Okazaki Fragment Maturation in Yeast

Abstract

To address the different functions of Pol ␦ and FEN1 (Rad27) in Okazaki fragment maturation, exonucleasedeficient polymerase Pol ␦-01 and Pol ␦-5DV (corresponding to alleles pol3-01-(D321A, E323A) and pol3-5DV-(D520V), respectively) were purified and characterized in this process. In the presence of the replication clamp PCNA, both wild-type and exo ؊ Pol ␦ carried out strand displacement synthesis with similar rates; however, initiation of strand displacement synthesis was much more efficient with Pol ␦-exo ؊ . When Pol ␦-exo ؊ encountered a downstream primer, it paused with 3-5 nucleotides of the primer displaced, whereas the wild type carried out precise gap filling. Consequently, in the absence of FEN1, Pol ␦ exonuclease activity was essential for closure of simple gaps by DNA ligase. Compared with wild type, Okazaki fragment maturation with Pol ␦-exo ؊ proceeded with an increased duration of nick translation prior to ligation. Maturation was efficient in the absence of Dna2 and required Dna2 only when FEN1 activity was compromised. In agreement with these results, the proposed generation of double strand breaks in pol3-exo ؊ rad27 mutants was suppressed by the overexpression of DNA2. Further genetic studies showed that pol3-exo ؊ rad27 double mutants were sensitive to alkylation damage consistent with an in vivo defect in gap filling by exonucleasedeficient Pol ␦.Efficient and faithful maturation of Okazaki fragments during DNA replication in eukaryotes depends on a coordinated degradation of the RNA primer strand by one or more nucleases along with gap-filling DNA synthesis by a replicative DNA polymerase followed by ligation of the remaining nick. Previous models based on a combination of biochemical and genetic studies have indicated a role for the flap 5Ј-endonuclease FEN1 and the nuclease/helicase Dna2 in carrying out degradation including the removal of a displaced flap and a role for DNA polymerase ␦ (Pol ␦) 1 to carry out DNA synthesis (1). However, biochemical experiments in the accompanying paper (3) indicate that the main degradative force is provided by FEN1. The activity of Dna2 becomes crucial only in cases where strand displacement proceeds to the extent that proteins inhibitory to FEN1 can bind to the displaced 5Ј-strand (2, 3). Many DNA polymerases have an intrinsic 3Ј-5Ј exonuclease activity, which corrects polymerase errors and prevents mutations. Recently, we provided genetic evidence for the action of the 3Ј-5Ј-exonuclease of Pol ␦ in the process of Okazaki fragment maturation in vivo (4, 5). This was indicated by synthetic lethality of rad27 (FEN1) mutants with several exodeficient mutants in Pol ␦ and by a dramatic increase in duplication mutations in viable pol3-exo Ϫ rad27 double mutants. We have suggested that the 3Ј-5Ј-exonuclease could be specifically involved in preventing the excessive formation of 5Ј-flaps by strand displacement synthesis.Okazaki fragment maturation is mediated by the concerted strand displacement of Pol ␦ and degradation of the displaced strand by the nucleas...

The human Bloom syndrome gene suppresses the DNA replication and repair defects of yeastdna2mutants

“…Therefore, it is conceivable that the hBLM͞scFEN1 interaction at the replication fork leads to either enhanced cleavage by scFEN1 or enhanced recruitment of scFEN1 to the flap, effectively raising the local concentration of the protein and reducing the need for scDna2. Suggesting that some component of the maturation system, such as a helicase, may be missing in vitro, pol ␦ cannot strand displace rapidly enough in a reconstituted model system containing purified pol ␦, proliferating cell nuclear antigen, RFC, FEN1, Dna2, and ligase to account for rates of Okazaki fragment maturation in vivo (25,26). Thus, hBLM might stimulate flap formation.…”

“…In vitro reconstitution of the processing reaction showed that, if the flap was Ͼ30 nt, then Dna2 was required to stimulate the endonucleolytic activity of FEN1; however, Dna2 had no effect if the flap was Ͻ30 nt (24,25). Thus, Dna2 is likely to be essential for Okazaki fragment maturation only in cases where FEN1 activity is somehow impaired (25,26).…”

“…In vitro reconstitution of the processing reaction showed that, if the flap was Ͼ30 nt, then Dna2 was required to stimulate the endonucleolytic activity of FEN1; however, Dna2 had no effect if the flap was Ͻ30 nt (24,25). Thus, Dna2 is likely to be essential for Okazaki fragment maturation only in cases where FEN1 activity is somehow impaired (25,26).Dna2 is conserved in primary sequence from yeast to human (23,27,28). In Xenopus, depletion of Xenopus laevis DNA2 (xDNA2) from egg extracts leads to inhibition of DNA replication, and Caenorhabditis elegans dna2 mutants appear to be replication-deficient (29), so the function of Dna2 in DNA replication is also conserved (27).…”

“…Yeast dna2 mutants are sensitive to x-rays, methyl methanesulfonate (MMS), bleomycin, and hydroxyurea (HU) (15,16). The mechanism by which Dna2 participates in base excision repair (MMS sensitivity) may be related to its role in DNA replication, because reconstitution of the repair reaction requires the same proteins as those involved in Okazaki fragment maturation (26). Some hypomorphic alleles of dna2 induce high levels of recombination in the yeast rDNA, and yeast dna2 mutants have short lifespans (12).…”

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

“…pol ␦ displaces relatively short flaps, which are cleaved by FEN1 as they are created, leaving a nick for LigI (7)(8)(9). FEN1 binds at the 5Ј-end of the flap and tracks down the flap cleaving only at the base (5,10,11).…”