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

描述(申请人提供)：在大肠杆菌中，基因组稳定性由甲基定向错配修复(MMR)途径维持，该途径将新复制的DNA分子中的错误碱基对或核苷酸插入/缺失等错误减少1000倍。人类中E.ColiMMR蛋白同源基因的缺陷与癌症发展速度的增加有关，由于E.ColiMMR是研究最多的DNA修复途径之一，阐明其分子机制有望为细胞内避免突变提供新的线索。MMR途径是当蛋白质MutS的复合体识别并结合到新复制的DNA分子上的复制错误(RE)，并与蛋白质MutL激活内切酶Muth时启动的。然后，Muth在半甲基化的d(GATC)位点上剪断新复制的链，该位点用于区分新的链和原始的链。这个位点可以远离1000多个碱基对，没有明显的定向偏向(5‘-或3’-从RE)，但3‘-到-5’解旋酶UvrD随后被加载到朝向RE的缺口处，并且与适当的核酸外切酶一起移除通过RE的新复制的链以便正确地重新合成。虽然单个MMR相关蛋白(MAP)的作用已经确立，但不同的MAP复合体如何有效地协调大跨度DNA的GATC到RE的切除仍然是争论的主题。在生理条件下，MutS以二聚体和四聚体络合物的平衡状态存在，后者最近被观察到与RES在DNA分子上形成环；尽管MutS四聚体的作用存在争议，但MutS四聚体对DNA的环化被认为是RE如何与远距离的d(GATC)位“耦合”-即如何有效地找到RE附近的d(GATC)位点，以及如何将切割限制在两个位点之间的DNA。这一关于MutS四聚体DNA环作用的假设将通过以下具体目标进行研究：(1)直接测量DNA和MAP复合体之间随着途径的进展而产生的序列和错误特异性结合力。将在单分子(S.M.)上研究MAP络合物对Res然后d(GATC)位点的搜索。使用力谱(FS)技术的水平和纳米技术FS设备。结合荧光显微镜将使我们能够确定特定MAP复合体的作用，解决辅助因素对它们行为的影响，并沿着DNA绘制它们的时空相互作用图。(2)阐明RE-to-d(GATC)“偶联”的机制及其与DNA切除的关系。DNA环是否以及如何改变d(GATC)划痕、偏向UvrD定向加载或切除终止的动力学/效率将通过原子力显微镜、拴系粒子运动实验和S.M.来解决。荧光分析。

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.