Originally we had:
Aim I. The functions of DNA uptake proteins
Q. 1. Which genes are needed for DNA uptake?
Q. 2. Which proteins contact DNA during uptake?
Under Q. 1 we proposed to create nonpolar knockout mutants of every competence gene (all 25 CRP-S genes and 2 other genes implicated in DNA uptake), and characterize their phenotypes. Under Q. 2 we proposed to systematically test whether they bound DNA, using His-tagging and crosslinking.
The RA has almost completed making the knockouts. Those of them with cotranscribed downstream genes (i.e. likely to have polar effects) are not nonpolar yet, because the selection for excision of the selective cassette turns out not to work in our lab strain (it's the standard lab strain Rd). But we expect she'll be able to manually screen for excision (or we can hire an undergrad to do it). We expect that this will be completed before the proposed start date of the grant. We now describe this in the Preliminary Results section of the proposal, and we've eliminated what was Q. 1.
We now also have a prioritized list of candidate DNA-contacting proteins, based on several attributes. One is the presence of protein-export signals and other clues in the sequence annotation. Another is what is known about the functions of homologs. Another is what is known from the available mutants; mutants are available for only some proteins, and for some of these the mutant is expected to be polar on another gene that could account for its phenotype.
Aim I. Identifying DNA uptake proteins
Q. 1. Which proteins contact DNA during uptake?
Rationale: Our in vivo crosslinking strategy can succeed where previous in vitro approaches have failed. We will begin these studies with the two top candidates on our list, secretin and pilin. In parallel we will use two simple steps to identify additional strong candidate proteins.
Methods: 1. Basic crosslinking assay:
- First put a His-tag on the gene in the chromosome. Test whether cells with this tag arre still able to take up DNA. But a test for DNA crosslinking might be worth doing even if the tag does interfere with function, if the protein is still assembled into a pore (secretin) or pilus (pilin). So we should also test retrieval of the tagged secretin or pilin after formaldehyde crosslinking,with and without reversing the crosslinks before running the SDS-PAGE gel. This will tell us whether the protein is assembled into its normal complex.
- Incubate the mutant cells with 32P-labelled DNA (probably the 222-bp USS-C fragment). The standard way to form DNA-protein crosslinks is by adding formaldehyde, but this has the BIG disadvantage of also forming protein-protein crosslinks. We hope to instead purchase photoaffinity nucleotides and incorporate one or more of these into the DNA we give to the cells. This will allow us to specifically induce DNA-protein crosslinks (no protein-protein crosslinks) by irradiating the cells with UV (do we need a UV laser? I think a colleague has one.) After a very short time (1 minute?) UV to form crosslinks.
- Wash the cells to remove the external DNA, then lyse them and load them on a Ni-NTA resin column to bind the His-tagged protein. Wash the column to remove everything else. Elute the protein and check whether (i) the expected protein has eluted and (ii) any radioactivity has eluted. If the protein is there but the radioactivity isn't, there was no crosslinking between this protein and the DNA. If radioactivity elutes with the protein, investigate further.
- This experiment needs several controls. The most important is probably a positive control for crosslinking of the DNA with a known protein. How about SSB - it binds single-stranded DNA in the cytoplasm? or DprA - it binds incoming DNA and protects it from nucleases? A good negative control will be cells that aren't UV'd, as will DNA without the photoreactive nucleotide, and cells without the protein tag.
- We will do simple transformation and DNA uptake assays on all the non-polar mutants. These are already standard in our lab, and can be done in one term by an undergraduate or M.Sc. student. Only proteins that are needed for normal DNA uptake will be retained as candidates.
- We will use formaldehyde crosslinking , followed by gel electrophoresis and HPLC-mass spectromtry to identify proteins that are crosslinked (directly or indirectly) to DNA. Any proteins not crosslinked will not be strong candidates. The DNA will be tagged with biotin so that it and all crosslinked proteins can be recovered by attachment to streptavidin-coated magnetic beads (Dynabeads), and after recovery the crosslinks will be reversed so the proteins can be identified. This is not a very specific test for uptake proteins, as it will also give proteins that bind incoming DNA in the cytoplasm, but missing proteins can be safely excluded.
Here;'s where the planning peters out a bit...
What do we do with these proteins once we've found them? We test them for in vitro DNA binding, using bandshift and Southwestern assays.
What if secretin and pilin don't crosslink to DNA???? Does this mean that they don't contact DNA at all? This would be quite surprising.