The role of the topoisomerases in theta-type DNA replication will be studied. This will be accomplished by reconstituting pBR322 DNA replication using purified proteins. Previous data on the replication of ColEl and pBR322 DNA, the findings, by our laboratory, that ColEl and pBR322 DNA contain two effector sites for E. coli replication factor Y, and that the effector sites from pBR322 DNA can act as rifampicin resistant dnaB-, dnaC-, and dnaG-dependent origins of complementary strand DNA synthesis when they are single-stranded, indicates that one should be able to reconstitute pBR322 DNA replication using DNA polymerase I, RNA polymerase, RNase H, the DNA polymerase III elongation system, SSB, the dnaB, C, and G proteins, replication factor Y and proteins i, n, and n"""""""", a topoisomerase(s), and a helicase(s). This would be the first theta-type DNA replication system reconstituted in this fashion. It will allow the determination of which topoisomerases are involved, and, through the creation of partial reactions which mimic the initiation, elongation, termination, and segregation stages of theta-type DNA replication, create an unparalled opportunity to determine how modulations of the shape of DNA influence and play a role in its replication. Information gained from these studied should be applicable to most other DNA replication systems and will serve as a guide when similar components are available for such studies in eucaryotic DNA replication systems. In addition, the proposed studies will define how the replication proteins interact in a system which is under topological constraints.

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National Institute of General Medical Sciences (NIGMS)
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Sloan-Kettering Institute for Cancer Research
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Kumar, Rupesh; Grosbart, Ma?gorzata; Nurse, Pearl et al. (2017) The bacterial condensin MukB compacts DNA by sequestering supercoils and stabilizing topologically isolated loops. J Biol Chem 292:16904-16920
Kumar, Rupesh; Nurse, Pearl; Bahng, Soon et al. (2017) The MukB-topoisomerase IV interaction is required for proper chromosome compaction. J Biol Chem 292:16921-16932
Chang, Elizabeth; Pourmal, Sergei; Zhou, Chun et al. (2016) N-Terminal Amino Acid Sequence Determination of Proteins by N-Terminal Dimethyl Labeling: Pitfalls and Advantages When Compared with Edman Degradation Sequence Analysis. J Biomol Tech 27:61-74
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Bigot, Sarah; Marians, Kenneth J (2010) DNA chirality-dependent stimulation of topoisomerase IV activity by the C-terminal AAA+ domain of FtsK. Nucleic Acids Res 38:3031-40

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