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.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM034558-02
Application #
3285800
Study Section
Biochemistry Study Section (BIO)
Project Start
1984-07-01
Project End
1986-06-30
Budget Start
1985-07-01
Budget End
1986-06-30
Support Year
2
Fiscal Year
1985
Total Cost
Indirect Cost
Name
Sloan-Kettering Institute for Cancer Research
Department
Type
DUNS #
064931884
City
New York
State
NY
Country
United States
Zip Code
10065
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
Bahng, Soon; Hayama, Ryo; Marians, Kenneth J (2016) MukB-mediated Catenation of DNA Is ATP and MukEF Independent. J Biol Chem 291:23999-24008
Nurse, Pearl; Marians, Kenneth J (2013) Purification and characterization of Escherichia coli MreB protein. J Biol Chem 288:3469-75
Hayama, Ryo; Bahng, Soon; Karasu, Mehmet E et al. (2013) The MukB-ParC interaction affects the intramolecular, not intermolecular, activities of topoisomerase IV. J Biol Chem 288:7653-61
Lee, Chong; Marians, Kenneth J (2013) Characterization of the nucleoid-associated protein YejK. J Biol Chem 288:31503-16
Perez-Cheeks, Brenda A; Lee, Chong; Hayama, Ryo et al. (2012) A role for topoisomerase III in Escherichia coli chromosome segregation. Mol Microbiol 86:1007-22
Hayama, Ryo; Marians, Kenneth J (2010) Physical and functional interaction between the condensin MukB and the decatenase topoisomerase IV in Escherichia coli. Proc Natl Acad Sci U S A 107:18826-31
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

Showing the most recent 10 out of 50 publications