For nearly three decades, the only biological function attributed to the intrinsic 3'y5' exonuclease (Exo) activity present in many DNA polymerases was the correction of errors during DNA synthesis so as to prevent point mutations. Our study has identified a novel biological function of the DNA polymerase d Exo: it can supplement, or backup, the Rad27/Fen1 5'-flap endonuclease to create ligatable nicks between adjacent Okazaki fragments during lagging strand replication. In yeast, Pold Exo deficiency and a partial rad27/fen1 defect (rad27-p) result in strong negative interactions as shown by synergistic increases in rates of large duplications as well as intra- and interchromosomal recombination. Strains combining various Pold Exo mutations and a rad27/fen1 deficiency required the wild type double strand break (DSB) repair genes, suggesting accumulation of DSBs. A negative interaction between Pold Exo and rad27/fen1 defects was observed with the double mutant strains as well as with single rad27 mutants overexpressing the exonuclease-deficient Pol3-01 protein. We proposed that increased rate of duplications and other synergistic interactions of Pol d Exo defect with a deficiency in Rad27/Fen1 can be explained by an increased capacity of the Pold Exo-deficient enzyme for strand displacement leading to increased flap formation in the lagging strand. Base excision repair, which like Okazaki fragment maturation is also dependent on the concerted action of Pold and FEN1, is likewise heavily compromised in a pol3-exo- rad27 double mutant as indicated by its extreme sensitivity to MMS. The role of strand displacement was assessed with purified wild type and mutant Pold holoenzymes. A new approach based upon self-annealed oligonucleotides allowed us to create primer-template substrates for strand displacement experiments with high efficiency. We found that Exo deficient Pold is more effective in displacement synthesis than the wild-type enzyme. Thus, Pol d Exo activity can serve not only for correcting replication errors, but also for avoiding excessive strand displacement during lagging strand replication, thereby preventing DNA double-strand breaks and genome rearrangements. Current models of Okazaki fragment maturation propose concerted strand-displacement by Pol delta__3'-5' degradation by Pol delta exonuclease and the degradation of the displaced strand by the 5'-endonuclease activities of FEN1 and Dna2. We have studied this process using both wild type Pol delta and an exo-deficient mutant of Pol delta with increased capacity for strand displacement. The increased stand displacement was primarily due to increased initiation events rather than elongation rate. In the presence of FEN1, Pol delta-exo- nick-translation is more efficient than wild type Pol delta. The optimal rate of maturation of a model Okazaki fragment in our system required that all factors were present stoichiometrically with DNA, except for DNA ligase which required a 10-fold excess. Under these conditions, nick translation past the RNA/DNA junction prior to ligation occurred for only ~5 nt with wild type Pol delta, and 8-10 nt with the exo- mutant enzyme. No significant role for Dna2 could be demonstrated in this maturation process, except when 5'-flaps of 30 nt were present, as previously demonstrated by others. We propose that the role of Dna2 in the maturation of Okazaki fragments is to rescue in rare cases when strand displacement by Pol delta has gone unaccompanied by the 5'-endonuclease activity of FEN1 and/or 3'-5' exonuclease activity of Pol delta. Complementary genetic experiments support this model. pol3-exo- mutations exhibit strong synergistic interactions with a partial FEN1 defect (rad27-p). These mutants also required the double-strand break (DSB) repair system for growth, suggesting accumulation of DSBs. Overexpression of Dna2 rescues the lethality caused by a DSB repair defect. Sensitized genetic systems and at-risk motif reporters have been used to develop a sensitive screen for environmental factors inhibiting DNA metabolic components that suppress genome instability. A variety of factors are now being examined. The screening addresses DNA polymerase proofreading and various types of DNA repair. Once factors have been identified in yeast, they will be assessed in cultured human cells using lines and facilities in this lab and through a collaborative effort with the Taylor lab. Genome wide screen has been performed for the genes involved in the repair of aberrant replication intermediates (breaks, stalled forks, etc.). Genes found are analyzed for their effects on sensitivity to DNA damage, prevention of hyper-instability caused by at-risk motifs (Alu inverted repeats), and interactions with known defects in DNA repair in order to identify pathways damaged by a mutation.
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