We propose to continue our studies of the replication of bacteriophage lambda DNA in an in vitro system that is reconstituted with 20 highly purified lambda and E. coli proteins. Our long range goal is to achieve a detailed mechanistic understanding of the biochemical events that occur during the initiation, propagation, termination, and regulation of lambda DNA replication. The viral O and P replication proteins promote the assembly of an ordered series of nucleoprotein structures at the lambda replication origin (ori- lambda) prior to priming and DNA chain elongation. Recent findings indicate that the first two of these structures, the O-some and the ori- lambda.O.P.DnaB complex, have the capacity to capture and hold alternate DNA conformations induced by a combination of negative DNA supercoiling and O protein binding to ori lambda. Moreover, the step leading to formation of these """"""""pre-open"""""""" complexes appears to be a key point of regulation of lambda DNA replication. We will characterize this step further (a) by defining its dependence on superhelical tension; (b) by defining the amino acid residues of the lambda O and P proteins and E. coli DnaB helicase that interact with single stranded DNA and/or with the A/T-rich region of ori lambda; and (c) by analyzing how transcriptional events distant from orilambda facilitate the DNA melting step. Using radiolabeled proteins, we will define the stoichiometries of the O, P, and DnaB proteins in pre-initiation structures assemble at orilambda. The fate of radiolabeled O, P and DnaB following initiation will be monitored. We will continue our efforts to use x-ray crystallography to define the tertiary structure of the DNA-binding domain of the lambda O initiator. In related experiments, we will use a genetic approach to define the amino acid residues of the N-terminal domain of O involved in specific recognition of origin DNA sequences. We will also select mutant orilambda DNA replication and identify the molecular step or steps in the initiation pathway affected by individual mutations. Biochemical studies of lambda DNA replication will continue to provide important insights into the biological mechanisms used in the initiation and regulation of chromosomal DNA replication. This knowledge will help guide studies of these vital processes in more complex organisms.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Research Project (R01)
Project #
Application #
Study Section
Biochemistry Study Section (BIO)
Project Start
Project End
Budget Start
Budget End
Support Year
Fiscal Year
Total Cost
Indirect Cost
Johns Hopkins University
Schools of Public Health
United States
Zip Code
Leng, Fenfei; Amado, Luciana; McMacken, Roger (2004) Coupling DNA supercoiling to transcription in defined protein systems. J Biol Chem 279:47564-71
Leng, Fenfei; McMacken, Roger (2002) Potent stimulation of transcription-coupled DNA supercoiling by sequence-specific DNA-binding proteins. Proc Natl Acad Sci U S A 99:9139-44
Gonciarz-Swiatek, M; Wawrzynow, A; Um, S J et al. (1999) Recognition, targeting, and hydrolysis of the lambda O replication protein by the ClpP/ClpX protease. J Biol Chem 274:13999-4005
Stephens, K M; McMacken, R (1997) Functional properties of replication fork assemblies established by the bacteriophage lambda O and P replication proteins. J Biol Chem 272:28800-13
Learn, B A; Um, S J; Huang, L et al. (1997) Cryptic single-stranded-DNA binding activities of the phage lambda P and Escherichia coli DnaC replication initiation proteins facilitate the transfer of E. coli DnaB helicase onto DNA. Proc Natl Acad Sci U S A 94:1154-9
Karzai, A W; McMacken, R (1996) A bipartite signaling mechanism involved in DnaJ-mediated activation of the Escherichia coli DnaK protein. J Biol Chem 271:11236-46
Learn, B; Karzai, A W; McMacken, R (1993) Transcription stimulates the establishment of bidirectional lambda DNA replication in vitro. Cold Spring Harb Symp Quant Biol 58:389-402
Mensa-Wilmot, K; Carroll, K; McMacken, R (1989) Transcriptional activation of bacteriophage lambda DNA replication in vitro: regulatory role of histone-like protein HU of Escherichia coli. EMBO J 8:2393-402
Dodson, M; McMacken, R; Echols, H (1989) Specialized nucleoprotein structures at the origin of replication of bacteriophage lambda. Protein association and disassociation reactions responsible for localized initiation of replication. J Biol Chem 264:10719-25
Alfano, C; McMacken, R (1989) Ordered assembly of nucleoprotein structures at the bacteriophage lambda replication origin during the initiation of DNA replication. J Biol Chem 264:10699-708

Showing the most recent 10 out of 15 publications