Mechanisms Of Genome Instability

近三十年来，归因于存在于许多DNA聚合酶中的固有3 'y 5'外切核酸酶（Exo）活性的唯一生物学功能是校正DNA合成期间的错误以防止点突变。我们的研究已经确定了DNA聚合酶d Exo的一种新的生物学功能：它可以补充或备份Rad 27/Fen 1 5 '-flap内切核酸酶，以在滞后链复制期间在相邻的Okazaki片段之间产生可连接的切口。 在酵母中，Pod Exo缺陷和部分rad 27/fen 1缺陷（rad 27-p）导致强烈的负相互作用，如大重复率以及染色体内和染色体间重组的协同增加所示。菌株组合各种Pod Exo突变和rad 27/fen 1缺陷需要野生型双链断裂（DSB）修复基因，这表明DSB的积累。在双突变菌株以及过表达核酸外切酶缺陷Pol 3 -01蛋白的单rad 27突变体中观察到Pod Exo和rad 27/fen 1缺陷之间的负相互作用。我们提出，增加的重复率和其他协同相互作用的Pol d Exo缺陷与Rad 27/Fen 1的缺陷可以解释为增加的能力的Pol d Exo缺陷酶链置换，导致增加皮瓣形成在落后的链。与Okazaki片段成熟一样，碱基切除修复也依赖于Pod和FEN 1的协同作用，在pol 3-exo-rad 27双突变体中同样严重受损，如其对MMS的极端敏感性所示. 链置换的作用进行了评估与纯化的野生型和突变体Pod全酶。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，Exo缺陷型Pod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，而且可以用于避免在滞后链复制期间的过度链置换，从而防止DNA双链断裂和基因组重排。 冈崎片段成熟的当前模型提出了通过Pol δ外切核酸酶的Pol δ_3 '-5'降解以及通过FEN 1和Dna 2的5 '-内切核酸酶活性的置换链的降解的协同链置换。我们已经研究了这个过程中使用野生型Pol δ和外切缺陷突变体的Pol δ链置换能力增加。增加的立场位移主要是由于增加的启动事件，而不是伸长率。在FEN 1存在下，Pol δ-外切-切口-翻译比野生型Pol δ更有效。在我们的系统中的模型冈崎片段的成熟的最佳速率要求，除了DNA连接酶需要10倍过量，所有的因素都存在与DNA的化学计量。在这些条件下，在连接之前，通过RNA/DNA接合处的切口平移对于野生型Pol δ仅发生约5 nt，而对于外切突变酶仅发生8-10 nt。 在这个成熟过程中，DNA 2没有显著的作用，除了当存在30 nt的5 '-瓣时，如先前其他人所证明的。我们提出，Dna 2在冈崎片段成熟中的作用是在极少数情况下进行拯救，此时Pol delta的链置换不伴有FEN 1的5 '-内切核酸酶活性和/或Pol delta的3'-5 '外切核酸酶活性。补充的遗传实验支持这一模型。pol 3-外切突变显示出与部分FEN 1缺陷（rad 27-p）的强协同相互作用。这些突变体也需要双链断裂（DSB）修复系统的增长，这表明积累的DSB。Dna 2的过表达挽救了由DSB修复缺陷引起的致死性。 敏感的遗传系统和风险基序报告已被用于开发一个敏感的筛选环境因素抑制DNA代谢成分，抑制基因组的不稳定性。目前正在审查各种因素。筛选解决DNA聚合酶校对和各种类型的DNA修复。一旦在酵母中鉴定出因子，将使用本实验室的细胞系和设施，并通过与泰勒实验室的合作，在培养的人类细胞中对它们进行评估。 已经对参与异常复制中间体（断裂、停滞的分叉等）修复的基因进行了全基因组筛选。分析发现的基因对DNA损伤敏感性的影响，预防由风险基序（Alu反向重复序列）引起的高度不稳定性，以及与DNA修复中已知缺陷的相互作用，以确定突变损伤的途径。

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.