrome is a familial genetic disorder associated with sunlight sensitivity and a high predisposition to cancers. The mutated gene, Bloom protein (BLM), encodes a DNA helicase that functions in genome maintenance via roles in recombination repair and resolution of recombination structures. We designed substrates representing illegitimate recombination intermediates formed when a displaced DNA flap generated during maturation of Okazaki fragments escapes cleavage by flap endonuclease-1 and anneals to a complementary ectopic DNA site. Results show that displaced, replication protein A (RPA)-coated flaps could readily bind and ligate at the complementary site to initiate recombination. RPA also displayed a strand-annealing activity that hastens the rate of recombination intermediate formation. BLM helicase activity could directly disrupt annealing at the ectopic site and promote flap endonuclease-1 cleavage. Additionally, BLM has its own strand-annealing and strand-exchange activities. RPA inhibited the BLM strand-annealing activity, thereby promoting helicase activity and complex dissolution. BLM strand exchange could readily dissociate invading flaps, e.g. in a D-loop, if the exchange step did not involve annealing of RPA-coated strands. Use of ATP to activate the helicase function did not aid flap displacement by exchange, suggesting that this is a helicase-independent mechanism of complex dissociation. When RPA could bind, it displayed its own strand-exchange activity. We interpret these results to explain how BLM is well equipped to deal with alternative recombination intermediate structures. Bloom protein (BLM)2 is a member of the RecQ family of 3Ј-5Ј helicases that assist in maintaining genome stability. Mutation or loss of function of the BLM protein causes Bloom syndrome (BS), an autosomal recessive disease characterized by sunlight sensitivity, proportional dwarfism, and a high predisposition toward many different types of cancer (1). Cells with BLM deficiency show increased chromosomal abnormalities, including hyper-recombination, elevated rates of sister chromatid exchange, and the abnormal accumulation of replication intermediates, resulting in an increase in the overall level of genomic instability (2-4). Knock-out of BLM in mice causes embryonic lethality, whereas some mutations produce live mice prone to tumorigenesis (5, 6).BLM plays a role in several critical genome maintenance pathways. Immunodepletion of Xenopus BLM inhibits the replication of DNA in reconstituted nuclei, suggesting that BLM is directly involved in DNA replication (7). Telomere proteins TRF2 and TRF1 colocalize with BLM in immortalized cells lines and regulate its helicase activity in vivo, signifying a role for BLM in telomere maintenance (8). BLM assists in the recovery of stalled replication forks and in the prevention of repeat expansion by stabilizing repeated sequences (9 -13). Additionally, BLM has been proposed to promote proper intermediate resolution and suppress crossovers in the homologous recombination pathwa...

Medium-chain acyl-CoA dehydrogenase deficiency (MCADD) is an autosomal recessive disorder that leads to a defect in fatty acid oxidation. ACADM is the only candidate gene causing MCAD deficiency. A single nucleotide change, c.985A>G, occurring at exon 11 of the ACADM gene, is the most prevalent mutation. In this study, we report a Caucasian family with multiple MCADD individuals. DNA sequence analysis of the ACADM gene performed in this family revealed that two family members showing mild MCADD symptoms share the same novel change in exon 11, c.1052C>T, resulting in a threonine-to-isoleucine change. The replacement is a nonconservative amino acid change that occurs in the C-terminal all-alpha domain of the MCAD protein. Here we report the finding of a novel missense mutation, c.1052C>T (p.Thr326Ile), in the ACADM gene. To our knowledge, c.1052C>T has not been previously reported in the literature or in any of the current databases we utilize. We hypothesize that this particular mutation in combination with p.Lys304Glu results in an intermediate clinical phenotype of MCADD.