DNA Replication, Repair, and Mutagenesis In Eukaryotic And Prokaryotic Cells

真核和原核细胞中的 DNA 复制、修复和诱变

• 批准号: 8736845
• 负责人: ROGER WOODGATE
• 金额: $ 188.65万
• 依托单位: EUNICE KENNEDY SHRIVER NATIONAL INSTITUTE OF CHILD HEALTH & HUMAN DEVELOPMENT
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/8736845
• 关键词: Amino Acids; Archaea; Bacteria; Base Pairing; Binding; C-terminal; Cell Mobility; Cells; Chimera organism; Complex; DNA; DNA Damage; DNA biosynthesis; DNA lesion; DNA polymerase V; DNA-Directed DNA Polymerase; Enzymes; Escherichia coli; Eukaryota; Eukaryotic Cell; Excision; Exhibits; Family; Genome; Genomics; Glycine; Human; In Vitro; Kinetics; Laboratories; Life; Light; Maps; Mediating; Molecular; Monoubiquitination; Mutagenesis; Mutation; Organism; Pathway interactions; Phenotype; Polymerase; Process; Prokaryotic Cells; Property; Proteins; Pyrimidine Dimers; RNA chemical synthesis; Rad30 protein; Resistance; Ribonuclease H; Ribonucleosides; Ribonucleotides; Scientist; Set protein; Site; Testing; Two-Hybrid System Techniques; UV induced; Ubiquitin; Ubiquitination; Ultraviolet Rays; Variant; Zinc Fingers; base; citrate carrier; endonuclease; in vivo; mutant; preference; protein complex; protein protein interaction; repaired; ribonuclease H1; ribonuclease HII; sugar

Scientists within the Laboratory of Genomic Integrity (LGI) study the mechanisms by which mutations are introduced into DNA. These studies span the evolutionary spectrum and include studies in bacteria, archaea and eukaryotes Most damage-induced mutagenesis in Escherichia coli is dependent upon the UmuD'C protein complex, which comprises DNA polymerase V (pol V). We recently discovered that pol V is also characterized by substantially reduced sugar selectivity. When the canonical Watson-Crick base pairing is preserved, purified pol V accompanied by accessory proteins, readily incorporates all ribonucleotides (ribonucleoside monophosphates, rNMPs) and catalyzes efficient and highly processive RNA synthesis in vitro in the presence of all four rNTPs. The ability of pol V to incorporate ribonucleotides is dramatically enhanced by a Y11A substitution at the conserved steric gate residue of UmuC. Interestingly, while UmuC_Y11A is highly inaccurate in vitro, it exhibits low mutability in vivo. Furthermore, despite the observation that the UmuC_Y11A variant catalyzed TLS past a T-T cyclobutane pyrimidine dimer (CPD) in vitro at least as efficiently as the wild-type enzyme, it conferred minimal UV-resistance to a delta umuDC strain. To explain these phenotypes, we suggested that the dramatic increase in rNMP incorporation promoted by UmuC_Y11A leads to the induction of downstream pathways involving rNMP processing. Specifically, that the rNMP-targeted repair pathways would not only reduce umuC_Y11A-dependent spontaneous and UV-induced mutagenesis, but also interfere with completion of TLS resulting in the observed decrease in UV resistance. The major enzymes initiating this pathway are ribonucleotide-specific endonucleases, Ribonuclease H (RNase H), which are present in organisms across all domains and are classified as types 1 and 2 based on sequence conservation and substrate preference. By taking advantage of the different capacities for ribonucleotide incorporation by the UmuC_Y11A pol V variant, we examined rNMP-processing pathways that cause phenotypic changes in strains expressing the pol V variants. While there was an 4-fold increase in the absolute number of Y11A-dependent mutations in the delta rnhA strain (lacking RNase HI) compared to the rnh+ strain, when expressed as a percentage of wild-type pol V-dependent mutagenesis, umuC_Y11A mutagenesis actually decreased from 7 to 5% of the wild-type levels. In contrast, in the isogenic delta rnhB strain (lacking RNase HII), the number of umuC_Y11A-dependent revertants increased approximately 5-fold compared to the rnhB+ strain and reached 40% of the level of mutagenesis observed with wild-type pol V. Our studies clearly demonstrated that RNase HII participates in a repair pathway that reduces the accumulation of rNMPs, as well as incorrect dNMPs incorporated into undamaged and damaged DNA by UmuC_Y11A. Based upon its in vitro properties, we expected pol V umuC_Y11A to be as mutagenic, if not more so, than the wild-type pol V, but even in the delta rnhB strain, Y11A-dependent mutagenesis was less than half of that observed with wild-type pol V, indicating that additional repair pathways act to reduce the mutagenic consequences of rNMPs incorporated by the highly error-prone umuC_Y11A. Indeed, in the isogenic delta rnhA delta rnhB strain umuC_Y11A spontaneous mutagenesis increased significantly to 72% of the level observed with wild-type pol V. Thus, our studies revealed that although RNase HI alone does not appear to participate in the removal of ribonucleotides incorporated by umuC_Y11A, in the absence of RNase HII, where there is likely to be a significant accumulation of ribonucleotides into DNA, RNase HI helps reduce the mutagenic burden of errant ribonucleotide incorporation into the E.coli genome. Our studies on the human TLS polymerases focused on DNA polymerase eta and iota. Both polymerases (pols) co-localize in replication factories in vivo after cells are exposed to UV-light and this co-localization is mediated through a physical interaction between the two TLS pols. The regions responsible for the interaction were previously loosely mapped to the C-terminal 200 amino acids of each protein. Although the two polymerases clearly co-localize at sites of DNA damage, the kinetics of their re-localization differs, suggesting that the two polymerases are not tightly associated in a living cell. Our studies begin to shed light on how such an interaction is facilitated and regulated. We identified the region in pol eta responsible for the interaction with pol iota as its ubiquitin binding zinc finger (UBZ) motif. Similarly, we demonstrated that the region responsible for the interaction between pol iota and pol eta is pol iotas ubiquitin binding motif (UBM). Since pol iota is also known to be monoubiquitinated in vivo, we hypothesized that the preferred partner of pol eta might actually be a ubiquitinated form of pol iota To test this hypothesis, we generated a chimera in which the N-terminus of ubiquitin was fused to the C-terminus of pol iota The mutant chimera lacked the two C-terminal glycine residues, and therefore only allowed for non-covalent interactions. The chimera interacted avidly with pol eta in the two-hybrid assays and this interaction was dependent upon I44 of ubiquitin (in the pol iota-Ub chimera). When expressed in human HEK293T cells the pol iota-Ub chimera was also able to pull-down considerably more pol eta than wild-type pol iota. We therefore concluded that the preferred partner for pol eta is a ubiquitinated form of pol iota The functional importance of the pol eta-iota interaction was clearly demonstrated by the fact that mutants of pol iota that are unable to interact with pol eta exhibit reduced accumulation of into replication factories. Conversely, the pol iota-Ub chimera, which exhibited a tighter interaction with pol eta showed an enhanced accumulation into replication foci. Given the complex set of protein-protein interactions that pol eta and pol iota are known to participate in, it seemed reasonable to predict that the ubiquitination status of the pols allows a cell a variety of ways to regulate the formation of TLS complexes. For example, monoubiquitination of pol eta is known to inhibit an interaction with ubiquitinated PCNA, but we demonstrated, it enhances its interaction with pol iota. Upon DNA damage, pol eta is de-ubiquitinated and this leads to a reduced ability to interact with pol iota, but a concomitant increased ability to interact with ubiquitinated PCNA, which helps explain why the polymerases exhibit different sub-cellular mobility in a living cell.

基因组完整性实验室（LGI）中的科学家研究了将突变引入DNA的机制。 这些研究跨越了进化光谱，包括细菌，古细菌和真核生物的研究 大肠杆菌中大多数损伤诱导的诱导诱导的诱变取决于UMUD'C蛋白复合物，该蛋白复合物包括DNA聚合酶V（POL V）。我们最近发现，POL V的特征也大大降低了糖选择性。当保留典型的Watson-Crick基底配对时，纯化的POL V并伴有辅助蛋白，很容易融合所有核糖核苷酸（核糖核苷单磷酸盐，RNMP），并在所有四个RNTP的情况下在体面的体面促进效率和高度处理的RNA合成。 POL V合并核糖核苷酸的能力通过在UMUC的保守空间栅极残基处的Y11A取代而大大提高。 有趣的是，尽管UMUC_Y11A在体外高度不准确，但体内表现出低的可突变性。此外，尽管观察到UMUC_Y11A变体催化TLS经过T-T环丁烷嘧啶二聚体（CPD）的体外效率至少与野生型酶一样有效，但它赋予了Deltaumudc菌株的最小UV-UV抗性。为了解释这些表型，我们建议由UMUC_Y11A促进的RNMP掺入的急剧增加导致诱导涉及RNMP处理的下游途径。具体而言，以RNMP为目标的修复途径不仅会降低UMUC_Y11A依赖性自发性和紫外线诱导的诱变，而且会干扰TLS的完成，从而导致观察到的紫外线耐药性降低。 启动该途径的主要酶是核糖核苷酸特异性内核酸酶，核糖珠酶H（RNase H），它们存在于所有域的生物体中，并根据序列保护和底物偏好而归类为1和2类型。 通过利用UMUC_Y11A POL V变体的核糖核苷酸掺入的不同能力，我们检查了RNMP处理途径，从而导致表达POL V变体的菌株的表型变化。 与RNH+菌株相比，Delta RNHA菌株中Y11a依赖性突变的绝对数量增加了4倍，而当野生型POL V依赖性诱变的百分比表示时，UMUC_Y11A mutagenesis实际上从野生型的7％降低到5％。相比之下，与RNHB+菌株相比，在等源性DELTA RNHB菌株（缺少RNase HII）中，UMUC_Y11A依赖性逆转的数量增加了约5倍，并达到了用野生型Pol V观察到的诱变水平的40％。 我们的研究清楚地表明，RNase HII参与了减少RNMP积累的修复途径，以及UMUC_Y11A纳入未损坏和损坏的DNA中的不正确的DNMP。 Based upon its in vitro properties, we expected pol V umuC_Y11A to be as mutagenic, if not more so, than the wild-type pol V, but even in the delta rnhB strain, Y11A-dependent mutagenesis was less than half of that observed with wild-type pol V, indicating that additional repair pathways act to reduce the mutagenic consequences of rNMPs incorporated by the highly error-prone UMUC_Y11A。 实际上，在等源性的delta rnha delta rnhb菌株UMUC_Y11A自发性诱变显着增加到野生型Pol V所观察到的水平的72％。因此，尽管RNase HI单独使用rnase HI似乎并未参与其中的核糖核苷酸含量，而在其中，可能会增加Is i is i is i is i i i is i imuc_y11a，而umuc_y11a可能会出现rn的含量，而rnase hi可能会出现，而rnase hi可能会出现umuc_y11a，而rnase hi可能存在，则rnase hi可能会出现rnase hi。核糖核苷酸进入DNA，RNase HI有助于减轻核糖核苷酸掺入大肠杆菌基因组的诱变负担。 我们对人类TLS聚合酶的研究集中在DNA聚合酶ETA和IOTA上。两种聚合酶（POLS）在细胞暴露于紫外线后体内复制工厂共定位，并通过两个TLS POL之间的物理相互作用介导这种共定位。 先前将负责相互作用的区域松散地映射到每种蛋白质的C端200氨基酸。 尽管两种聚合酶显然在DNA损伤部位共定位，但它们重新定位的动力学有所不同，这表明两个聚合酶在活细胞中没有密切相关。 我们的研究开始阐明这种相互作用是如何促进和调节的。 我们确定了负责与POL IOTA相互作用的POL ETA中的区域，为其泛素结合锌指（UBZ）基序。 同样，我们证明了负责POL IOTA和POL ETA之间相互作用的区域是Pol iotas ubiquitin结合基序（UBM）。 Since pol iota is also known to be monoubiquitinated in vivo, we hypothesized that the preferred partner of pol eta might actually be a ubiquitinated form of pol iota To test this hypothesis, we generated a chimera in which the N-terminus of ubiquitin was fused to the C-terminus of pol iota The mutant chimera lacked the two C-terminal glycine residues, and therefore only allowed for非共价相互作用。 嵌合体与两个杂交测定法中的POL ETA相互作用，这种相互作用取决于泛素的I44（在pol iota-ub Chimera中）。 当在人类HEK293T细胞中表达时，Pol Iota-ub Chimera也能够比野生型Pol Iota更重要。 因此，我们得出的结论是，Pol Eta的首选合作伙伴是Pol Iota的泛素化形式 Pol Eta-iota相互作用的功能重要性通过以下事实清楚地证明了这一事实，即无法与Pol Eta相互作用的突变体显示出降低的复制工厂的积累。相反，与POLETA表现出更严格的相互作用的POL IOTA-UB嵌合体显示出增强的复制灶的积累。 鉴于已知POL ETA和POL IOTA参与的复杂蛋白质 - 蛋白质相互作用，因此预测POLS的泛素化状态似乎可以通过多种方式调节TLS复合物的形成。例如，已知POL ETA的单泛素化抑制与泛素化PCNA的相互作用，但我们证明了它增强了其与Pol Iota的相互作用。在DNA损伤后，POL ETA被去泛素化，这导致与POL IOTA相互作用的能力降低，但同时增加了与泛素化PCNA相互作用的能力，这有助于解释为什么聚合酶在活细胞中表现出不同的亚细胞迁移率。