This project will use a genetic and biochemical approach to study the molecular mechanism by which mutations are introduced during DNA replication and DNA repair. An in vitro system able to replicate bacteriophage T7 DNA and encapsulate the product into phage heads will be used to produce infective phage in vitro. This system is also able to repair some types of DNA damage. The reactions will be perturbed in ways that might reduce the fidelity of DNA replication and repair, and the phage produced in vitro will be examined for mutations. In vitro mutagenesis will be examined by: altering the pools of DNA precursors, including analogues of normal precursors, and using damaged DNA as a template. The location and base sequence near to the mutation site will be considered. Site directed mutagenesis will be investigated by attempting to recombine mutagen treated T7 DNA restriction fragments into intact T7 genomes in vitro. Also, T7 genomes damaged by ultraviolet radiation or alkylating chemicals will be repaired in vitro, and measurements of mutation induction consequential to DNA repair will be made. Special attention will be given to inducible repair modes that require new protein synthesis for error-free (adaptive response) or error-prone (SOS-response) DNA repair. The intent is to determine the mechanisms by which mutations are introduced during constitutive and inducible modes of DNA repair. In another part of this study heteroduplex T7 DNA will be encapsulated and examined for mismatch repair, and the in vitro DNA replication system will be used in an effort to defect mismatch repair in vitro. To assist in these mutagenesis studies in further improvements in the T7 DNA packaging system are planned. This will involve isolation and study of structures important in T7 bacteriophage assembly and an effort to understand the mechanism of DNA encapsulation. The long term goal of this project is to provide basic knowledge that will allow more meaningful judgments and more accurate extrapolations to be made concerning health hazards caused by genetic damage inflicted by environmental insults.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM034614-03
Application #
3285930
Study Section
Microbial Physiology and Genetics Subcommittee 2 (MBC)
Project Start
1984-08-01
Project End
1988-08-31
Budget Start
1985-09-01
Budget End
1986-08-31
Support Year
3
Fiscal Year
1985
Total Cost
Indirect Cost
Name
Temple University
Department
Type
Schools of Medicine
DUNS #
City
Philadelphia
State
PA
Country
United States
Zip Code
19122
Yang, Y; Masker, W (1997) Double-strand breaks increase the incidence of genetic deletion associated with intermolecular recombination in bacteriophage T7. Mol Gen Genet 255:277-84
Yang, Y; Masker, W (1996) Instability of repeated dinucleotides in bacteriophage T7 genomes. Mutat Res 354:113-27
Yang, Y; Masker, W (1996) Deletion during recombination in bacteriophage T7. Mutat Res 349:21-32
Kong, D; Masker, W (1994) Deletion between direct repeats in T7 DNA stimulated by double-strand breaks. J Bacteriol 176:5904-11
Kong, D; Masker, W (1993) Deletion between directly repeated DNA sequences measured in extracts of bacteriophage T7-infected Escherichia coli. J Biol Chem 268:7721-7
Masker, W; Crissey, M A (1993) The effect of the 3'-->5' exonuclease of T7 DNA polymerase on frameshifts and deletions. Mutat Res 301:235-41
Scearce, L M; Masker, W (1993) Deletion between direct repeats in bacteriophage T7 gene 1.2. Mutat Res 288:301-10
Pierce, J C; Masker, W (1992) Frameshift mutagenesis in bacteriophage T7. Mutat Res 281:81-7
Masker, W (1992) In vitro repair of double-strand breaks accompanied by recombination in bacteriophage T7 DNA. J Bacteriol 174:155-60
Scearce, L M; Pierce, J C; McInroy, B et al. (1991) Deletion mutagenesis independent of recombination in bacteriophage T7. J Bacteriol 173:869-78

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