Initiation of chromosomal DNA replication in a novel bacterium

You are studying the initiation of chromosomal DNA replication in a novel bacterium that grows at 75°C. To this end you have isolated temperature sensitive replication mutants in twelve different proteins/genes derived from this strain. You call these mutations hot1-hot12. Your hope is to use these mutations to identify proteins that act during initiation of DNA replication.

1) How could you reduce the number of mutants to investigate? Describe the experiment that you would perform to focus on initiation factors and which type of mutants you would choose for further investigation.

Question continued: Using your approach described above you narrow the field to 3 candidate mutations (hot4, hot6, hot9). You decide to take two approaches to study the proteins encoded by these mutations.

(1) You will clone the genes that are mutated and over-express the proteins
they encode.

(2) Your technician will use biochemical complementation to purify the activity
that complements the ability of cell extracts derived from the mutant cells
to replicate a plasmid containing the chromosomal origin. You quickly clone the genes that complement the mutant strains and purify the proteins they encode. You decide to first test whether one of more of the proteins associate with the origin using a Gel Shift Assay. You get the results shown on the diagram:

2) Based on the results above, what can you conclude about the association of the three factors with the origin?

Question continued: After several months of biochemistry, your technician assures you that he has purified factors that complement each of the three mutant extracts. He tests each of the fractions in the same assay and he gets the following results.

3) How can you explain the difference between your technician's results and the results that you obtained with the proteins you purified.

Question continued: Based on your ideas concerning the different results obtained by you and your technician, you ask your technician to test whether the hot4 complementing factor also complements the defects observed in extracts derived from any other extract. He finds that the hot4 complementing factor also complements the defects in extracts derived from hot 8 and hot 12, two mutations that you eliminated in your initial analysis of the mutants (re: Question 1).

4) How can you explain the ability of the hot4 complementing factor to complement replication in extracts derived from mutations that you have determined to effect either initiation (hot4) or elongation (hot 8 and hot 12).

5) Assuming that your technician has purified the hot4 complementing
factor to homogeneity, briefly describe how you would test your hypothesis
experimentally.