Analysis of the interactions between the 5' to 3' exonuclease and the single-stranded DNA-binding protein from bacteriophage T4 and related phages.
Author: Laurence S. Boutemy
Format: NOOK Study eTextbook
DNA replication and repair is one of the most important cellular processes, since preserving the integrity of the DNA genome is essential to all forms of life. Many proteins are involved in the DNA replication process, and their interaction ensures that the DNA is duplicated and repaired in a coordinated and efficient manner. Bacteriophage T4 is a very good model to study DNA replication, since it encodes all the proteins required at the replication fork, proteins which have been extensively characterized. However, how these proteins interact and coordinate the replication process is still largely unknown. One of these interactions that appears to govern the rate and efficiency of the lagging strand synthesis occurs between the 5' to 3' exonuclease RNase H and the single-stranded DNA-binding 32 protein. The interaction between these two proteins is the focus of this work.;RNase H and the 32 protein, as well as a number of mutants and truncations, were cloned, expressed and purified. These proteins were then used to form different variants of the RNase H + 32 protein complex, which were characterized through biophysical and structural studies. A crystal structure was obtained for the RNase H + 32-B truncation. This structure, along with the results obtained from the biophysical experiments, provides valuable information on how these two proteins interact to coordinate the lagging strand DNA replication.;Finally, the study of the interaction between RNase H and the 32 protein from bacteriophage Rb 69, a phage related to bacteriophage T4, was also initiated.