DNA damage repair enzymes

[bioquest]

Are there any known enzymes that will fix oxidative dna damage without correcting mismatched bases? Any enzymes that are thought to work like that? thanks

[Ygggdrasil]

Yes. Photolyase (http://en.wikipedia.org/wiki/Photolyase) is one such example that fixes thymine dimers (by using the energy from light which creates thymine dimers in the first place. Pretty cool!). Another example is 8-oxoguanine glycosylase (http://en.wikipedia.org/wiki/Oxoguanine_glycosylase) which repairs oxidized guanine nucleotides. I'm sure there are other examples, but these are the first two on the top of my head.

[bioquest]

well I read that


Photolyase is present and functional in prokaryotes, is present in lower eukaryotes (as yeast) where it is thought to have a minor role, and it has not been found in human cells. However, many higher eukaryotes, including humans, possess a homologous protein called cryptochrome that is involved in light-sensitive regulatory activities such as modulating circadian rhythms.

but even though it's not found in human cells it could be introduced into them, through genetic manipulation or endosomes or something right, I mean could it be?

[Ygggdrasil]

Probably. You'd probably have to modify the enzyme to tell human cells to send the protein into the nucleus (remember prokaryotes have no nucleus so they lack nuclear localization signals that target them to the nucleus), but in theory there should be nothing that would prevent you from getting these proteins to work in humans.

[bioquest]

I know photolyse won't work; What are the other enzymes that would fix oxidative damage but not mismatched bases? 8-oxoguanine glycosylase can't fix all the oxidative dna damage right?

[Mike H]

8-oxoguanine glycosylase is a member of the larger DNA glycosylase family, which contains a number of related enzymes that do similar chemistry on different base pairs. There's even a uracil-DNA glycosylase, which sounded a bit strange to my ear the first time I heard of it, but it does exist.

Nucleotide excision repair (a more general and flexible alternative to more specific DNA repair mechanisms, as I recall) involves a number of proteins in eukaryotes, of which I can remember about two, if I'm lucky, on a good day. You may be interested in investigating that more carefully, as I am of no use on the subject.

[bioquest]

what about the enzymes in this plant 4767-year-old bristlecone pine although I heard some of the cells in it were only a couple of years old

[bioquest]

okay I just realized only the bark was alive in that plant or something/that I was misinformed but

Anyone have insight on what DNA repair enzymes/systems you would add to something to extend it's longevity/help with aging. overexpressing some of them seemed to cause more problems with mismatched bases, so I wanted to know about potential non human dna repair enzymes/systems that could be added to a mouse

[Mike H]

Many of these mechanisms are found in a number of organisms across all domains of life. They are not exactly the same, of course - for instance, nucleotide excision repair (NER) in eukaryotes involves more proteins than in prokaryotes - but the basic notion of NER is conserved.

I'm not aware of anyone trying to add a completely new DNA repair pathway to an organism, but DNA repair research is a rather large world to keep track of, especially if you're not in the field (such as myself). You might find the DNA Repair Lectures (http://asajj.roswellpark.org/huberman/DNA_Repair/DNA_Repair.htm) site at the Roswell Park Cancer Institute useful as a starting point for navigating through the field. I would suggest starting with the references there, at least with regard to finding people who are currently involved with the cutting edge research and go from there.

Good luck!

[bioquest]

Does photolyse fix oxidative dna damage?

[Mike H]

The links provided have very clear explanations of what photolyase does, far more so than what someone who is not a specialist (aka me) can provide. The page on direct repair of DNA damage on the Roswell Park website mentions something that is of relevance to your earlier question about introducing a foreign photolyase to an organism. It takes a simple Google search to find the (freely available) paper which discusses the answer to this question.

I get the impression that we're being asked to spoon-feed homework answers. It may be uncouth to say so, but I can't shake the feeling.....

[bioquest]

well but what I mean is, could photolyse solve the oxidative dna damage problem, or would it only fix some of the damage? I'm not in school yet so its not homework answers I mean I know it's called the homework help forums

[Ygggdrasil]

There are many types of oxidative damage that can occur on DNA. As such, there are many enzymes to recognize and fix oxidative damage. Photolyase will fix only a certain type of oxidative DNA damage (thymine dimers) and will not fix other types.

[bioquest]

Is there any known one enzyme that would repair all oxidative dna damage/is there any known combination of enzymes that would solve the oxidative dna damage problem if overexpressed or something? (Without causing higher problems in mismatched bases) What are the ones, if any, that would do it without fixing or attempting to fix/making mistakes in fixing mismatched bases?

ty

[Mike H]

Base excision repair mechanisms typically rely upon specific DNA glycosylases for proper functioning, so they're hardly an extremely general mechanism. The direct repair methods are usually only there for particular types of DNA damage (such as photolyase), so they will only revert back a particular sort of damage. Nucleotide excision repair relies upon structural perturbations to the DNA upon damage, so it's a bit more general, but it is not foolproof either. There are other mechanisms which come into play under certain circumstances, but are not otherwise active.

There's no one single DNA repair mechanism that I'm aware of that can repair everything with the necessary fidelity to the original genome. This is why there are so many different systems -even mismatch repair - to overlap one another and make sure that your genes stay intact.

[bioquest]

Is there any known thing- any combination or anything of enzymes etc I say combination because you said "there's no one single DNA repair mechanism that can repair everything with the necessary fidelity to the original genome" that if overexpressed or something or just used the way it is would solve/almost solve or anything the oxidative dna problem? without creating a higher problem with mismatched bases? (Since apparently it would see problems that weren't there and try to fix what didn't need to be fixed) I mean I guess it could/would cause other problems I was just asking about the ones that wouldn't cause increased problems with mismatched bases

I mean, are the answers to these no/unknown? with our current knowledge?

So direct reversal methods wouldn't solve the oxidative dna damage problem or anything, they would just work on some types of oxidative damage?

So there's not a group of enzymes, with for example something like photolyse in it, that could solve/mostly solve or anything the oxidative dna damage issue and not cause more problems with mismatched bases?

[Mike H]

From the little I've read since undergrad molecular biology, one would probably have to find a way to upregulate (overexpress) EVERY repair mechanism in order to ensure that adequate fidelity is ensured. The question is, given the baseline rate of mutations in eukaryotes (which is a value I don't remember), just how much upregulation would be required to suppress even that? If you need a huge jump in upregulation for a minimal effect in taking care of baseline mutation, it might not be worth it, which will make sense by the end of this post. I get the impression it could be one of those things where 10% effort will get you 90% of the desired results, but that last 10% of the desired results is extremely costly.

Another issue is how to actually initiate DNA repair mechanisms. I know that people have suggested that there are redox chemistry-based triggers, triggers related to varying points of the cell cycle, interactions of other genes which end up providing a mechanism for initiating DNA repair....the question becomes, if one has to start messing around with the redox chemistry within the cell, or mucking around with the various biochemical events in the cell cycle, you might set off DNA repair at a higher rate, but at what cost to the rest of the cell?

Direct repair methods reverse specific chemical (oxidative) modifications, they're not general ways of cleaning up DNA. Photolyase reverses the production of cyclobutane pyrimidine dimers. O6-alkylguanine alkyltransferase reverses the alkylation of guanine. They are not more general methods like the various types of excision repair pathways.

There might be hints or even partial answers to potential questions to your answers, but they're probably strewn about in the research literature, alluded to but not really strongly asserted since it's too early to say. I did get curious about the idea of transplanting a foreign photolyase into mice - Google "mouse foreign photolyase" one of these days, and you'll find the paper which describes an experiment that did just that. What I've presented is pretty much the textbook knowledge of DNA repair (as that is all I am actually familiar with myself). It would make for an interesting literature review or even meta-analysis, though.

[bioquest]

it's only the enzymes, nothing else that repairs DNA right

But when combined do the direct reversal methods reverse every type of oxidative dna damage? (When combined) or do they not cover all the types?

If you overexpressed all the dna repair enzymes, and that caused a higher amount of incorrectly fixed mismatched bases, would the dna repair enzymes also eventually correct the mismatched bases? Or would the incorrectly fixed mismatched bases still be a big issues

[Mike H]

Well, it depends on the mechanism as to what does the work of DNA repair. For something like photolyase, it's just the photolyase as I recall - it breaks the bonds between the two pyrimidine bases, and that's pretty much all there is to say. But if you look at the nucleotide excision repair pathway (see here (http://asajj.roswellpark.org/huberman/DNA_Repair/ner.html)), there are a lot of proteins involved in the process. The base excision pathway (see here (http://asajj.roswellpark.org/huberman/DNA_Repair/ber.html)) makes note that you need the appropriate DNA N-glycosylase, followed by an AP endonuclease, followed by DNA polymerase to make the entire repair.

My understanding is that the direct reversal methods are not able to reverse every type of oxidative damage. To quote the Roswell Park website,

The huge variety of DNA-reactive chemicals in our environment combined with the huge variety of alterations that can be produced by radiation and by oxidative and free radical attack on DNA can generate so many types of damage that coping with all types of damage by evolutionary development of damage-specific DNA glycosylases would be difficult if not impossible.

If that's being said about the base excision repair pathway (which uses DNA glycosylases), I would think it applies even more strongly in the case of direct DNA repair.

As for your last two questions, not sure. Sounds like it would make for a good set of experiments, though, if they haven't already been done.

[bioquest]

Could/Would the increase of problems with mismatched bases etc caused by the enzymes be permanent- would they only be able to fix those errors after 2 generations/a limited amount of time or could they fix them anytime?

is that known? My friend said they only repair mismatched bases after 2 cell generations or she said something like that indicating that after an amount of time has passed, they don't correct the mistake anymore, but maybe if all the enzymes were overexpressed they would eventually fix all the mistakes?

[Mike H]

I am not familiar enough with the DNA repair area to say anything about such questions. I even checked my old biochem text since I'm doing some book reorganization, no such luck. These questions appear to be a step or two above, at the very least, the default undergraduate treatment.

If you could get a citation or two out of your friend regarding these phenomena she describes, it would be very helpful in either answering your question or aiding you in interpreting the articles/sources. We might even get a journal club-esque conversation going on about this topic, which could be interesting. But I, at least, am not familiar enough with this area to go ahead without some citations/references.

[Ygggdrasil]

Another issue to consider. It seems the cell has taken advantage of the effects of oxidative DNA damage and the repair pathways to regulate the expression of genes. Therefore, overexpression of DNA repair genes, as with any genetic manipulation, could have unintended consequences.

See also:
http://blogs.nature.com/thescepticalchymist/2008/02/chemiotics_we_had_to_destroy_t.html

[bioquest]

I was wondering if the radiation resistant abilities of bacteria, insects etc might lead to the solution/somewhat solution to oxidative dna damage in a mammal (I read that it's the oxidation resistance of the (dna repair?) protein(s) in Deinococcus radiodurans that help it to survive radiation, I don't know if that's because the bacteria has a high level of antioxidents in it or for some other reason, but maybe the radiation resistant protein could help with oxidative dna damage, if for example it's not resistant to oxidation just due to antioxidents?)

Then I read this though, this quote is from here http://www.madsci.org/posts/archives/2003-12/1072227809.Zo.r.html they also give past speculation on reasons for the radiation resistance and some information on why some of the reasons people thought the radiation resistance existed is wrong and list some general things that aren't behind the radiation resistance

"it is clear that insects are resistant to ionizing radiation and that this resistance is an inherent property of their cells. But it is not clear exactly what the basis of this cellular resistance is, although the dominant theory is that it relates to the relatively small amount of DNA in insect cells" would this go for bacteria and other things as well, do you think then that the radiation resistance of things and/or the radiation resistant protein in Deinococcus radiodurans would lead to a solution/somewhat solution to the oxidative dna damage problem? Why/why not?"

Yah I think people would definitely be interested in dna damage and repair to have discussions, to join a club on it or something. Um, they'd probably be way more caught up on it than I am though

I also thought this was interesting

quote from http://www.madsci.org/posts/archives/2003-12/1072227809.Zo.r.html
"The radiation resistance is inherent to the cells, since cells derived from insects are also radiation resistant when grown in cell culture. For example, a dose of 60 Gy is required to produce a 80% kill of insect cells, while doses of 1-2 Gy are sufficient to generate this level of killing in mammalian cells"

does that maybe mean it's not the antioxidents alone that are responsible for radiation resistance and that that could help with solving the oxidative dna damage issue somewhat? What do you think is responsible for the radiation resistance based on the above paragraph? thanks

Also how much would the body destroying any cancer cells that arose, if the body had that ability, help with the dna damage problem?

[Mike H]

Ygggdrasil - Thanks for that link, it made for very interesting reading.

bioquest - First, take a look at these three links. http://en.wikipedia.org/wiki/List_of_number_of_chromosomes_of_various_organisms and http://en.wikipedia.org/wiki/Deinococcus_radiodurans and http://en.wikipedia.org/wiki/Chromosome Read them? No? Read them first. Do it. Done yet? Good, now we can proceed.

Let me talk about the insect thing first. If you look at the list of chromosomes of various organisms, you'll see for yourself that everyone's favorite insect model organism, Drosophila melanogaster (the common fruit fly), only has 8 chromosomes, which is, as I recall, indicative of most insect genomes (barring such things like insect polyploidy). The human genome contains 46 chromosomes, which is more than five times as large as the Drosophila genome. That means humans have to run DNA repair on a much larger genome than do insects. At the site you posted, it is noted that

There was speculation in the 1950's that the number of chromosomes influenced radiosensitivity, and that insects had fewer chromosomes. There may be some truth in this speculation, as we now know that (in general) the less DNA a cell has the more resistant it is to ionizing radiation. Since DNA is the critical target for cell killing (in mammalian as well as insect cells), it is logical that the less DNA there is, the harder it is to hit. While the density of ionizations produced by X-rays is sufficient that animal size could not explain resistance, the density of ionizations is low enough that total cellular DNA content could explain resistance........ So it is clear that insects are resistant to ionizing radiation and that this resistance is an inherent property of their cells. But it is not clear exactly what the basis of this cellular resistance is, although the dominant theory is that it relates to the relatively small amount of DNA in insect cells.

The take-home message from this, in my reading of it, is that the enhanced ability of insect cells to resist radiation damage is most likely due to its smaller genome. It has a lot less DNA to repair compared to mammals. To give a non-scientific analogy, think of mowing and landscaping a yard in the summer when you've got plenty of rain, so it needs to be done regularly. You're going to do a neater and better job if it's only one acre versus tending a huge 10 acre area. You're going to miss more spots, more poorly done edges, show more signs of fatigue with a 10 acre area. It has nothing to specifically do with particular aspects of insect biochemistry/DNA repair - it just means that there are fewer genes for radiation to effect in insects (8 chromosomes' worth or so versus 30 or more in various mammals) and that the insect DNA repair mechanisms have a lot less ground to cover than a typical mammalian system. It's a matter of scale, not necessarily antioxidants or specialized DNA repair mechanisms.

With regard to D. radiodurans, it has two chromosomes (chromosomes are probably not the best term to use with respect to bacteria, but that's a semantic issue I'd prefer not to get into at the moment, thank you very much). What little I know I about D. radiodurans' resistance to radioactivity is summed up in the Wikipedia article. I'm not an expert with D. radiodurans (never worked with it, only had a few mentions of it in a few classes long ago, and read the occasional news snippet about it since then), but again, you're dealing with a very small genome, there are a few copies of it in each individual D. radiodurans bacterium, and its likely relevant ability to endure very dry conditions. Here, sure, you apparently have accelerated DNA repair mechanisms (not least due to multiple copies of its genome) and high concentrations of manganese which supposedly serves as an antioxidant. I'm not sure if trying to reengineer the human species to have a couple of copies of its genome is advisable. At the very least, you'd have one heck of a time getting it past a review board. If you suggest that maybe there's a way to increase manganese intake/absorption in humans to provide a similar benefit, I would suggest you read up on the nutritional biochemistry of manganese. It is a necessary trace element, to serve as a cofactor in a number of metalloproteins, but too much can lead to undesirable side effects.

I would be sure to read the link that Ygggdrasil so kindly provided. It may be that efforts to improve one's health may turn out to be counterproductive, as there might be processes at work that one can't easily fathom.

Also, I think you might find the NIH's free online bookshelf here - http://www.ncbi.nlm.nih.gov/sites/entrez?db=books - to be an interesting and useful resource. Biochemistry, molecular and cell biology, genetics, cancer research, and more, all easily searchable.

[bioquest]

Is the DNA repair protein in the bacteria resistant to radiation though, (i read that it was I can look online for more info) or would that just be because of the antioxidents in the bacteria?

Also it would be impossible to use antioxidents in a way where they were never free radicals in the human body right? (assuminng free radicals they wouldn't be necessary for a human to survive although someone said to me they would be, I'm not sure)

[Mike H]

From the Wikipedia article on D. radiodurans in response to your first question:

Michael Daly has suggested that the bacterium uses manganese as an antioxidant to protect itself against radiation damage.[10] In 2008 his team showed that high intracellular levels of manganese(II) in D. radiodurans protect proteins from being oxidized by radiation, and proposed the idea that "protein, rather than DNA, is the principal target of the biological action of [ionizing radiation] in sensitive bacteria, and extreme resistance in Mn-accumulating bacteria is based on protein protection".[11]

It's an open question, but this hypothesis is not an unreasonable one. It could be that in D. radiodurans the presence of manganese(II) serves as antioxidant protection for the proteins. More research is needed.

I'm not sure what you're trying to get at with your second question. I can say the following - free radicals are essential in certain roles, so they are necessary, at least based on our current understanding of them. In the link that Ygggdrasil posted, antioxidant use, at least in one case, led to an increased incidence of lung cancer. It's not that they necessarily acted as free radicals, but perhaps prevented necessary free radicals from doing their jobs. (I haven't read that article which is linked there, and I'm probably not going to, given the backlog of papers I should be reading at the moment.)

[bioquest]

If the DNA repair protein in the bacteria is radiation-resistant but not because of the antioxidents, how could that help with dna repair in a mammal? Is it possible that all we need for good dna repair is something like oxidative/radiation resistant repair proteins/enzymes

[bioquest]

k, never mind, I just read what you posted I misread it a bit before sorry that was really stupid of me, I was kind of sleepy last night

But I think it would be cool to have a club or journal club-esque conversation or something where people discuss dna damage and repair, I mean I think it's a relevant, interesting, important subject to find out about and discuss and I think a lot of people would be interested in it. Since for one thing I don't know how you'd extend human life without solving the dna damage issue, unless you could regenerate whatever you needed to in the body using outside stem cells, (or using something like nanotechnology or I don't know) like embryonic ones or cells from epilepsy patients if that thing I read regarding the mcknight brain institute was correct. I don't see any real solution to the dna damage issue other than overexpressing the enzymes so I'm really interested in that, if anyone wants to talk with that about me. At first I was like, I'm going to make it my goal to find out all the unknown stuff about genes and proteins (obviously not by myself)...and then I narrowed it down to what I'm interested in which is overexpressing dna repair enzymes and now I'm interested in just that, but I'd still like to learn about other things and about genes and proteins

[bioquest]

My last question on it though is, are there any theoretical way(s) that could be used now that would make it so that there would be no free radicals in the brain/any specific organ that existed long enough to do damage, that could be used in a way the mammal/brain would survive?

Also could the ability to regenerate as fast as biologically possible theoretically solve the oxidative dna damage or would cells still get oxidative dna damage?

[Mike H]

My last question on it though is, are there any theoretical way(s) that could be used now that would make it so that there would be no free radicals in the brain/any specific organ that existed long enough to do damage, that could be used in a way the mammal/brain would survive?

Also could the ability to regenerate as fast as biologically possible theoretically solve the oxidative dna damage or would cells still get oxidative dna damage?

Short answer: I don't know.

Long answer: Asking what is or isn't theoretically possible starts to bring this discussion into ground bordering if not infringing on the overly speculative, especially when at least one of its participants (me) has not a whit of actual, useful experience in working with either eukyarotic systems or DNA repair mechanisms. If there is someone with more knowledge, who can make an educated guess based on detailed knowledge and actual experience, that's a different story. The thing to remember is that someone could follow up on an experimental anomaly this week in a lab somewhere, publish the results late next year when they've followed through, and 10 years from now we all look back and say, "Wow, that discovery really jumped us way ahead of where we thought we'd be at the moment." Or they could not, and people miss some observations or make some understandable but ultimately incorrect conclusions, and suddenly we're far behind the curve as it was originally imagined.

I don't mean to discourage you from asking questions, but these are questions which demand a far more extensive knowledge of the field in question and its literature than is typically passed along to non-specialists in the field, at least from what I can tell.

I would think about perhaps going back, really delve into the links that have been shared in this thread (and the citations/references contained within those links), and work your way through things in a more methodical and less rushed manner. Right now I feel like you're asking us to run up to the peak of Mount Everest ("It's only five and a half miles, everyone, it's not that far of a distance!") with you, instead of making our way slowly through the various base camps at different altitudes. Read up on DNA repair. Read up on what triggers/initiates DNA repair. Read up on antioxidant mechanisms, on the roles that free radicals do carry out in biological systems. Read up on alternate hypotheses on how to extend an organism's life like caloric restriction. (No, I have no information on it beyond the Wikipedia entry. Sorry.) Read up on how scientists actually overexpress or silence gene expression in cells. Take notes, draw yourself diagrams, sketch out pathways, really take your time and go step by step.

[bioquest]

Are there synthetic DNA repair enzymes that are being used/made or anything that could solve the oxidative dna damage issue? I mean I know they've made synthetic DNA

[Mike H]

Are you talking about known DNA repair proteins that were the focus of site-directed mutagenesis studies to modify their behavior or proteins designed from scratch? If the former, I have not a clue. I imagine that literature would be quite an effort to map and analyze. If the latter, there are only a few published results of proteins being designed from scratch to do actual chemistry (not just ligand or cofactor binding) and none of them, as I recall, had anything to do with DNA repair.

You should probably look up "protein engineering" and "protein design"/"de novo protein design" for a better idea about the latter, if that's what you were asking about in your question. It's a very interesting but very challenging field.

[bioquest]

are there 2000 year old trees or anything, or is it just the bark or something from those that are alive after thousands of years? I thought there was a type of tree that lived to 2000

[bioquest]

I mean, is it just the bark on trees like this http://www.earthsky.org/teachers/article/giant-sequoia that live a really long time, or is it the tree itself, like could the dna damage ability etc of these trees be transferred to animals and could the animal then live a really long time, or it is just the bark or something on the trees that live a really long time?

it says here
The oldest big trees are about 3000 years old.

http://www.icogitate.com/~tree/species/sequoiadendron.giganteum.htm

[bioquest]

Flooding cells with antioxidents/things that kill free radicals wouldn't stop all oxidative dna damage because the mitochondria/free radical production and the mitochondrial dna are so close together right?

Also sorry if this is an ocd question but you wouldn't be able to change a human into a different organism than a human using genetic manipulation or something right?

[Mike H]

Some antioxidants (such as certain vitamins) are known to be lipid soluble and therefore prone to accumulation and subsequently become toxic since they can't be excreted as quickly as water soluble compounds. So why would anyone want to flood an organism with them? Derivatives of vitamin A are known to serve as hormones which end up involved in regulating gene transcription. Things are never as simple as they seem, which seems to be a point that is overlooked.

Also, this interest in somehow quenching all oxidative chemistry in the cell is getting a little odd. We oxidize carbohydrates and fats in our cells for energy. We oxidize foreign substances to ultimately aid in us excreting them. We oxidize various unreactive compounds in our cells to subsequently functionalize them with new chemical moieties. There are entire journals dedicated to free radicals in chemistry and biology, both their important roles and their damaging effects. The NIH link to free books I shared earlier is a resource you should be focused on fully exploiting - there's solid material in genetics, molecular & cell biology, and biochemistry that should be worked through before trying to tackle the immense research literature out there in DNA repair, free radicals in biology, and the like.

I really don't have anything else knowledgeable to say about any of these topics at this point.

[bioquest]

So, when you say you'd have to overexpress every repair mechanism, would that solve the problem of higher problems with mismatched bases since one of those would also fix those? Could you go into more detail about what you meant when you said every repair mechanism? Also what stuff is there to repair dna other than enzymes?

the little I've read since undergrad molecular biology, one would probably have to find a way to upregulate (overexpress) EVERY repair mechanism in order to ensure that adequate fidelity is ensured.

[Mike H]

Regarding your questions, it's impossible to say with any degree of certainty. (I'm starting to sound like a broken record here.....) We're still learning about what various mechanisms trigger DNA repair, there are still plenty of questions about how genes get turned on and off, and it's impossible to speculate about your proposed experiments since - as has been noted before - data trumps theory every time, so it's very possible there are other pathways which link together processes that we might not expect. Also, my point stands about having to overexpress being a potentially inefficient idea stands, as I described in my earlier post.

As for what I meant by every repair mechanism, I meant exactly that - every mechanism listed and described to varying degrees of detail in the Wikipedia article, the Roswell Park lectures, and in the books on the online bookshelf at NIH. Not to mention all of the citations/references listed in those sources! As for what proteins (including enzymes) are involved in DNA repair, all of that information is contained in those sources that have been very kindly linked to in this thread.

You should be thankful that we've provided you with such a pool of references to dig into this topic more deeply. Unless someone who actually specializes in DNA repair/free radical biochemistry shows up, you have pretty much reached a generalist's knowledge limit in this field. If you want more information/insight, you either have to contact someone who does this stuff for a living or become something of an expert yourself. Sorry.

[bioquest]

I am really thankful you were helping me
Sorry if I sounded stupid

[Mike H]

It's not a matter of sounding stupid at all, it's a matter of recognizing that the questions you're asking are good ones that can't be answered simply by possessing a relatively general knowledge base in biochemistry, molecular biology, and the like. Your questions that layer synthetic biology, gene therapy, and other current areas that are very much still in a research stage on top of one another make it very difficult to answer speculations that involve them all to one degree or another. That's why I've been suggesting to you to take advantage of the resources and links that we've provided. The specialized books on DNA repair and other fields that are cited in these links build upon that baseline of knowledge, as does the research literature. And you may still come to the point where the only way to really obtain any sort of answer to some of these questions is to do the experiments. I don't know what your career intentions are, but if you really are interested in these questions that you've put forth, either you or someone else will have to run those experiments, most likely in multiple model organisms at that.

It's part and parcel of becoming familiar with a field of inquiry - I spent the first six months of working in a lab in grad school mostly reading papers and getting some simple basic experiments worked out for subsequent studies. There's no way to circumvent it.

[bioquest]

Delivering lots of the dna repair enzymes would not be enough to overexpress them right?- You'd probably have to genetically modify a mouse or something to overexpress the enzymes?

[Mike H]

Saying you want to deliver DNA repair proteins sounds as if you want to pump an organism full of DNA repair proteins.

Saying you want to overexpress DNA repair proteins sounds as if you want to coax the cell into making more DNA repair proteins.

They're not the same thing. If you want to inject an animal full of proteins, that does not necessitate doing any sort of genetic manipulation. If you want to overexpress the proteins, you either need to genetically engineer it or find an alternate way to induce overexpression. This ties back to my point about being able to figure out what triggers DNA repair mechanisms, and how it all ties together.

I can't believe I did this (I'm just too pleasant for my own good at times), but there is a very recent review on regulation of DNA repair during the cell cycle that came out this year. The citation is:

Dana Branzei and Marco Foiani. (April 2008) "Regulation of DNA repair throughout the cell cycle." Nature Reviews Molecular Cell Biology. 9(4): 297 - 308.

This is mostly full of citations from work done after 2001 (which is after the last time I studied any of this material in even the slightest bit of detail). I found it on the first page of a Google search for "DNA repair regulation." It contains over 150 citations, which I am sure will serve as a superb starting point for some more modern information on DNA repair in cells, and will probably make some of the earlier responses obsolete or even incorrect in this thread.

Have fun!

[bioquest]

Is adding tons of the protein to the cell with endosomes or something, a temporary way to induce overexpression of the protein?

Saying you want to deliver DNA repair proteins sounds as if you want to pump an organism full of DNA repair proteins.
Saying you want to overexpress DNA repair proteins sounds as if you want to coax the cell into making more DNA repair proteins.
They're not the same thing. If you want to inject an animal full of proteins, that does not necessitate doing any sort of genetic manipulation. If you want to overexpress the proteins, you either need to genetically engineer it or find an alternate way to induce overexpression.

It just seems like having a cell make it's own proteins and having a cell have tons of proteins delivered to it seems really similiar

[Mike H]

It is a matter of semantics, but one which is important to grasp. I'm oversimplifying here, but I think it captures the basic differences.

Delivery indicates that you are adding something externally to the cell, to increase the concentration of some (macro)molecule within the cell.

Overexpression indicates that you are coaxing the cell to synthesize more of a particular (macro)molecule either by genetic means or other methods, to increase the concentration of some (macro)molecule within the cell.

In the first case, you need to make sure that what you deliver is stable to be administered in a suitable way (oral, inhalation, intravenously, direct injection) and can then be routed to the desired area without being degraded or inducing an immune response. Even when it reaches the target area, it has to be able to enter the cell without too much difficulty. You may want to make sure that you avoid any feedback mechanisms which shut down natural production of the (macro)molecule upon an increased concentration of the desired (macro)molecule if that's important to you - this is the issue, for example, with steroid use, where externally taken steroids shut down the body's natural production of them, and why getting off steroids can be problematic for some people since they're not producing enough (or any) naturally.

In the second case, you need to make sure you can induce overexpression of the desired (macro)molecule in its functional form without causing negative effects upon the cell. This may require extensive genetic manipulations or delicate chemical induction methods. It may require both. Or it could require that you expose the organism to particular conditions that "shock" the organism into some desired response.

Again, I reiterate my suggestion to peruse standard undergraduate-level textbooks available at the NIH Online Bookshelf to further your broad-spectrum background in biochemistry, molecular/cell biology, and genetics. Any such discussion you want to get into regarding this topic or related ones will depend on such knowledge.

[Ygggdrasil]

Overexpression specifically means to inducing a cell to produce more of a specific protein. So, no, although injecting tons of a specific protein into a cell will increase the amount of protein in the cell, it will not induce overexpression of the protein. To induce overexpression, you would have to perform some genetic manipulation or inject the DNA for the specific protein.

Overexpression (having the cell make its own proteins) v. delivering proteins are slightly different. Overexpression is harder to accomplish, but can also lead to more permanent elevation of the amounts of protein. Delivering proteins raises the amount of protein only temporarily because proteins are constantly being degraded by the cell.

[bioquest]

Do we only know how to overexpress some repair mechanisms (as opposed to them all?) through genetic manipulation? I mean I read once that they overexpressed dna repair enzymes in mice, but I don't know if they did that with each repair mechanism. I'll try to read as much as I can about it, it seems like overexpressing each one of them might be a bit complex?

[Ygggdrasil]

Causing cells to overexpress all of the DNA repair enzymes would be a very tough task. Overexpressing one gene is hard enough to accomplish, and performing it on a large set of proteins would be a very difficult and time consuming task.

That said, there may be some tricks and shortcuts. Some proteins are known to sense DNA damage and cause the expression of a variety of proteins that help the cell to respond to DNA damage (e.g. DNA repair enzymes, proteins that halt the cell cycle, and even proteins that induce programmed cell suicide if the damage is too severe). One such protein is the very famous tumor suppressor protein p53, sometimes referred to as the "guardian of the genome." The wikipedia article on p53 isn't that great (http://en.wikipedia.org/wiki/P53), but you can probably find some decent resources on p53 in a molecular biology text.

[bioquest]

It would probably be possible to deliver the proteins etc to the cells though rather than make the cells produce more of them?
Maybe it's possible to deliver every type of DNA repair protein/mechanism to the cells so that they have a large amount of them (rather than make the cells produce more themselves) and see what happens. But if the proteins that fix mismatched bases are identical to the ones that fix oxidative damage wouldn't you just get an increase in mismatched bases? So basically I was wondering if the enzymes were the same/was wondering about those things thanks for the previous information though, I'll try to do all the reading

[Mike H]

Ygggdrasil's earlier point about the constant turnover of proteins by the cell stands - even if a large amount of desired proteins could be delivered to a cell, they would eventually be degraded. It would be a one-time-only increase, unless you're thinking to supplement these proteins regularly to the organism. The other aspect is that there is the entire timing thing that is discussed in the article that I cited regarding the regulation of DNA repair at different points in the cell cycle, which adds another level of complexity.

Mismatch repair uses mostly different proteins, although as I recall certain ones like single-strand binding proteins are used in both cases, and there may some fine point about different DNA polymerases being used, although I can't remember whether that's organism-dependent, someone will probably want to check me on that. It would be good to read up on that, especially as many of these mechanisms were first worked out in bacteria and then in eukaryotes.

Is there a citation to back up this claim that overexpression of proteins involved in mismatch repair results in more mismatched base pairs, or is this all going off a vague mention somewhere that no one can really remember? Because I can very easily imagine someone did something with a specially bred model organism that may have been the product of years of breeding that was used to answer a particular set of similar but not directly overlapping questions, and thus makes generalizing the observations about something it wasn't really intended to figure out to all organisms a bit of a stretch, at least in my book. Hope that made sense.....

[bioquest]

yah I was thinking about supplementing the proteins regularly to the organism
Do you think it would be impossible right now to genetically manipulate a mouse/mammal to overexpress the protiens?

I just read somewhere that overexpressing enzymes caused higher problems with mismatched bases but that's not very specific about which enzymes or anything and then my friend said they were the same and I thought the dna repair enzymes worked together, but I guess the proteins could still be different?

[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 the DNA damage theory of aging (http://en.wikipedia.org/wiki/DNA_damage_theory_of_aging) 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 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/Mutations (http://en.wikipedia.org/wiki/Mutation):

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)