Forces and Long-Distance Coupling along DNA in the Mismatch Repair (MMR) Pathway

错配修复 (MMR) 途径中沿 DNA 的力和长距离耦合

• 批准号: 8783242
• 负责人: Eric Alan Josephs
• 金额: $ 5.15万
• 依托单位: DUKE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-09-01 至 2016-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8783242
• 关键词: Address; Affect; Atomic Force Microscopy; Base Pair Mismatch; Base Pairing; Behavior; Binding; Biological Assay; Biomedical Research; Cells; Chromatin Loop; Cleaved cell; Complex; Coupled; Coupling; DNA; DNA Repair Pathway; DNA-Protein Interaction; Defect; Development; Diffuse; Disputes; Distant; Ensure; Enzymatic Biochemistry; Equilibrium; Escherichia coli; Excision; Exonuclease; Fluorescence; Fluorescence Microscopy; Genome; Genome Stability; Genomics; Hereditary Disease; Hereditary Nonpolyposis Colorectal Neoplasms; Homologous Gene; Human; Individual; Kinetics; Left; Life; Light; Maintenance; Malignant Neoplasms; Maps; Measures; Methods; Mismatch Repair; Modeling; Molecular; Motion; Mutation; Nucleotides; Organism; Pathway interactions; Phosphodiesterase I; Physiological; Predisposition; Proteins; Replication Error; Research; Role; Scanning; Site; Techniques; dimer; endonuclease; helicase; insertion/deletion mutation; insight; particle; protein complex; public health relevance; research study; single molecule; tool

DESCRIPTION (provided by applicant): In E. coli, genomic stability is maintained by a methyl-directed mismatch repair (MMR) pathway which reduces errors such as mismatched base-pairs or nucleotide insertions/deletions in newly- replicated DNA molecules by 1000-fold. Defects in the homologues of E. coli MMR proteins in humans are associated with increased rates of cancer development, and as E. coli MMR is among the best-studied DNA repair pathways, elucidation of its molecular mechanisms promises to shed new light into mutation avoidance within the cell. The MMR pathway is initiated when complexes of protein MutS identify and bind to a replication error (RE) on a newly-replicated DNA molecule and, with protein MutL, activate endonuclease MutH. MutH then nicks the newly-replicated strand at a hemi-methylated d(GATC) site that serves to discriminate between the new and original strands. This site can be over 1000 base-pairs away with no apparent directional bias (5'- or 3'- from the RE), but 3'-to-5' helicase UvrD is then loaded at the nick toward the RE and with the appropriate exonuclease removes the newly-replicated strand through the RE to be re- synthesized correctly. While the roles of individual MMR-associated proteins (MAPs) are well- established, how different MAP complexes efficiently coordinate the GATC-to-RE excision across large spans of DNA is still the subject of debate. Under physiological conditions MutS exists in an equilibrium of dimeric and tetrameric complexes, the latter having been recently observed on ''looped'' DNA molecules with REs; although the role of MutS tetramers is disputed, the looping of DNA by MutS tetramers is proposed to be how a RE is 'coupled' to a distant d(GATC) site- that is, how a d(GATC) site near the RE can be efficiently found and how excision is limited to the DNA between the two sites. This hypothesis regarding the role of DNA looping by MutS tetramers will be investigated by the following specific aims: (1) To directly measure the sequence- and error-specific binding forces between DNA and MAP complexes as the pathway progresses. The search for REs then d(GATC) sites by MAP complexes will be investigated at the single-molecule (s.m.) level using force spectroscopic (FS) techniques and a nanotechnological FS apparatus. Combination with fluorescence microscopy will allow us to determine the roles of specific MAP complexes, resolve the effects of co-factors on their behavior, and map their spatio-temporal interactions along DNA. (2) To elucidate the mechanistic details of RE-to-d(GATC) ''coupling'' and its relationship to DNA excision. Whether and how DNA looping alters the kinetics / efficiency of d(GATC) nicking, biased directional loading of UvrD, or excision termination will be addressed via atomic force microscopy, tethered particle motion experiments, and s.m. fluorescence assays.

描述（由申请人提供）：在大肠杆菌中，基因组稳定性通过甲基指导的不匹配修复（MMR）途径维持，该途径减少了新抑制的DNA分子中的错误碱基对或核苷酸插入/缺失等误差。人类大肠杆菌MMR蛋白的同源物缺陷与癌症发展速率的增加有关，并且由于大肠杆菌MMR是研究最佳的DNA修复途径之一，因此阐明其分子机制有望在细胞内避免新的光转向突变。当蛋白质MUT的络合物识别并结合新复制的DNA分子上的复制误差（RE）时，就开始使用MMR途径。然后，Muth在半甲基化的D（GATC）部位上划分了新恢复的链，该链可区分新的和原始的链。该站点可以超过1000个碱基对，没有明显的方向性偏置（从RE到RE的5'-或3'-），但是然后将3'至5'Helicase UVRD装载在Nick朝向RE的nick上，并使用适当的外核酸酶移除，将新核酸酶拆下，可以正确地合成新的RE，以正确地合成。尽管已经确定了单个MMR相关蛋白（MAP）的作用，但不同的MAP复合物如何有效地协调大量DNA的GATC至RE切除，仍然是争论的主题。在生理条件下，Muts存在于二聚体和四聚体配合物的平衡中，最近在带有RES的“循环” DNA分子上观察到了后者。尽管有争议MUTS四聚体的作用，但建议通过MUTS四聚体对DNA进行循环，认为RE是如何“耦合”到遥远的D（GATC）位点的方式 - 即，在RE附近的D（GATC）位点如何有效地找到DNA，以及如何在两个站点之间进行切除，将其限制在两个位点之间的DNA之间。以下特定目的将研究有关MUTS四聚体循环DNA循环作用的这一假设：（1）直接测量DNA和MAP复合物之间的序列和误差特异性结合力随着途径的进展而进行。将使用力光谱（FS）技术和纳米技术FS设备在单分子（S.M.）水平上研究RES RES Res d（GATC）位点。结合荧光显微镜将使我们能够确定特定地图复合物的作用，解决副因素对其行为的影响，并沿DNA绘制其时空相互作用。 （2）阐明Re-to-to-to-D（GATC）'耦合''的机械细节及其与DNA切除的关系。 DNA循环如何改变D（GATC）缺口的动力学 /效率，UVRD的偏置定向载荷或切除终止将通过原子力显微镜，束缚粒子运动实验和S.M.来解决。荧光测定。