Structure-Function Studies Of DNA Replication Fidelity

今年，我们的团队经常与其他人合作，就DNA合成保真度的10个方面发表了文章。(1)我们测定了在几种序列背景下Pol nu插入、错插和错配延伸的动力学。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POLS Delta和epsilon在芽殖酵母复制中的作用是否在分裂酵母中进化保守。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。建立了两种Pol z功能的小鼠模型：B细胞特异性条件性敲除株和编码L2610F Pol z的敲入株，Pol z缺陷的B细胞免疫球蛋白基因突变频率降低，而L2610F小鼠的突变频率显著增加。数据表明，Pol z在SHM中起到了直接作用。(4)我们合作表明，在缺乏RNase H1和RNase H2的情况下，rNMPs在酵母基因组中的积累会导致复制压力和毒性。依赖于MMS2的模板切换和依赖于POLZ的旁路都在克服DNA复制过程中rNTP误掺入的有害影响方面发挥了作用。(5)我们与Robert E.London合作，用核磁共振技术描述了含有单个核苷酸的Dickerson DNA十二聚体的溶液结构。(6)研究了Pol lambda在DNA合成过程中rNTP的掺入情况。这些结构-功能研究提高了对聚合酶如何利用含有核糖核酸的底物进行DNA合成的理解，这可能与当rNTP：dNTP比率较高时非增殖细胞中发生的修复合成有关。(7)研究了酵母菌Pol epsilon的核酸外切酶活性对新插入核苷酸的校对能力。结果表明，Pol epsilon确实校对了新插入的rNMPs以增强基因组的稳定性，但校对错误的糖的效率远远低于校对错误的碱基。(8)我们参与了一项研究，该研究描述了用从萌芽酵母中纯化的酶对核苷酸切除修复途径进行完全的生化重建。(9)我们确定了当酵母Pol Zeta绕过酵母基因组中的自发损伤时在体内产生的突变的特异性。结果表明，当Pol Zeta对内源性、螺旋扭曲的病变进行突变旁路时，它可以进行一段短的过程性、容易出错的合成，以产生真正令人惊叹的串联双突变和簇状突变阵列。这一特性可能与Pol Zeta的多阶段致癌和进化保护有关。结果还表明，串联碱基对替换和多个密集突变的簇可能是有用的生物标记物，表明Pol Zeta的TLS在人类肿瘤形成的环境突变中所起的作用。(10)在与K.Garrish和P.bushel的合作中，我们比较了野生型酵母菌株和rhn201&8710；菌株中的mRNA表达。RNH201的缺失改变了349个基因的RNA表达，改变了1.5倍(Q值为0.01)，其中123个基因上调，226个基因下调。差异表达基因(DGs，右侧)包括那些参与应激反应和基因组维护的基因，这与RNaseH2在复制过程中移除DNA中包含的核苷酸的作用一致。上调的基因包括几个编码RNA聚合酶I和III亚基的基因，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RNaseH2在解决rRNA和tRNA基因转录过程中形成的R-环的作用。几个DEG参与端粒的维持，支持RNaseH2在解析端粒形成的RNA-DNA杂合体中的作用。大量的DEG编码核酸酶、解旋酶和参与dsRNA病毒反应的基因，观察到的结果可能与在RNaseH2缺陷人类中引发先天性免疫反应的核酸物种有关。

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.