Structure-Function Studies Of DNA Replication Fidelity

今年，我们的团队经常与其他人合作，发表了DNA合成保真度的10个方面。 (1)我们确定了动力学的Pol nu插入，错误插入和错配延伸在几个序列的情况下。 我们发现，这种容易出错的人类酶的低保真度主要是由于插入正确的dCTP的催化效率低，而不是错误插入dTTP的效率高。(2)我们测试了在芽殖酵母中Pos delta和Pos delta在复制中的作用是否在裂殖酵母中进化保守。 总体而言，数据表明情况确实如此（3）我们检验了Pol zeta参与SHM的假设。 产生两种Pol z功能的小鼠模型：B细胞特异性条件性敲除品系和编码L2610 F Pol z的敲入品系。 Pol z缺陷型B细胞免疫球蛋白基因座突变频率降低，而L2610 F小鼠突变频率显著增加。 这些数据表明，在SHM的直接作用的Pol z。 (4)我们合作表明，在RNA酶H1和RNA酶H2都不存在的情况下，酵母基因组中rNMP的积累会导致复制应激和毒性。 MMS 2依赖性模板转换和Pol z依赖性旁路都在克服DNA复制期间rNTP错误掺入的这些有害影响中发挥作用。 (5)我们与罗伯特·E。伦敦使用NMR描述含有单个核糖核苷酸的Dickerson DNA十二聚体的溶液结构。 (6)我们研究了在DNA合成过程中如何通过Pol lambda进行rNTP掺入。 这些结构-功能研究提高了对聚合酶如何使用含核糖的底物进行DNA合成的理解，这可能与rNTP：dNTP比率高时在非增殖细胞中发生的修复合成有关。 (7)我们研究了酵母Pol-1的外切核酸酶活性校正新插入的核糖核苷酸的能力。 结果表明，Polymorphism确实校正了新插入的rNMP以增强基因组稳定性，但校正不正确的糖的效率远远低于校正不正确的碱基。 (8)我们的研究描述了一个完整的生化重建的核苷酸切除修复途径与酶纯化芽殖酵母。 (9)我们确定了当酵母Pol zeta绕过酵母基因组中的自发病变时体内产生的突变的特异性。 结果表明，当Pol zeta对内源性螺旋扭曲病变进行诱变旁路时，它可以进行一个短轨道的进行性、易错合成，以产生一系列真正令人惊讶的串联双突变和簇突变。 这一特性可能与多阶段癌变和Pol zeta的进化保守性有关。 结果还表明，串联碱基对取代和多个紧密间隔的突变簇可能是有用的生物标志物，以指示TLS通过Pol zeta在人类肿瘤形成的环境诱变中的作用。 (10)与K合作。Garrish和P. Bushel，我们比较了野生型酵母菌株与rhn 201菌株中的mRNA表达。 RNH 201的缺失使349个基因的RNA表达改变了1.5倍（q值<0.01），其中123个上调，226个下调。 差异表达基因（DEG，在右边）包括那些参与应激反应和基因组维持，与RNA酶H2在复制过程中去除掺入DNA的核糖核苷酸的作用一致。 上调基因包括编码RNA聚合酶I和III亚基的几个基因，以及许多参与核糖体RNA加工和核糖体生物发生以及tRNA修饰和加工的基因，支持RNA酶H2在解析rRNA和tRNA基因转录期间形成的R环中的作用。 几个DEG参与端粒维持，支持RNA酶H2在解决端粒处形成的RNA-DNA杂交中的作用。 大量DEG编码参与应答dsRNA病毒的核酸酶、解旋酶和基因，观察结果可能与在RNA酶H2缺陷型人类中引发先天免疫应答的核酸种类相关。

This year our group, often in collaboration with others, published on 10 aspects of DNA synthesis fidelity. (1) We determined the kinetics of Pol nu insertion, misinsertion and mismatch extension in several sequence contexts. We found that the low fidelity of this error-prone human enzyme is primarily due to low catalytic efficiency for inserting correct dCTP rather than high efficiency for misinserting dTTP. (2) We tested whether the roles of Pols delta and epsilon in replication in budding yeast are evolutionarily conserved in fission yeast. Overall, the data indicate that this is the case (3) We tested the hypothesis that Pol zeta participates in SHM. Two mouse models of Pol z function were generated: a B-cell specific conditional knock-out strain and a knock-in strain encoding L2610F Pol z. Pol z-deficient B-cells had reduced mutation frequency at immunoglobulin loci, whereas L2610F mice had markedly increased mutation frequency. The data indicate a direct role for Pol z in SHM. (4) We collaborated to show that accumulation of rNMPs in the yeast genome in the absence of both RNase H1 and RNase H2 causes replication stress and toxicity. Both MMS2-dependent template switching and Pol z-dependent bypass have roles in overcoming these deleterious effects of rNTP misincorporation during DNA replication. (5) We collaborated with Robert E. London to describe the solution structure of the Dickerson DNA dodecamer containing a single ribonucleotide using NMR. (6) We investigated how rNTP incorporation occurs during DNA synthesis by Pol lambda. These structure-function studies improved an understanding of how polymerases use ribo-containing substrates for DNA synthesis, which may be relevant for repair synthesis occurring in non-proliferating cells when rNTP:dNTP ratios are high. (7) We investigated the ability of the exonuclease activity of yeast Pol epsilon to proofread newly inserted ribonucleotides. The results indicate that Pol epsilon does proofread newly inserted rNMPs to enhance genome stability, but proofreading of an incorrect sugar is substantially less efficient than is proofreading of an incorrect base. (8) We contributed to a study describing a complete biochemical reconstitution of the ribonucleotide excision repair pathway with enzymes purified from budding yeast. (9) We determined the specificity of mutations generated in vivo when yeast Pol zeta bypasses spontaneous lesions in the yeast genome. The results indicate that when Pol zeta performs mutagenic bypass of endogenous, helix-distorting lesions, it can perform a short track of processive, error-prone synthesis to generate a truly amazing array of tandem double and clustered mutations. This property may be relevant to multistage carcinogenesis and the evolutionary conservation of Pol zeta. The results also suggest that tandem base pair substitutions and clusters of multiple, closely spaced mutations may be useful biomarkers to indicate a role for TLS by Pol zeta in environmental mutagenesis underlying tumor formation in humans. (10) In collaboration with K. Garrish and P. Bushel, we compared mRNA expression in a wild type yeast strain to that in a rhn201&#8710; strain. Deleting RNH201 alters RNA expression of 349 genes by 1.5-fold (q-value <0.01), of which 123 are up regulated and 226 are down regulated. Differentially expressed genes (DEGs, on right) include those involved in stress responses and genome maintenance, consistent with a role of RNase H2 in removing ribonucleotides incorporated into DNA during replication. Up-regulated genes include several that encode subunits of RNA polymerase I and III, and many involved in ribosomal RNA processing and ribosomal biogenesis and in tRNA modification and processing, supporting a role for RNase H2 in resolving R-loops formed during transcription of rRNA and tRNA genes. Several DEGs are involved in telomere maintenance, supporting a role for RNase H2 in resolving RNA-DNA hybrids formed at telomeres. A large number of DEGs encode nucleases, helicases and genes involved in response to dsRNA viruses, observations that could be relevant to the nucleic acid species that elicit an innate immune response in RNase H2-defective humans.