Exonucleases mapping

Bam HI cleaves a six-bp palindromic sequence at the phosphodiester bonds between two guanosine nucleosides. Formation of an intrastrand crosslink between the two adjacent guanosine nucleosides inhibits digestion by the enzyme. Another method, termed exonuclease mapping, involves digestion of the... 

Exonuclease III j Removes nucleotides from 3 ends of DNA. DNA sequencing mapping of DNA-protein interactions. 

Deletion Mutants - Another approach has been the construction of deletion mutants by the judicious use of restriction enz3mies and appropriate exonucleases. The sites of the deletions were mapped in some Instances they were widely separated but still affected a single protein. The functional ability of the mutants was then determined. The results, while at an early stage, are in agreement with the conclusions reached with the temperature-sensitive mutants. The early proteins must be functional to effect DNA replication, stimulation of host cell DNA synthesis and cell transformation. Interestingly, not all three of the late proteins appear to be required for virus production. Much more will surely come from these studies. 

Two clones, carrying a 5.2 kb insert in inverse orientations were isolated and mapped by restriction enzyme analysis. The two clones were used to construct deletion clones with the exonuclease Ill/mung bean nuclease system. Deletion clones containing the psbG homologons region were sequenced. 

When the ts lethality was investigated, it was found that these cells were filamentous. In enteric bacteria filamentous growth is characteristically observed upon induction of the SOS system in response to DNA damage. It was determined that the xthR cells were in fact constitutively SOS-induced. Examination of genetic loci that mapped in the "pncX region revealed that the xthA locus in E. coll which encodes exonuclease HI mapped in this... 

The ts lethality can be reversed by second site mutations, which map at loci different from the original xthSi locus. Preliminary results suggest that one of these second site mutations may be at the structural gene for exonuclease in, i.e., at the xthh locus. The levels of exonuclease HI assayed in extracts of the revertant are below detectable limits and the revertant is sensitive to hydrogen peroxide. 

The structural gene for Exo III is xthA (7,36), which is located at the 38-min position on the E. coli map. A number of xth mutants ts and deletion) have been isolated, all of which simultaneously affect the AP endonuclease, 3 -phosphatase, and exonuclease activities. 

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