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Professor, Biochemistry Telephone: 215-707-3973 Fax: 215-707-7536 Email: wmasker@temple.edu
Department of Biochemistry
Our laboratory studies the biochemistry of how normally accurate molecular mechanisms sometimes lead to mutations during the replication or repair of DNA. Of particular interest is the mechanism by which deletions take place between directly repeated sequences of DNA. A second area of investigation is the question of how double strand breaks in DNA are repaired and why repair of these breaks is frequently accompanied by genetic rearrangements in the vicinity of the break. Our approach to these problems has been to use one of the simplest available DNA replication systems, that of bacteriophage T7, as a model system. Good in vitro systems have been developed to carry out most of the steps of T7 DNA replication, recombination, and repair. By packaging DNA in a second in vitro reaction a high yield of infective phage can be produced from DNA recovered from the DNA replication or repair reactions. The geneotype of these phage reflect mutational events that took place during the prior in vitro reactions.
To study deletion in T7 we interrupted the ligase gene with an insert of nonsense DNA sequence that is bracketed by direct repeats. The insert inactivates the gene and renders the phage incapable of growth on a selective host. Deletion between the direct repeats eliminates the insert and allows normal growth on the selective host. This system allows us to perform either in vivo or in vitro deletion studies. This system is currently being used to determine why a double strand break placed between the direct repeats markedly increases the frequency of deletion between the repeats. We are investigating the possibility that errors during recombination lead to deletion between direct repeats and that repair of double strand breaks via recombination may sometimes lead to deletion if the double strand break forms near a pair of direct repeats.
A second project in our laboratory deals directly with how double strand breaks are repaired. In T7 most double strand breaks are repaired via recombination with homologous DNA molecules present in the same cell or reaction. These recombinational repair events proceed without normal DNA replication and, therefore, are not the result of formation of new replication forks via invasion of a partial genome into an intact homologue. We have developed an in vitro system for repair of double strand breaks in T7 that is independent of the packaging system and used this to show that molecules of donor DNA are physically incorporated into a gap formed at the site of the double strand break. Currently we are attempting to determine the exact mechanism of this type of repair and to identify the roles of enzymes involved in double strand break repair in T7.
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Warren Masker, PhD