Transfer mtDNA to nucleus?

#31

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Posted 29 June 2004 - 03:52 AM

Easy there, wolfram. I said we need to improve DNA repair function not the enzyme itself.

The method that you describe is often used in big pharmaceuticals as part of their drug discovery programs. As you stated it involves exposing an organism to accelerated evolution via exposure to a mutagen. I do not want to discount your idea, which is a legitimate approach to solving the problem of enhanced DNA repair, namely the discovery of a DNA repair enzyme with increased efficiency. There are other organisms that already have superior DNA repair enzymes, such as Conan the Bacterium that is practically impervious to lethal doses of radiation. A simpler approach is to incorporate these existing enzymes.

But let's not miss the point here. I said that it is simple to do and it is. We do not need a better DNA enzyme (yet) to improve function. We just need more of it. The function of the enzyme is determined by its ability to repair a number of DNA damaged sites over a period of time. By increasing enzyme concentration we increase its activity over time. If we have sufficient enzyme present we should be able to reduce DNA damage to levels approximating zero. It sounds simplistic and it is. Thus we need to increase the rate of synthesis of DNA repair enzyme in the cell. We can do this by increasing the amount of regulatory transcription factor associated with the enzyme's expression or simply by adding more copies of the gene itself into the cell. There are no known side effects with DNA repair increase enzyme outside of not having enough which predisposes you to premature aging and cancer.

#32

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Posted 30 June 2004 - 05:37 AM

Most studies that have been conducted on DNA repair involve knockout mutants that result in loss of function of DNA repair enzymes and also complementation studies where function is rescued by restoring the gene. There are, however, a handful of studies that are beginning to emerge that demonstrate the validity of increasing DNA repair function by overexpressing DNA repair genes.

Here are two examples of how an increase in DNA repair enzymes inside mitochondria can substantially reduce intramitochondrial oxidative stress and subsequently reduce the incidence of oxidative stress induced apoptosis.

Conditional targeting of the DNA repair enzyme hOGG1 into mitochondria.
J Biol Chem. 2002 Nov 22;277(47):44932-7.

An earlier study showing how a vector was constructed containing the gene for OGG1 (a powerful DNA repair enzyme) and using the mitochondrial localization sequence from the gene for superoxide dismutase the transfected gene was able to target the recombinant protein into mitochondria and increase DNA repair function resulting in increased survival during oxidative stress. An inducible promoter was also attached to the recombinant protein to enable control of overexpression.

Endonuclease III and endonuclease VIII conditionally targeted into mitochondria enhance mitochondrial DNA repair and cell survival following oxidative stress.
Nucleic Acids Res. 2004 Jun 15;32(10):3240-7.

In this important study, the endonuclease III and endonuclease VIII DNA repair enzymes from the bacterium e. coli were transfected to HeLa cells and localised to the mitochondria using a mitochondrial targeting sequence. The enzymes were overexpressed and it was found that the HeLa cells were more resistant to menadione exposure. This is the first time bacterial DNA repair enzymes were transfected to a human cell line showing how enzymes from bacterial cells, which have no mitochondria, could be made to work inside human mitochondria organelles and increase DNA repair rate above basal levels resulting in increased survival when exposed to oxidants.

There are numerous known DNA repair and DNA protection proteins that could putatively decrease the rate pf DNA damage to levels far below what is normal in a cell. Not all DNA repair proteins are beneficial, however. For example RAD51, when overexpressed will induce increased rate of chromosomal crossing over resulting in gene shuffling, due to its DNA pairing function. At this stage this is the only example of DNA repair enzyme that could be harmful when overexpressed. Consequently, the potential therapeutic capability of a DNA repair gene needs to be determined by its mode of action.

A survey of the literature on DNA repair overexpression studies presently yields only cell lines as the model organism. It is thus we eager anticipation that we await the results on multicellular vertebrate organisms and the effects that these increased repair pathways will have on aging.

Edited by prometheus, 30 June 2004 - 05:56 AM.

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#33 olaf.larsson

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Posted 12 July 2004 - 10:39 PM

"In this important study, the endonuclease III and endonuclease VIII DNA repair enzymes from the bacterium e. coli were transfected to HeLa cells and localised to the mitochondria using a mitochondrial targeting sequence. The enzymes were overexpressed and it was found that the HeLa cells were more resistant to menadione exposure. This is the first time bacterial DNA repair enzymes were transfected to a human cell line showing how enzymes from bacterial cells, which have no mitochondria, could be made to work inside human mitochondria organelles and increase DNA repair rate above basal levels resulting in increased survival when exposed to oxidants"

Very fascinating that something like this can really work! Now lets find the bacteria with the best set of dna repair enzymes (Dienococcus radiodurens? ) and insert them to a mouse.

#34

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Posted 13 July 2004 - 01:32 AM

You've got the drift. It is a very simple experiment since we know the gene in d. radiodurans and we could direct it to both nuclear and mitochondrial DNA. We also need more information on the other DNA repair pathways, including their enzymes, substrate specificity, co-factors and rate limiting steps. Some repair enzymes when over-expressed, end up causing other mutations. So not all enzyme overexpression is beneficial. The more specific an enzyme is in substrate selection the less likely it will affect healthy DNA.

Dr. Mark Kelley from IU is has been researching DNA overexpression for some time including transfer of DNA repair enzymes from other species such as drosophila to mammalian cells and overexpression of DNA repair targeted to mitochondria.

#35 olaf.larsson

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Posted 13 July 2004 - 02:53 AM

I thought that DNA repair in mito was directly linked to the replication. So I didnt thought that one could improve repair by making the concentration of repair enzyme higher. Its amazing to see that it is possible to take an enzyme from a bacteria and insert it to human cells and see that it works and acctually performs a funktion without interfering or blocking or beeing blocked by something else.

Now a question Promoteus, what education do you have?
And why it seems like there only is two of us here on this forum. Where are the other peoples, in the members forum???

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#36

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Posted 13 July 2004 - 03:39 AM

I have an MS in cell biology and specialized in neuroscience. As for only being the two of us here, I suspect others may not share a need to participate other than occasionally reading the messages posted. Sometimes people lose interest when it becomes too technical. Other times you can only go so far with theoretical biology before you have to start getting some empirical data to substantiate hypotheses. Don't let the interest factor of others dampen your enthusiasm. There are not too many scientists here I suspect, even though there are a few widely read individuals. There are some sharp thinkers though. Even so, without the right grounding it is easy to take paths that lead nowhere. But by the same token individuals with very little formal training have been known to make groundbreaking discoveries. Keep up the brainstorming! This topic was initiated by you.

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