Student Theses and Dissertations

Date of Award

1989

Document Type

Thesis

Degree Name

Doctor of Philosophy (PhD)

RU Laboratory

Model Laboratory

Abstract

We would like to understand how proteins and enzymes interact with DNA. We describe here our studies of the Mrr methylation dependent restriction system, DNA single- and double-strand break repair in E. coli, and substrate recognition by the EcoRI endonuclease. Many species of bacteria make restriction-modification systems to destroy foreign DNA that enters the cell. These systems usually consist of an endonuclease that cleaves a specific DNA sequence and a methylase which modifies the DNA to protect the host chromosome. We observed that when foreign site-specific methylases are expressed in E. coli, the SOS DNA repair response is induced. This DNA damage is inflicted by E. coli restriction enzymes that cleave adenine (Mrr) or cytosine (McrB) methylated DNA. The genes encoding four of the five known E. coli restriction systems lie clustered together, perhaps to coordinate or sequester the cellular defense system. Several of these restriction systems differ between E. coli species, suggesting that their action may establish species boundaries. The EcoRI endonuclease cleaves DNA molecules at the sequence GAATTC. This enzyme is well characterized biochemically, the sequence of its gene is known, and the X-ray crystal structure of an EcoRI-DNA complex has been solved at 3 A resolution. (McClarin, et al., 1986). EcoRI serves as a paradigm for other restriction enzymes and as a model of DNA-protein interactions. We took a genetic approach to study the EcoRI endonuclease. We first asked if EcoRI DNA double-strand breaks are repaired in E. colt'. To this end, a series of temperature-sensitive EcoRI endonuclease alleles were isolated. Temperature shifts with these alleles revealed that in vivo DNA scission induces the E. coli SOS DNA repair response. However, neither SOS induction nor recombination are required to repair these lesions. DNA ligase is required and may suffice to repair EcoRI breaks in the E. coli chromosome. An in vivo DNA scission assay was devised based on the finding that DNA breaks induce the SOS response. SOS induction was monitored with strains carrying the lactose operon fused to an SOS inducible promoter. After DNA scission, these strains produce 3-galactosidase and form blue colonies on X-Gal medium. 'With this blue colony phenotype as a screen, two approaches were taken to isolate EeoRI mutants altered or disrupted in substrate specificity. First, amino acids (E144, R145, R200) implicated in substrate binding by the crystal structure were subjected to site directed mutagenesis. Of 50 of the 60 possible substitutions, several alleles retain weak endonuclease activity which, in vivo and in vitro, is of wild-type specificity. Therefore the simple hydrogen bond model proposed from the crystal structure is insufficient to explain substrate recognition and additional interactions must participate in the substrate-enzyme complex. In the second approach, mutants of an EcoRI TS allele were isolated which conditionally induce the SOS response and impair cell growth in spite of the normally protective methylase. In vitro, these mutant proteins exhibit enhanced cleavage activity at EcoRI* sites, sequences which differ by one nucleotide from the normal recognition site and are also cleaved by the wild-type enzyme under altered buffer conditions. Four of the five mutations of this type lie at the DNA-protein interface and may directly alter or disrupt substrate recognition. One other (H114Y) lies far from the binding and cleavage sites. This mutation falls three amino acids away from a previously described mutation, E111G (King, et al., 1986, 1988), which severely impairs cleavage activity without altering DNA binding. These two mutations support a model whereby DNA scission by the EcoRI endonuclease is allosterically activated upon substrate binding: we suggest that the E111G mutation inhibits this conformational change while the H114Y mutation renders it more facile such that additional DNA sequences act as allosteric effectors and trigger cleavage.

Comments

A thesis submitted to the Faculty of The Rockefeller University in partial fulfillment of the requirements for the degree of Doctor of Philosophy

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