:: Sandler Lab Rotation Projects ::Our lab is interested in the processes of DNA replication and homologous recombination. These are basic process that go on in all organisms and highly conserved evolutionarily. We have two model systems to: In E. coli, we ask cutting edge questions about how the two processes are connected and coordinated. The genes of we study are called the primosome assembly genes. These include: priA, priB, priC, dnaT, dnaC, dnaB and dnaG. The role of the primosome assembly proteins in the cell are thought to be loading the replisome (defined as all proteins at the replication fork) at a recombinational intermediate. This is necessary to restart replication forks that have collapsed due to DNA damage and have since been repaired by recombination. Currently we have defined two sets of pathways that the cells use to do this. One is PriA-dependent and involves PriABC DnaT and the other is PriA-independent needing at least PriC and Rep. All these proteins form complexes that identify the proper DNA substrate and then help DnaC load DnaB on to the DNA. The dnaC gene can mutate to suppress the need for priA, priB, priC and dnaT. So a f ocus in our studies is examining the dnaC gene. This work has been recently funded by the American Cancer Society. The other system asks how recombination occurs in extreme environments: from the very hot to the very cold. We do this by looking for RecA-like genes in samples of organisms taken from these environments. We then analyze these sequences with computers
for structure and phylogeny. The more interesting genes are overexpressed, proteins purified and studied enzymologically and structurally. Besides gaining insight in to how recombination occurs under these different environments, we will form a homologous
series of protein structures that can serve as a database to understand and predict how protein structure adapts to extreme environments. We are also initiating a genetic study of recombination in Sulfolobus sofataricus. This is a hyperthermophill
ic achaean. This work is funded by a Hatch grant. Available projects:LAB ROTATIONS1. One type of project looks at engineered strains of E. coli that have a programmed nick in the chromosome so that replication fork collapse can be monitored by Southern Blot analysis. 2-D gel electrophoresis will allow studies of the structures of these collapsed replication forks. Question as to how the forks are fixed and then restarted will be asked through using this system in a variety of genetic mutants. 2. Another type of project asks where in the cell does repair of replication forks take place. This is answered by making fusions of the Green Fluorescence Protein to replication and recombination proteins. The fate of these fusion proteins in the cell as a function of time and or DNA damage is then monitored by computer-aided microscopy. 3. A third type of project uses a genomics approach to understand global changes in genetic expression caused by the absence of replication restart proteins. Other types of projects are also possible. Please come and talk with me.
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Department
of Microbiology
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