Mechanisms Of Genome Instability

近三十年来，许多DNA聚合酶中固有的3‘Y5’外切酶(Exo)活性的唯一生物学功能是纠正DNA合成过程中的错误，以防止点突变。我们的研究发现了DNA聚合酶dExo的一种新的生物学功能：在滞后的链复制过程中，它可以补充或支持Rad27/Fen15‘-Flat内切酶，在相邻的Okazaki片段之间产生可连接的缺口。 在酵母中，Pold Exo缺乏和部分rad27/fen1缺陷(rad27-p)导致强烈的负相互作用，表现为大复制率以及染色体内和染色体间重组的协同增加。结合各种Pold Exo突变和rad27/fen1缺陷的菌株需要野生型双链断裂(DSB)修复基因，这表明DSB积累。Pold Exo与rad27/fen1缺陷之间存在负相互作用，与双突变株和过表达核酸外切酶缺陷Pol3-01蛋白的单株rad27突变株均存在负相互作用。我们认为，Pold Exo缺陷与Rad27/Fen1缺陷的复制率增加和其他协同作用可以通过Pold Exo缺陷酶对链置换的能力增加导致滞后链中的瓣形成增加来解释。碱基切除修复，与Okazaki片段的成熟一样，也依赖于Pold和FEN1的协同作用，同样在pol3-exo-rad27双突变体中严重受损，这表明它对MMS非常敏感。 用纯化的野生型和突变型Pold全酶评价链置换的作用。一种基于自退火寡核苷酸的新方法使我们能够高效地创建用于链置换实验的引物-模板底物。我们发现外源缺失的Pold在置换合成中比野生型酶更有效。因此，Pol d Exo活性不仅可以用于纠正复制错误，还可以避免在滞后链复制过程中过度的链移位，从而防止DNA双链断裂和基因组重排。 目前的Okazaki片段成熟模型认为，通过Pol Delta外切酶降解Pol Delta_3‘-5’来协调链置换，通过FEN1和DNA2的5‘-内切酶活性来降解被移位的链。我们使用野生型Pol Delta和外源缺失的Pol Delta突变体研究了这一过程，该突变体具有更大的链置换能力。林分位移的增加主要是由于起始事件的增加，而不是延伸率的增加。在FEN1存在的情况下，Pol Delta-exo-Nick翻译比野生型Pol Delta更有效。在我们的系统中，模型Okazaki片段的最佳成熟率要求所有因素都以化学计量的形式存在于DNA中，除了DNA连接酶，它需要10倍的过剩。在此条件下，野生型Pol Delta在连接前仅发生了~5nT的缺口平移，而外源突变酶仅发生了8~10nT的缺口转移。 DNA2在这一成熟过程中没有明显的作用，除非有30个核苷酸的5‘-瓣存在，正如其他人所证明的那样。我们认为，DNA2在冈崎片段成熟过程中的作用是在极少数情况下拯救Pol Delta的链置换，而不伴随FEN1的5‘-内切酶活性和/或Pol Delta的3’-5‘外切酶活性。互补的基因实验支持这一模型。Pol3-exo突变与部分FEN1缺陷(rad27-p)显示出很强的协同作用。这些突变体还需要双链断裂(DSB)修复系统才能生长，这表明DSB的积累。DNA2的过度表达挽救了DSB修复缺陷造成的致命性。 敏化的遗传系统和高危基序记者已被用来开发一种敏感的筛选，以寻找抑制DNA代谢成分的环境因素，这些因素抑制基因组的不稳定性。目前正在对各种因素进行审查。筛查涉及DNA聚合酶校对和各种类型的DNA修复。一旦酵母中的因素被确定，它们将在培养的人类细胞中使用这个实验室的线路和设施进行评估，并通过与泰勒实验室的合作努力。 已经对参与异常复制中间产物(断裂、停滞的叉子等)修复的基因进行了全基因组筛查。分析发现的基因对DNA损伤的敏感性，防止由高危基序(Alu反向重复)引起的超不稳定，以及与DNA修复中已知缺陷的相互作用，以确定被突变破坏的途径。

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.