neocarzinostatin for Double Stranded Breaks in worms?

Model Organism: Worms Methods & Reagents
1

Hi all,

Just wondering if anyone has had any success using neocarzinostatin to induce DSBs in worms? I know people use it in cell culture and mouse embryos and that it gives a decent amount of single stranded breaks as well. I’m hoping to use it as I don’t have access to an X-ray irradiator.

Any advice is greatly appreciated.

Thanks

2

I’m not familiar with neocarzinostatin and couldn’t rapidly find anything much.

A lot depends on what you’re trying to do: are you doing assays of DNA damage response? Are you trying to cause genomic rearrangements?

Various chemicals that aren’t specific for double-strand breaks can be used to cause them for various practical approaches; in particular, this has been done to randomly generate small deletions (to then be screened using PCR), to integrate extrachromosomal transgenes, and to cause translocations. These include EMS, MMS, and trimethylpsoralen. The details matter: the dosage used is often high, there is a lot of sickness, and there are a lot of other mutations induced. Of course, if you specifically need double-strand breaks then none of these are for you.

3

Wouldn’t it be easier to just use a Stratalinker? Probably more readily available too

4

Hi,

Thanks for the responses so far. My purpose is to specifically introduce double stranded breaks for to determine what variation of DNA damage my mutant is sensitive to upon exposure. I’ve got access to a UV irradiator for single strand breaks, but cannot induce DSBs. Neocarzinostatin has been used in different organisms for introducing DSBs, but you do get a lot of single stranded breaks.

Nonetheless, thanks for the input.

5

Yeah, getting access to a gamma-irradiator can be problematic. When I was in the DNA repair field, I never came across anyone using neocarzinostatin. Since you’re trying to determine what type of DNA damage to which your mutant might be sensitive, I’d recommend:

-camptothecin, a topoisomerase I poison that inhibits the enzyme and prevents its release from DNA, creating capped single-ended DSBs
-doxorubicin or etoposide, topoisomerase II poisons that will create DSBs
-interstrand cross-linking agents (probably cisplatin and TMP/UVA; HN2 is good, but very nasty to work with); these are more complicated lesions as they tend to be a mixture of adducts to one strand and cross-links to both strands.
-hydroxyurea to arrest replication forks

Protocols for most of those agents exist in worms; I never came across a doxorubicin or etoposide protocol, so it might be worth developing one. Finding which set of DNA damaging agents to which your mutant is sensitive can be really informative. For example, camptothecin and cross-linking agents would block replication forms and require replication to generate DSBs. If you do see sensitivity to replication blocking lesions, TMP/UVA is particularly informative: in the absence of UVA, you will be assaying the effect of a TMP monoadduct on one strand. Following UVA you’ll be interstrand cross-links which will block forks and require complex, multi-pathway repair. It’s a bit cleaner than other cross-linking agents which also form intra-strand cross-links.

I published sensitivity assays to camptothecin, cisplatin, TMP/UVA and HN2 back in the day:
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1933397/

Monica Colaiacovo also had a good set of protocols:
https://www.ncbi.nlm.nih.gov/pubmed/26807430

6

if you want to try something different, you could get some americium 421 from a smoke detector and make your own alpha particle irradiator.
(of course you don’t want to exactly emulate the radioactive boyscout).

7

Thanks Jordan and Imu1,

This is really helpful. I’ll go with camptothecin, it’s inexpensive and easy to obtain.

Thanks once again.