Repair Mechanisms of the Cell – Part 1

The following article reproduces the article "Repair Mechanisms in the Cell. 1. Nucleic Acids – a Fragile Information Storage" by Dr. Boris Schmidtgall, published in the Studium Integrale Journal (31st Year ┃ Issue 1; May 2024). Quotes without further source attribution originate directly from the aforementioned original article.

It is an everyday experience that everything earthly is subject to decay. Accordingly, repairs are among our most frequent actions. It is a fundamental principle that as the complexity of a device increases, so does the effort required for its repair. Extensive sets of tools as well as strategic considerations are often needed to carry out complex repairs:

1. Recognition / Assessment: The damage must be correctly assessed and an appropriate procedure selected. It must be possible to distinguish between the undamaged and the damaged state, and the actor must know various procedures "that lead to success with different types of damage."

2. Gaining Access: Often, the damaged area is hidden by other components and must first be made accessible for repair. This requires precise knowledge of the larger context of the system.

3. Reinforcing the Destruction: It is often necessary to turn a partial break into a complete one so that subsequent healing can succeed. This is known from both building renovations and bone fractures. – "The targeted breaking of a damaged part is followed by joining or reconstruction with subsequent restoration." Precision is particularly important here to avoid further damage and irreversible destruction.

4. Restoration: The repair of a complex damaged unit requires profound technical knowledge, skill, and experience. Nevertheless, almost everyone has experienced that improperly executed repairs can also increase the damage.

Repairs are often demanding tasks; therefore, it would save us a lot of effort if our technical inventions could repair themselves. With our inventions, this is still a long way off, but that is exactly what happens in biological cells:

Various complexes of biological nanomachines perform diverse and highly complex repairs. These are addressed in a three-part article series; this first part focuses on the repair of nucleic acids. Nucleic acids are DNA and RNA. The former represents the blueprint of the respective organism and consists of the commonly known letters A, T, C, and G. RNA is similar in nature but essentially serves as a transmitter and regulator of the "flow of information from DNA to proteins."

Causes of Damage to Nucleic Acids:

Nucleic acids are relatively stable with regard to their chemical rate of decay, but from a chemical standpoint, they are in an unstable state. In other words: these molecules in the cell are constantly subject to an irreversible tendency toward decay, even if that decay proceeds rather slowly.

The resulting damage would lead to the rapid death of the individual were it not for enzymes that constantly repair the damage. The causes of this damage can be of a biological or non-biological nature. These include, among others, electromagnetic radiation such as UV radiation, which can change the structure and bonds of DNA and RNA; through oxidation (i.e., reaction with so-called reactive oxygen species), the shape as well as the chemical and biological properties of nucleic acids are altered; water can cleave chemical bonds in DNA, and double- and single-strand breaks can occur due to faulty copying processes.

For a more detailed discussion of these and other causes, see the original article and the corresponding box on page 33.

Damage to nucleic acids influences almost all processes in the cell. Various scientists estimate the number of DNA damages in humans per day and cell at 10⁴–10⁵ (Lindahl 1993; Yousefzadeh et al. 2021). While reliable data for a comparable estimate is still lacking for RNA, the repair effort there is likely to be similarly enormous.

"Consequently, all organisms must possess highly efficient, functionally very broad-based, and constantly available repair mechanisms in order to survive." If damage is very severe or even irreparable, programmed cell death (apoptosis) occurs. This selfless behavior aims to preserve the individual.

Repair Mechanisms for DNA Damage:

Cells respond to damaged nucleic acids with "a multi-stage, highly complex process" that is still largely misunderstood today (Huang & Zhou 2021). Briefly summarized:

1. Perception and Signal Transduction: "Sensor proteins interact with the damaged site and transmit a signal that triggers various measures aimed at repairing the damage."

2. Implementation: These measures include halting the cell cycle and activating appropriate repair processes – or triggering programmed cell death.

The second step of this process implies a weighing of the repair effort followed by decision-making. If a repair is then carried out, a choice of the repair program appropriate to the respective damage is also necessary.

All living beings are equipped with the following repair tools:

1. Recognition factors: Proteins that identify the damage.

2. Helicases: Enzymes that unwind the DNA helix.

3. Nucleases: Enzymes that cut out damaged parts of the genetic material.

4. DNA polymerases: Enzymes that re-synthesize [reproduce] the removed part of the genetic material.

5. DNA ligases: Enzymes that link the newly synthesized strand with the still intact genetic material.

Repair of Double-Strand Breaks (DSB):

Double-strand breaks represent the greatest danger to cells. It is particularly demanding to bring the separated strand ends into direct proximity to one another for re-linking – a particularly difficult task in the very dense cellular environment (Vogt & He 2023).

E. coli bacteria served as the first research objects for DSB repairs. As early as 1974, the hypothesis was formulated under the designation "SOS response" that "DNA strand breaks in bacteria trigger a response with the goal of repairing the damage (Radman 1974)." Many further details are known today about this highly complex process (Lima-Noronha et al. 2022). The RecA protein is an exceptionally important and multifunctional control element that triggers the SOS response.

RecA recognizes the exposed single strands and binds to them. This causes the cleavage of the LexA gene, which prevents the production of a protein and protects the DNA. In this process, a group of regions in the bacterial genome are exposed that are responsible for activating the repair program. This includes increased production of the RecBCD protein complex. This large protein complex specifically unwinds the DNA at the DSB site and prepares the DNA for the restoration of the damaged section using a DNA polymerase (a molecular machine for DNA production) by making cuts at specific locations.

Furthermore, with SulA, a protein is produced that prevents cell division so that the repair can be completed without passing on the damaged genetic material. In addition, the production of DNA polymerases is increased by the SOS signal.

The repair process most frequently performed by cells is called "homologous recombination" (HR) and proceeds as follows:

The previously formed DNA-RecA complex (unit) searches for a double-stranded DNA (dsDNA) that structurally matches the damaged DNA section. This undamaged dsDNA then serves as a copy template for the reproduction of the damaged DNA. After completion, a DNA ligase [machine for linking DNA strands] connects the new and the intact old sections with each other.

Homologous recombination consumes a lot of energy and is a very low-error repair process. This is due to the polymerases active in the repair process, which recognize incorrectly incorporated nucleotides and replace them with the correct nucleotides. However, HR cannot always be carried out, for example, if the DNA is too severely damaged or if no suitable copy template is available.

For this case, organisms are equipped with DNA polymerases that can realize repairs even without a copy template. With these, however, the susceptibility to errors for incorrectly incorporated letters and loss of information is higher.

"The model presented here is based on research work on E. coli bacteria but is in principle applicable to many other bacteria (Lima-Noronha et al. 2022)." The repair process is considerably more complex in organisms with a cell nucleus (eukaryotes), but there are some similarities regarding the enzymes. In contrast, the signal that triggers the repair measures is completely different, as is its transduction (Huang & Zhou 2021; Niida & Nakanishi 2006).

In Archaea (single-celled microorganisms without a nucleus, one of two domains of prokaryotes), many repair elements have been discovered that are similar to those for DSB. However, significantly less is known about the entire repair system and the information underlying it in their case. According to current knowledge, Archaea living under harsh conditions (hot springs, Dead Sea, etc.) possess the same repair tools as other Archaea. This fact is so far not understood and surprising, as their living conditions lead to significantly higher mutation rates (Marshall & Santangelo 2020).

Repair of Base Mismatches (mismatch repair, MMR):

Base mismatches are a frequent result of faulty DNA duplication (replication) and are encountered approximately every 10⁶ DNA building blocks. In eukaryotes and bacteria, the mechanism for restoring the correct base pairings is reasonably well studied (Modrich 2016). The difficulty here lies in simultaneously determining both the mismatch and the strand "that contains the incorrectly incorporated nucleotide."

E. coli bacteria accomplish this task with the help of the proteins MutL and MutS. They form a unit, bind to the DNA, and slide over it (Modrich 2016). If the MutS-MutL complex encounters a mismatch, another protein, MutH, comes into action, which can distinguish the newly copied strand from the template thanks to the still missing methyl marker and inserts a cut specifically on the non-methylated strand.

This ingenious procedure ensures that the cut is placed on the newer copy. To explain: a double strand consists of two single strands, one of which is the copy template and the other is the copy. The copy template is older and marked with a methyl group ("methylated") at certain points. Based on the absence of this marker, the copied and thus probably faulty strand can be identified (Marshall & Santangelo 2020).

The helicase UvrD unwinds the DNA helix and identifies the cleavage site. Thereupon, the strand part with the incorrectly incorporated nucleotide is removed by an exonuclease, newly produced by a DNA polymerase, and linked by a ligase to the single strand serving as a template. According to current knowledge, E. coli bacteria require at least eight different proteins for the repair of mismatches.

Eukaryotes possess a set of tools for repair similar to E. coli bacteria; however, they have no direct equivalent to MutH, whose function is instead performed by other enzymes. In contrast, among prokaryotic microorganisms of the domain Archaea, equivalents to MutS/MutL have only been detected in a few species (e.g., Methanosaeta thermophila). "In many Archaea, MMR is carried out by NucS/EndoMS nucleases, which differ significantly in structure from MutL/MutS. There are, however, also Archaea in which neither MutL/MutS nor EndoMS have been identified (Marshall & Santangelo 2020)."

Base Excision Repair (BER):

If a nucleobase is structurally altered, for example, through reaction with so-called reactive oxygen species (oxidation), it must be removed. The repair mechanism responsible for this is very similar across all living beings (Wallace 2014).

Structurally altered nucleobases are recognized as defects by DNA glycosylases. Subsequently, they sever the chemical bond between the sugar component of the DNA and the defective nucleobase. This creates a "hole" (abasic site) in the DNA. Another enzyme (AP-specific lyase) cleaves off the remaining sugar residue; following this, an exonuclease removes the damaged strand section, a DNA polymerase produces a new and intact copy, and finally, a ligase performs the bond formation once again.

"An interesting aspect of this repair mechanism is that both in humans and in bacteria, eleven different glycosylases are present, four of which are responsible for removing misplaced uracil or thymine, six for oxidatively modified nucleobases, and one for incorrectly methylated nucleobases (Wallace 2014)." To date, no species with smaller sets of glycosylases are known.

Nucleotide Excision Repair (NER) and Ribonucleotide Excision Repair (RER):

DNA cross-links caused by UV radiation are resolved "by recognizing and excising the affected nucleotides." DNA cross-links are problematic because they block the transcription into mRNA and the replication of the DNA. To resolve them, certain enzymes make cuts on both sides of the DNA double helix.

In bacteria, the multifunctional enzyme complex UvrA2/B/C/D is responsible for this. This is followed, "as in the cases described above, by the four-step process of unwinding, removal, new synthesis, and ligation." In Archaea, bacteria, and humans, NER is designed very similarly. Halobacteria became extremely sensitive to UV light after the genes for the production of NER enzymes were knocked out (Marshall & Santangelo 2020).

A ribonucleotide is a single building block of RNA. Occasionally, these are "erroneously" integrated into the DNA. This is due, among other things, to the fact that ribonucleotides are several orders of magnitude more abundant in the cell than DNA building blocks. This vast excess is necessary for various functional reasons, but it also inevitably entails that DNA polymerases occasionally "grab the wrong one" during DNA duplication.

Furthermore, during DNA replication, short RNA segments are used to initiate DNA production; they act as a "jump-start," so to speak. In this process, however, it can happen that the ribonucleotides are not completely removed and are subsequently integrated into the DNA.

In some Archaea, the average incorporation of single RNA building blocks is "one ribonucleotide per approx. 1,000 DNA building blocks" (Marshall & Santangelo 2020). All organisms use a similar repair mechanism for this error, which is why it is described as "universally conserved." Proponents of evolutionary theory interpret this as evidence of common descent, but it also illustrates the extensive immutability of the repair mechanism.

"The nuclease RNaseH2 specifically introduces a bond cleavage at either the 3' end (bacteria, eukaryotes) or the 5' end of the ribonucleotide. Subsequently, restoration occurs here as well through unwinding, removal, new synthesis, and ligation."

Unique Repair Processes in Eukaryotes:

Eukaryotes are equipped with considerably more complex repair sequences than bacteria and Archaea, even though they utilize similar enzymes. Summarized in a few words:

At the site of damage, the protein complex "9-1-1 complex" is formed, which activates a signal that is then transmitted via a chemical signaling chain. The two proteins ATM and ATR play a particularly important role here, as they "forward" the signal to over 100 proteins. In the next step, effector proteins (e.g., p53) receive the signal and initiate the restoration (Niida & Nakanishi 2006).

This also includes halting the cell cycle, which first enables a complete repair before cell division (Huang & Zhou 2021; Niida & Nakanishi 2006). Repair enzymes, as in bacteria, initiate production and trigger corresponding repair programs – or programmed cell death, should the repair effort be too great.

Eukaryotes and the other two groups of organisms differ primarily in signal transduction, while the repair programs of eukaryotes described in this article resemble those of bacteria and Archaea in many aspects.

Repair of RNA Damage:

RNA molecules can be damaged by oxidation and biochemical attacks from other organisms, as well as by the addition of a water molecule causing bond cleavage (hydrolysis). During the last 20 years, many remarkable enzymes for repairing damaged RNA molecules have been described. However, only tRNA molecules and ribosomes are repaired, not small RNA molecules.

Some repair enzymes can perform two opposing tasks simultaneously by breaking and repairing specific bonds (Burroughs & Aravind 2016). Furthermore, RNA repair systems possess not only the ability to repair damaged RNA molecules but can also prevent future attacks by subsequently marking (methylating) the repaired site with methyltransferases, preventing further cleavages (Shuman 2023).

In bacteria and Archaea, the repair systems for RNA molecules consist of various coordinated enzymes. "In eukaryotes, the individual functional modules are combined into a single, giant multi-enzyme complex, which is referred to as a 'Swiss Army knife protein' because of its multifunctionality (Burroughs & Aravind 2016)."

All domains of life are equipped with RNA repair enzymes, and their components are very similar in all species, which is why they are also considered "highly conserved."

Discussion: Explaining the Origin of Repair Systems through Evolution?

The fact that DNA and RNA are chemically unstable and susceptible to damage in many different ways gives scientists pause. According to De Laat et al. (1999), it is a "fundamental problem that genetic information is stored in a form that is not chemically inert."

Given the extent of the nucleic acid damage mentioned in this article from UV radiation and water alone, current speculations regarding the hypothetical "RNA world" as an evolutionary intermediate step toward the emergence of the first cells appear nonsensical.

At the same time, it is clearly evident that alleged early single-celled organisms "could not have survived without an extensive 'toolbox' of highly efficient recognition, signaling, and repair enzymes." Hardly any researcher dares to attempt a detailed modeling of an evolutionary origin of repair mechanisms. It is simply asserted without justification that they arose evolutionarily.

Concurrently, it is frequently noted that repair mechanisms from bacteria to humans are "extremely conserved." In terms of evolutionary theory, they would therefore have remained largely unchanged for about 3.5–4 billion years of evolution:

"Furthermore, several DNA repair systems are deeply conserved in every or at least in two of the domains of life [...], even in prokaryotes" (Burroughs & Aravind 2016).

It follows inevitably that almost all highly complex repair enzymes must have arisen very quickly, if not practically simultaneously, in early evolution. "However, no one has the courage to put such an irrational assumption on paper."

Furthermore, many repair processes operate on the basis of the molecular energy currency ATP—comparable to a cordless screwdriver that requires electricity to function. This dependency on ATP also requires constant ATP production, which represents another major challenge for evolutionary origin hypotheses.

Thus, one would have to assume that both an already extraordinarily complex energy metabolism, including ATP production, and an extremely complex and finely tuned repair process emerged at the same time. It seems futile to model a realistic, step-by-step sequence of an evolutionary origin for this, while an intelligent plan presents itself almost forcefully as the cause.

Also incomprehensible from an evolutionary perspective is the great difference in signal transduction between bacteria/Archaea and eukaryotes. In E. coli bacteria, the repair program is started by exposing the DNA via DNA-binding proteins, whereas in eukaryotes, the signal is transmitted via a chemical signaling chain. These are fundamentally different processes at the biochemical level.

In the specialist literature, no concrete proposals can be found for the step-by-step transition of repair mechanisms from Archaea/bacteria to eukaryotes, including the required selection pressures.

A step-by-step emergence of nucleases poses an additional significant problem. Simple and poorly-aimed nucleases would destroy nucleic acids very quickly. Therefore, all nucleases must have cut very precisely only at the correct positions from the beginning. Moreover, strict control of nuclease activity must always be ensured—comparable to a scalpel that must be kept in a sheath (Spivak 2015).

At the same time, however, rapid activation of nuclease production must also be guaranteed, which again enormously overtaxes a gradual early evolutionary process.

In the case of the exactly coordinated and synchronized cooperation of various enzymes, the idea of a meticulously programmed unit of nanorobots suggests itself. It is therefore not surprising when many scientists formulate their hypotheses as if someone had established a goal:

"DNA repair mechanisms have evolved to recognize general distortions of the DNA backbone rather than the actual change, enabling the recognition of a wide spectrum of DNA lesions." (Marshall & Santangelo 2020; emphasis added)

Such goal-oriented formulations are incompatible with an unconscious and undirected evolution, but are entirely consistent with intelligent design. Finally, the similarity of repair enzymes in all domains is better understood through functional necessity than through common descent. Just as different screwdrivers are created with similar shapes due to their similar functions, "so too are enzymes whose purpose is the repair of nucleic acids."

Special thanks to Dr. Boris Schmidtgall for proofreading this article.

Literature:

• Burroughs MA & Aravind L (2016) RNA damage in biological conf licts and the diversity of responding RNA repair systems. Nucleic Acids Res. 44, 8525–8555.

• De Laat et al. (1999) Molecular mechanism of nucleotide excision repair. Genes & Development 13, 768–785.

• Huang RX & Zhou PK (2021) DNA damage repair: historical perspectives, mechanistic pathways and clinical translation for targeted cancer therapy. Signal Transduction and Targeted Therapy 6, 254.

• Lima-Noronha M et al. (2022) Sending out an SOS – the bacterial DNA damage response. Genet. Mol. Biol. 45, 3.

• Lindahl T (1993) Instability and decay of the primary structure of DNA. Nature 362, 709-715.

• Marshall CJ & Santangelo TJ (2020) Archaeal DNA Repair Mechanisms. Biomolecules 10, 1472, doi:10.3390/biom10111472.

• Modrich P (2016) Mechanisms in E. coli and Human Mismatch Repair. Angew. Chem. Int. Ed. 55, 8490–8501.

• Niida H & Nakanishi M (2006) DNA damage checkpoints in mammals. Mutagenesis 21, 3–9.

• Radman M (1974) Phenomenology of an inducible mutagenic DNA repair pathway in Escherichia coli: SOS repair hypothesis. In: Prakash L (ed) Molecular and environmental aspects of mutagenesis. Charles C. Thomas, Springfield, pp 128–142.

• Shuman S (2023) RNA repair: hiding in plain sight. Annu. Rev. Genet. 57, 461–489.

• Spivak G (2015) Nucleotide excision repair in humans.

  DNA repair 36, 13–18.

• Vogt A & He Y (2023) Structure and mechanism in non-homologous end joining. DNA repair 130, 103547.

• Wallace SS (2014) Base excision repair: A critical player in many games. DNA repair 19, 14–26.

• Yousefzadeh M et al. (2021) DNA damage - how and why we age? eLife 2021, 10:e62852