DNA damage repair enzymes

[bioquest]

I know for sure though that I read somewhere that overexpressing enzymes caused more problems with mismatched bases in mice but it didnt' say which enzymes and I don't remember where I read it- I know for sure that I read this though and I think it was a legitimate source I read it in. but it could have been any enzymes they overexpressed right- I just thought they worked together to repair dna damage when they did it

I would want to supplement them to a mouse regularly but I'd prefer to geneticallly modify it to overexpress them would that be possible does anyone know how to do that? What I mean is, is anyone a high level geneticist or knows one- or do you think genetically modifying a mouse or something to overexpress them all would be impossible?

I really appreciate you guys talking to me about this and Im really glad I got to talk with other people who were at least somewhat interested in the same thing

 

[Mike H]

My interest in DNA repair is pretty much non-existent, I think being asked to regurgitate mechanistic details of the various pathways did me in as an undergraduate. But that's perhaps another story for another day.....

While I've never worked with mice, most of what I've seen reports of in the literature are work with various transgenic mice, either with selected genes knocked out or overexpressed. It's typically not the whole pantheon of proteins that are involved in a process, especially in what I've seen of proteins being overexpressed in mice - it's a protein or two involved in metabolism that's overexpressed in muscle tissue, as an example, not every single protein involved in the various energy-producing pathways in muscle. Proposing to do it in all tissue types sets the bar even higher. It would make for a neat opening scene in a sci-fi novel at the moment, but reengineering entire chunks of a mammal's genome, I'd say, is a bit beyond our current capabilities. That's what you're really getting at - it's not just about DNA repair, but about manipulating everything from the cell cycle (remember that citation I listed about the regulation of DNA repair in the cell cycle) to other proteins that are involved in a number of processes that work with DNA (helicases, polymerases, single-stranded DNA binding proteins, ligases and so on), and all without killing the organism.

Here's another question - what would you do to support the cell's demand for energy to overexpress of all of these DNA repair proteins? More carbohydrates, fats, and lipids would have to be oxidized for energy (outside of suggesting that we reengineer the eukaryotic cell to obtain energy from alternate processes that have yet to be devised, heh), generating even more potentially toxic metabolic byproducts. This is perhaps related to the caloric restriction idea I've mentioned earlier here and elsewhere, where it seems restricting calories (but not necessarily nutrients) may be of benefit in extending longevity.

Another thing to keep in mind is potential roles of other macromolecules in DNA repair. It's been suggested that various chemical modifications to the histones may be an influence in not only gene expression and regulation but also DNA repair. It's been suggested that there's a "histone code" which suggests that the various types of modifications to the histones acts as a certain sort of code itself, but it's still a very active area of research from what I know.

One factor to consider is that if aging is caused by an accumulation of genetic errors over the years, you'd have to keep these functions upregulated for quite some time to garner any real benefit. Basically such that you're spending a lot of effort and energy that might be disproportionate to what it may be worth, and - not surprisingly - you'd have to test to make sure that years of upregulation (overexpression) in and of itself was not dangerous due to unexpected consequences.

I am, to be honest, a bit skeptical about the overly optimistic prospects some present for life extension. I do think that the potential maximum human lifespan has not been reached, and I do think that distinct improvements in the quality of life as one gets older is definitely possible. (80 is the new 40 - that's what I hope they're saying when I'm 75. Heh.) I do think that if we're really serious about pursuing such ends, we need to do so responsibly (we need to make sure that the global society is no longer shackled to fossil fuels but rather has switched over to solar power so there's plenty of energy for our expanding population) and equitably (EVERYONE needs to be able to benefit from such life-extension/quality-of-life-improvement technologies and methods, not just an elite few).

The ultimate point to all of this is that there's a lot of things to read and understand, not just about DNA repair in particular, if you really want to understand what's going on, and especially if you intend to pursue research in this area. Of course, depending on what comes from the research into the evolution of aging, the path might get even steeper if it turns out there might be good evolutionary reasons for aging.......

 

[bioquest]

well my friend who said the enzymes were the same...she says the enzymes that repair mismatched bases and oxidative damage are different but then she said that they cause the same thing to happen/have the same purpose and so there would be an increased problem with mismatched bases

I mean I know that aging's a pretty complex thing to try to understand...and a lot of people have told me they think 120 or 150 is the limit to lengthening the human lifespan but I mean they didn't say they were 100% or anything..and I know if we could extend life we'd have to deal with the consequences such as overpopulation and the problems caused by that

 

[Mike H]

The standard textbook presentation would go something like this.

- Mismatch repair corrects errors in DNA replication in newly replicated DNA, not damaged DNA that has long since been produced. A particular mechanism involving what I'm going to call Mut proteins (at least in E. coli, there's something analogous in eukaryotes by my understanding) identify mismatches using the fact that the parent strand is methylated while the daughter strand is unmethylated. It cuts out the section where the mismatch occurs and then a DNA polymerase and ligase fill in the gap. Its purpose is to catch the rare but occasional error in the replication process.

- Excision repair corrects damage to the DNA well after it's been produced. It does not rely upon the same recognition method as in mismatch repair since the DNA has long since been methylated in this situation. Yes, it can cut out a stretch of nucleotides as happens in mismatch repair, and yes, polymerase and/or ligase is needed to fill in the gap. Its purpose is to repair damage so damage/mutation is avoided.

Yes, there may be some overlap in the proteins used in the cleanup stage (there are a couple of different DNA polymerases, I can never keep them straight in my mind), but the substantive work of identifying and snipping out the offending nucleotide(s) are being done by very different sets of proteins. They may have a similar general strategy for fixing their individual problem, but they identify different problems in different ways. I don't remember all of the details of the biochemistry involved, that's something you would be better served by going to a textbook as they usually have the chemical structures all sketched out very neatly.

Sure, in the abstract, both are being used to preserve the fidelity of genetic information as it is passed along from generation to generation. If that was what was meant, that's fine. But, in the specifics, I'm not entirely sure how an increase in the concentration of proteins involved in excision repair would cause DNA replication to somehow become more error-prone. That's what gets me. I'm sure someone can come up with some sort of explanation for why an increased concentration of proteins involved in mismatch repair might backfire, but it sounds kind of wonky at first pass.

I would also ask your friend for citations. It's impossible to compose any sort of response to what amounts to second-hand information without them.

 

[bioquest]

Would you eventually be screwed due to mismatched dna if you solved all other types of dna damage/potential causes of aging?
So mismatch repair would never repair old errors...even if you overexpressed all proteins/repair mechanisms and you'd possibly eventually be screwed due to old mismatch errors if they were serious/numerous? (ie due to overexpressing another protein or something) yes next time I get any info from my friend I'll ask for citations or something

 

[Mike H]

What's the criterion for "screwed" exactly? If one could somehow manage to minimize the effect of oxidative DNA damage as much as you seem to want to do, and that the only errors are likely going to be the ones that slip past the relatively faithful replication process and mismatch repair, you've essentially cleaned the slate clear of oxidative damage. I'm not sure that point has been made entirely clear - the chance that a particular nucleotide could be copied incorrectly during a round of replication is extremely tiny, something like one in a billion. Mismatch repair enhances the fidelity of the replicated DNA by a factor of 100, as I recall. You should probably try and work out the numbers for yourself regarding the fidelity of DNA replication, the quality of mismatch repair and the various other repair pathways, and see what would happen if you only had the occasional slip-up in replication and mismatch repair. The numbers I've given are off the top of my head. And I'm not sure how you'd suppress spontaneous genetic mutations either, most of which tend to be neutral ones. I'm not sure *why* you'd want to suppress these, as they - by definition - don't have any known effect on an organism.

Mismatch repair, at least in its standard default role, corrects for mistakes that escape proofreading during replication. It leaves correcting damage/errors post-replication to the other pathways which have been mentioned earlier in this thread a number of times. Some of the proteins involved in mismatch repair are somehow involved in other processes that involve DNA, but that's an active area of research which is not something I'm familiar with - I will leave you to investigate that on your own.

There's another potential issue that this made me remember - oxidative stress, as I may have mentioned before (I can't remember), can trigger apoptotic pathways to kill off the cell before it becomes cancerous/infected/otherwise bad for the organism as a whole. Being able to respond to one's environment may be essential and initiate mechanisms to aid the organism in its survival.

While I know this isn't a homework problem or assignment, I do hope you start showing more initiative in following up on the suggestions for reading. What I mentioned about mismatch repair in this reply and my previous one about its role is very clearly laid out in textbooks, for instance. I don't know where you are in your education, but I can assure you that if you do want to pursue research in these fields, you will have a lot of reading to do on your own.

 

[bioquest]

Yah, I know that not having cells with oxidized cells/that would get killed would cause problems with cancer, I thought making something have white blood cells that are resistant to cancer like with par4 gene therapy might help with that

I was just wondering if problems with mismatched bases could be fatal it just seemed like it could be a problem if the mechanism only fixed new wrong mismatched bases and not old ones but what you said made things a lot clearer, sort of

 

[Mike H]

I understand that the question here was how to reduce oxidative stress (as manifested in damage to DNA) that may arise from normal metabolism, but if that starts interfering with various mechanisms intended to allow an organism to adapt to and survive stressful situations.....it's like keeping a small clutch of trees safe while you ignore the fact the rest of the forest is burning. One needs to keep an eye on the big picture. And I'm not sure counting on gene therapy just yet (which is still, overwhelmingly, in research) is a sure-fire way out. Maybe in 20 years gene therapy might be reliable and robust enough, though, although I imagine reengineering a mammalian genome will still be a work in progress.

It may be helpful to think about what a mutation actually is, and that what genes code for (most of the time) are proteins. Whether leucine gets coded for by one codon or another doesn't matter, as it will not affect the protein with regard to its structure or folding or function. If leucine was mutated into another hydrophobic amino acid, and it was just serving a structural role (not involved in the protein's function - catalysis, protein-protein interaction, etc.), it would be neutral. Mutation is what causes variation, which is what natural selection acts upon in evolution. Mutation is not some sort of horrific phenomenon that always causes death, despair, and woe. The Wikipedia article on http://en.wikipedia.org/wiki/DNA_damage_theory_of_aging" [Broken] even points out that while related, mutation and DNA damage can't be simply considered the same.

I'm going to have to bow out of this discussion at this point, as I've long since reached my limit of understanding without becoming an expert on DNA repair myself. I can't imagine that I can say anything that a couple of texts and plenty of review articles can't do a hundred times better, with a wider perspective, and in more detail. If, however, you have questions about the material presented in the texts we've linked you to, I imagine that many more of us can help you since it's far less speculative in nature and on firmer scientific ground.

Disclaimer: This post was written while infectious and on decongestants, to say nothing of the fact that genetics was years ago for me. Corrections welcomed....

 

[bioquest]

Is it dna damage (oxidative, deletions, etc) + mutations/dna mutations that cause aging/problems? In that context what would the mutations mean? I understand you're saying some of them would have neutral effects?

 

[Mike H]

RTFM. Or, in this case, RTFReferences.

The first person to suggest that DNA damage, as distinct from mutation, is the primary cause of aging was Alexander (1967). By the early 1980s there was significant experimental support for this idea in the literature (Gensler & Bernstein, 1981. By the early 1990s experimental support for this idea was substantial, and furthermore it had become increasingly evident that oxidative DNA damage, in particular, is a major cause of aging (Bernstein & Bernstein, 1991; Ames & Gold, 1991; Holmes et al., 1992; Rao & Loeb, 1992; Ames et al., 1993).

-- From http://en.wikipedia.org/wiki/DNA_damage_theory_of_aging" [Broken] Bold text highlighted by me to make the hypothesis regarding what causes aging (at least in this presentation of the theory) clear. It is DNA damage, NOT mutation, that seems to be the culprit in aging (at least as it is presented on Wikipedia).

Below, selected quotes from http://en.wikipedia.org/wiki/Mutation" [Broken]:

Mutations create variation within the gene pool....The majority of these mutations will have no effect

Neutral mutations are defined as mutations whose effects do not influence the fitness of an individual. These can accumulate over time due to genetic drift. It is believed that the overwhelming majority of mutations have no significant effect on an organism's fitness. Also, DNA repair mechanisms are able to mend most changes before they become permanent mutations

A neutral mutation is a mutation that occurs in an amino acid codon (presumably within an mRNA molecule) which results in the use of a different, but chemically similar, amino acid. This is similar to a silent mutation, where a codon mutation may encode the same amino acid (see Wobble Hypothesis); for example, a change from AUU to AUC will still encode leucine, so no discernible change occurs (a silent mutation).

Silent mutations are DNA mutations that do not result in a change to the amino acid sequence of a protein. They may occur in a non-coding region (outside of a gene or within an intron), or they may occur within an exon in a manner that does not alter the final amino acid sequence. The phrase silent mutation is often used interchangeably with the phrase synonymous mutation; however, synonymous mutations are a subcategory of the former, occurring only within exons.

As noted above, it is DNA damage, not mutations per se, that is responsible for aging. Your focus on mutations being some sort of major problem or crisis makes no sense. Most of them are neutral in effect - they are neither beneficial nor deleterious.

You really need to read the references that are linked here or where we point you in the general direction. They're all quite clear, lucid and can answer your questions, at least in my opinion. I'm kind of disappointed - here we've pointed you to a resource that, if you had to buy all those books yourself, would run you a bill in the thousands of U.S. dollars (the NIH Online Bookshelf), those Wikipedia articles must have taken some time to prepare by people (especially the better-referenced and written ones), and still it seems you can't find any of this information on your own.

 

[bioquest]

When you said overexpressing every repair mechanism did you mean only the ones that belonged to the species/type of animal they belonged to- you weren't saying you should add repair mechanisms from other species/animals were you? If I was adding proteins or something via endosomes to increase those repair mechanisms what else would I have to add other than proteins ie what repair things were you referring to other than the proteins? I want to do all the reading I'm just trying to understand a bit better about what you were saying about that relating to that specifically

also did you say overexpress every repair mechanism, to deal with just the oxidative types of dna damage, or to deal with all/some more types of dna damage? (if so what types) ty

 

[Mike H]

I meant all of the naturally occurring DNA repair mechanisms to that organism, not foreign ones. That is all. I had no thought of adding anything to the organism via liposomes or injections or any such means. I simply meant that you would need to upregulate/overexpress every naturally occurring DNA repair mechanism native to that organism.

There are other types of DNA damage than just oxidative types. They are described in the vast literature on DNA replication and repair with a great deal of care and knowledge.

 

[bioquest]

Were you only talking about overexpressing the known, not unknown proteins/mechanisms that do dna repair though?

if I already asked that sorry, I'll go through the thread again, my computer is just loading really slowly right now so I didn't go through the thread to see if I already asked that

 

[Mike H]

How can someone discuss overexpressing unknown proteins or initiating a process through unknown mechanisms by definition, as they're unknown? It doesn't make sense.

Anyway, now I'm really done. This thread has gone on for three weeks, and the amount of material which we've cited or have pointed you in the direction of will last you months, if not longer, given the citations/references at the various websites and papers. I wish you luck with your work, though.

 

[bioquest]

yah sorry I was being OCD and I need to stop being ocd

 

[bioquest]

so does this mean, that the proteins that would repair mismatches and the proteins that would repair oxidative dna damage do not cause exactly the same thing when they repair the dna? You said they may have a similiar general strategy for the fixing their individual problem quote from earlier in the thread:

"there may be some overlap in the proteins used in the cleanup stage (there are a couple of different DNA polymerases, I can never keep them straight in my mind), but the substantive work of identifying and snipping out the offending nucleotide(s) are being done by very different sets of proteins. They may have a similar general strategy for fixing their individual problem, but they identify different problems in different ways. I don't remember all of the details of the biochemistry involved, that's something you would be better served by going to a textbook as they usually have the chemical structures all sketched out very neatly."

Would other proteins, not just the ones that do the actual repair (such as signalling proteins etc) have to be overexpressed too? we're talking about overexpressing all known dna repair proteins, not just the ones that solve oxidative damage but the ones that solve mismatched bases etc too right?

 

[bioquest]

Is it possible to transfer hundreds of proteins/tons of proteins (how many?) at a time via endosomes, nanoparticles, viruses, combination of them etc? (Ie before the first proteins of that much disappeared) I know it would be temporary...but also what would be the most efficient delivery methods/combinations of them and why? thanks (to deliver base/nucleotide excision repair proteins + possibly others, specifically)

 

[bioquest]

my friend messed me up about mismatch repair but um..what i was wondering is can the issue of DNA damage contributing to aging be solved by replacing the genes that control etc dna repair with undamaged genes? Would that be more efficient (to solve the problem etc of dna damage contributing to aging) than just increasing the amount of ubiquitin proteins/proteins that clean up degraded proteins? Even if you could sequence a cell; there would be too much dna damage that contributes to aging in the cell to fix it all with gene therapy right?