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

描述（由申请人提供）：在E。在大肠杆菌中，基因组稳定性通过甲基指导的错配修复（MMR）途径来维持，该途径将新复制的DNA分子中的错误（例如错配碱基对或核苷酸插入/缺失）减少1000倍。E.大肠杆菌MMR蛋白在人类中与癌症发展率的增加相关，并且作为大肠杆菌MMR蛋白在人类中与癌症发展率的增加相关。大肠杆菌MMR是目前研究最多的DNA修复途径之一，其分子机制的阐明有望为细胞内的突变避免提供新的线索。当MutS蛋白复合物识别并结合新复制的DNA分子上的复制错误（RE），并与MutL蛋白一起激活核酸内切酶MutH时，MMR途径启动。MutH然后在半甲基化的d（GATC）位点切割新复制的链，该位点用于区分新链和原始链。该位点可以远离超过1000个碱基对，没有明显的方向偏差（从RE的5 '或3'），但是3 '至5'解旋酶UvrD然后在朝向RE的切口处加载，并且用适当的核酸外切酶通过RE去除新复制的链以正确地重新合成。虽然单独的MMR相关蛋白（MAP）的作用已经很好地建立，但是不同的MAP复合物如何有效地协调跨大跨度DNA的GATC至RE切除仍然是争论的主题。在生理条件下，MutS以二聚体和四聚体复合物的平衡存在，后者最近在具有RE的“环状"DNA分子上被观察到;尽管MutS四聚体的作用存在争议，但MutS四聚体对DNA的成环被认为是RE如何与远距离d（GATC）位点“偶联”的-即，如何有效地发现RE附近的d（GATC）位点以及如何将切除限制在两个位点之间的DNA。关于MutS四聚体在DNA成环中的作用的这一假设将通过以下具体目标进行研究：（1）随着途径的进展，直接测量DNA和MAP复合物之间的序列特异性和错误特异性结合力。在单分子（s.m.）水平使用力光谱（FS）技术和纳米技术FS设备。结合荧光显微镜将使我们能够确定特定的MAP复合物的作用，解决辅因子对其行为的影响，并绘制它们的时空相互作用沿着DNA。(2)阐明RE-to-d（GATC）“偶联”的机制细节及其与DNA切除的关系。DNA成环是否以及如何改变d（GATC）切口的动力学/效率，UvrD的偏置定向加载或切除终止将通过原子力显微镜，拴系粒子运动实验和s.m.荧光测定。

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.