The research focuses on the mechanism of viral DNA packaging and on the construction of phage T4 in vitro packaging-derived cloning vectors. Our work is especially directed toward 1) understanding the structures and functions of the multifunctional phage T4 packaging (prohead and terminase) proteins. The 3- dimensional basis for enzymatic (DNA translocating) and structural (prohead form-determining) roles of the major capsid protein gp23*), of a minor processed enzymatic product (gp23**), and of capsid gene mutants will be determined. The assembly of the DNA entrance (also the prohead initiation) vertex as an integral membrane protein will be examined using overexpression vectors; the role of a catalytic gene product and host systems in the membrane insertion will be determined. Specific packaging mutations in both these prohead proteins will be selected following gene-directed mutagenesis. The DNA terminase proteins which interact with the DNA entrance vertex protein in the prohead have been overexpressed and purified-their mechanisms of action in DNA translocation and concatemer cutting will be investigated. lambda and lambda-pBR322 derivative DNAs can be packaged into T4 heads in vitro. cos and/or P1 DNAs packaged into T4 and recircularized with the homologous site-specific recombination systems should allow development of T4-hybrid megacosmid vectors. We have proposed a novel """"""""spiral-fold"""""""" model for packaged phage DNA. Morphological and (with phage ) chemical work can definitively establish this model. Packaged kinked or non-B form DNA will be detected, and its interaction with the DNA binding site in the major capsid protein and its minor processed enzymatic product (gp23**) will be probed. We will determine what DNA structures can be packaged in vivo and in vitro; e.g. whether nicks and heteroduplex loops are excluded from packaged DNA in vitro, and whether the terminase proteins act to discriminate against such structures in coupled DNA repair processes.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Method to Extend Research in Time (MERIT) Award (R37)
Project #
5R37AI011676-17
Application #
3480622
Study Section
Microbial Physiology and Genetics Subcommittee 2 (MBC)
Project Start
1977-07-01
Project End
1992-03-31
Budget Start
1991-04-01
Budget End
1992-03-31
Support Year
17
Fiscal Year
1991
Total Cost
Indirect Cost
Name
University of Maryland Baltimore
Department
Type
Schools of Medicine
DUNS #
003255213
City
Baltimore
State
MD
Country
United States
Zip Code
21201
Mullaney, Julienne M; Black, Lindsay W (2014) Bacteriophage T4 capsid packaging and unpackaging of DNA and proteins. Methods Mol Biol 1108:69-85
Thomas, Julie A; Black, Lindsay W (2013) Mutational analysis of the Pseudomonas aeruginosa myovirus KZ morphogenetic protease gp175. J Virol 87:8713-25
Dixit, Aparna Banerjee; Ray, Krishanu; Thomas, Julie A et al. (2013) The C-terminal domain of the bacteriophage T4 terminase docks on the prohead portal clip region during DNA packaging. Virology 446:293-302
Wu, Weimin; Thomas, Julie A; Cheng, Naiqian et al. (2012) Bubblegrams reveal the inner body of bacteriophage ?KZ. Science 335:182
Black, Lindsay W; Rao, Venigalla B (2012) Structure, assembly, and DNA packaging of the bacteriophage T4 head. Adv Virus Res 82:119-53
Thomas, Julie A; Weintraub, Susan T; Wu, Weimin et al. (2012) Extensive proteolysis of head and inner body proteins by a morphogenetic protease in the giant Pseudomonas aeruginosa phage ?KZ. Mol Microbiol 84:324-39
Dixit, Aparna Banerjee; Ray, Krishanu; Black, Lindsay W (2012) Compression of the DNA substrate by a viral packaging motor is supported by removal of intercalating dye during translocation. Proc Natl Acad Sci U S A 109:20419-24
Dixit, Aparna; Ray, Krishanu; Lakowicz, Joseph R et al. (2011) Dynamics of the T4 bacteriophage DNA packasome motor: endonuclease VII resolvase release of arrested Y-DNA substrates. J Biol Chem 286:18878-89
Ray, Krishanu; Sabanayagam, Chandran R; Lakowicz, Joseph R et al. (2010) DNA crunching by a viral packaging motor: Compression of a procapsid-portal stalled Y-DNA substrate. Virology 398:224-32
Rao, Venigalla B; Black, Lindsay W (2010) Structure and assembly of bacteriophage T4 head. Virol J 7:356

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