**3.2 Poliovirus and coronavirus**

*DNA - Damages and Repair Mechanisms*

**3.1 Copy-choice recombination**

damage [9, 34].

**3. Repair of RNA genomes by copy-choice recombination**

**Figure 2** indicates how an accurate undamaged progeny single-stranded genome

can be generated from a damaged parental genome by strand-switching (copychoice) recombination. As shown in this **Figure 2**, (1) during synthesis of a progeny strand by a replicative polymerase, a damage in the (green) template strand (strand being copied) blocks polymerase progression. (2) If another (orange) homologous template is available, the polymerase may switch templates, thereby bypassing the damage. (3) The newly synthesized strand may then release from the second template strand. (4) The newly synthesized strand can return and pair with the original template. (5) The polymerase may then complete the replication using the original template. (6) These steps can generate a new recombinant genome without

**26**

**Figure 2.**

*Copy-choice recombination.*

Poliovirus (Family *Picornaviridae*; Genus *Enterovirus*) is a positive ssRNA ((+) ssRNA) virus that can undergo genetic recombination when there are at least two ssRNA viral genomes in the same host cell. RNA recombination is considered to be a major driving force in determining the course of poliovirus evolution [35]. RNAdependent RNA polymerase (RdRp), an enzyme encoded in the viral genome, catalyzes genome replication. Kirkegaard and Baltimore [34] presented results strongly supporting a copy-choice mechanism for RNA recombination for poliovirus. By this mechanism the RdRp switches between (+)ssRNA templates during synthesis of the progeny negative strand (−)ssRNA (**Figure 2**). Recombination in RNA viruses is considered to be an adaptive mechanism for maintaining genome integrity [36].

To regenerate the next generation of (+)ssRNA strands, the (−)ssRNA strands are also copied and this may also be accompanied *infrequently* by strand switching [34].

When cells are infected by two or more viruses containing genome damage the viruses may undergo multiplicity reactivation. Polioviruses are able to undergo

#### **Figure 3.**

*Coronavirus. Modified from https://commons.wikimedia.org/wiki/File:3D\_medical\_animation\_coronavirus\_ structure\_vie.png with license https://www.scientificanimations.com/CC BY-SA (https://creativecommons.org/ licenses/by-sa/4.0)*

multiplicity reactivation [37]. That is, when polioviruses were irradiated with UV light and then allowed to infect host cells at a multiplicity of two or greater, viable progeny are produced at UV doses that inactivate the virus in single infections. As noted above, multiplicity reactivation occurs in various different virus systems, and has been shown to be a form of recombinational repair [27, 28].

Coronaviruses (Family *Coronaviridae*) (see **Figure 3**) are (+)ssRNA enveloped viruses. The genome size of coronaviruses ranges from about 26 to 32 kilobases, one of the largest among RNA viruses. They have characteristic club-shaped spikes that project from their surface, which in electron micrographs create an image reminiscent of the solar corona, from which their name derives.

RNA recombination appears to be a major driving force in the evolution of (+) ssRNA coronaviruses. Recombination contributes to genetic variability within a coronavirus species, the capability of a coronavirus species to jump from one host to another and, infrequently, the emergence of a novel coronavirus [38]. The mechanism of recombination in coronaviruses likely involves template-switching during genome replication [38]. Also, the (+)ssRNA plant carmoviruses and tombusviruses frequently undergo recombination by RdRp template-switching (copy-choice) [39]. A key step in the evolution of repair in the RNA world appears to have been the emergence of template-switching (copy-choice) recombination as a major mechanism for dealing with genome damage.

#### **4. Reverse transcription of the RNA genome to DNA in HIV**

Human immunodeficiency virus (HIV (Family *Retroviridae*) (**Figure 4**) is a positive single-stranded RNA ((+)ssRNA) virus. Each HIV virus particle encapsidates two (+)ssRNA genomes.

During infection of a host cell, genome replication is catalyzed by reverse transcriptase, an RNA-dependent DNA polymerase [40]. During reverse transcription, recombination between the two genomes can occur [9]. The reverse transcriptase can switch between the two parental RNA genomes by copy-choice recombination [40], and such events may occur throughout the genome. Thus the two infecting genomes from each virus can cooperate to form a complementary negative single-strand DNA copy that has recombined information from the two parental RNA genomes. Recombination is necessary for efficient HIV replication and the maintenance of genome integrity [40]. During each replication cycle, from 5 to 14 recombination events may occur per genome [41]. The recombination events are "clustered" so that one recombination event is correlated with another that is close by. This clustering is apparently caused by correlated template-switches, known as high negative interference, during minus-strand DNA synthesis [42]. That is, once a switch is made from template *a* to template *b*, then another switch is made very soon (not at some random time) back to template *a*. Template-switching in HIV is considered to be a repair mechanism for salvaging damaged genomes that is essential for maintaining genome integrity [9, 40].

After the first single strand DNA copy is synthesized, another round of replication generates a duplex DNA molecule which can integrate into the host DNA genome to form a provirus [9].

#### **4.1 HIV recombination can sometimes produce genetic variation**

Recombination of the viral genomes can introduce genetic variation among progeny HIV that contributes to the evolution of resistance when humans are treated with anti-retroviral therapy [43]. Viral genome recombination may also

**29**

**Figure 4.**

*Origin of DNA Repair in the RNA World DOI: http://dx.doi.org/10.5772/intechopen.93822*

play a role in overcoming the immune defenses of the human host. The sequence of events necessary to produce genetic variation by recombination that is adaptively

*Human Immunodeficiency virus (HIV). Top image indicates outer conformation of the virion. Lower image shows the two RNA genomes present within the virion, the reverse transcriptase and other components of the virion. Top image: https://commons.wikimedia.org/wiki/File:HIV.png BruceBlaus/CC BY-SA (https:// creativecommons.org/licenses/by-sa/4.0) Bottom image: https://commons.wikimedia.org/wiki/File:HI-Virion-en.png US National Institute of Health (redrawn by en:User:Carl Henderson) / Public domain.*

For an adaptive benefit of genetic variation to be realized, the two RNA genomes contained in an individual infecting virus particle would have to be derived from separate progenitor viruses of differing genetic constitution. In general, only viruses that have packaged two genetically different RNA genomes can produce a recombinant genome with a genotype distinctly different from that of its parents [44] . For this to occur multiple events are required [44]. These events are: (1) A human host cell would need to be infected by two viruses of genetically different lineages, and the genomes of these two different viruses would have to produce progeny genomes. (2) Two different progeny RNA genomes produced from such an infection would have to be co-packaged into the same progeny virus particle. (3) When this progeny virus infects a new host cell, template-switching would have to occur during reverse

beneficial to HIV are considered next.

*DNA - Damages and Repair Mechanisms*

multiplicity reactivation [37]. That is, when polioviruses were irradiated with UV light and then allowed to infect host cells at a multiplicity of two or greater, viable progeny are produced at UV doses that inactivate the virus in single infections. As noted above, multiplicity reactivation occurs in various different virus systems, and

Coronaviruses (Family *Coronaviridae*) (see **Figure 3**) are (+)ssRNA enveloped viruses. The genome size of coronaviruses ranges from about 26 to 32 kilobases, one of the largest among RNA viruses. They have characteristic club-shaped spikes that project from their surface, which in electron micrographs create an image reminiscent

RNA recombination appears to be a major driving force in the evolution of (+) ssRNA coronaviruses. Recombination contributes to genetic variability within a coronavirus species, the capability of a coronavirus species to jump from one host to another and, infrequently, the emergence of a novel coronavirus [38]. The mechanism of recombination in coronaviruses likely involves template-switching during genome replication [38]. Also, the (+)ssRNA plant carmoviruses and tombusviruses frequently undergo recombination by RdRp template-switching (copy-choice) [39]. A key step in the evolution of repair in the RNA world appears to have been the emergence of template-switching (copy-choice) recombination as a major mecha-

Human immunodeficiency virus (HIV (Family *Retroviridae*) (**Figure 4**) is a positive single-stranded RNA ((+)ssRNA) virus. Each HIV virus particle encapsi-

During infection of a host cell, genome replication is catalyzed by reverse transcriptase, an RNA-dependent DNA polymerase [40]. During reverse transcription, recombination between the two genomes can occur [9]. The reverse transcriptase can switch between the two parental RNA genomes by copy-choice recombination [40], and such events may occur throughout the genome. Thus the two infecting genomes from each virus can cooperate to form a complementary negative single-strand DNA copy that has recombined information from the two parental RNA genomes. Recombination is necessary for efficient HIV replication and the maintenance of genome integrity [40]. During each replication cycle, from 5 to 14 recombination events may occur per genome [41]. The recombination events are "clustered" so that one recombination event is correlated with another that is close by. This clustering is apparently caused by correlated template-switches, known as high negative interference, during minus-strand DNA synthesis [42]. That is, once a switch is made from template *a* to template *b*, then another switch is made very soon (not at some random time) back to template *a*. Template-switching in HIV is considered to be a repair mechanism for salvaging damaged genomes that is

After the first single strand DNA copy is synthesized, another round of replication generates a duplex DNA molecule which can integrate into the host DNA

Recombination of the viral genomes can introduce genetic variation among progeny HIV that contributes to the evolution of resistance when humans are treated with anti-retroviral therapy [43]. Viral genome recombination may also

**4.1 HIV recombination can sometimes produce genetic variation**

has been shown to be a form of recombinational repair [27, 28].

**4. Reverse transcription of the RNA genome to DNA in HIV**

of the solar corona, from which their name derives.

essential for maintaining genome integrity [9, 40].

nism for dealing with genome damage.

dates two (+)ssRNA genomes.

genome to form a provirus [9].

**28**

#### **Figure 4.**

*Human Immunodeficiency virus (HIV). Top image indicates outer conformation of the virion. Lower image shows the two RNA genomes present within the virion, the reverse transcriptase and other components of the virion. Top image: https://commons.wikimedia.org/wiki/File:HIV.png BruceBlaus/CC BY-SA (https:// creativecommons.org/licenses/by-sa/4.0) Bottom image: https://commons.wikimedia.org/wiki/File:HI-Virion-en.png US National Institute of Health (redrawn by en:User:Carl Henderson) / Public domain.*

play a role in overcoming the immune defenses of the human host. The sequence of events necessary to produce genetic variation by recombination that is adaptively beneficial to HIV are considered next.

For an adaptive benefit of genetic variation to be realized, the two RNA genomes contained in an individual infecting virus particle would have to be derived from separate progenitor viruses of differing genetic constitution. In general, only viruses that have packaged two genetically different RNA genomes can produce a recombinant genome with a genotype distinctly different from that of its parents [44] . For this to occur multiple events are required [44]. These events are: (1) A human host cell would need to be infected by two viruses of genetically different lineages, and the genomes of these two different viruses would have to produce progeny genomes. (2) Two different progeny RNA genomes produced from such an infection would have to be co-packaged into the same progeny virus particle. (3) When this progeny virus infects a new host cell, template-switching would have to occur during reverse

transcription to generate a recombinant DNA copy. (4) The recombinant DNA would then need to integrate into the DNA genome of the infected cell. (5) The recombinant provirus would next have to be able to produce replication-competent virus progeny for the impact of the recombination to be observed.

How often cells in HIV patients are infected by more than one HIV (doubleinfection) is not known, and it is unknown how often mixed packaging occurs under natural conditions [44, 45]. As discussed above, from 5 to 14 strand-switching recombination events occur in each infection cycle. These events, in most cases, occur between genomes with the same genetic constitution. Thus it is apparent that although recombination can, under some circumstances, produce variation that is adaptive, the great majority of recombination events do not produce significant adaptive variation.

#### **4.2 Recombination as a repair process**

Infection by HIV results in chronic ongoing inflammation associated with reactive oxygen species production [46]. Thus a strategy for dealing with oxidative damages to the HIV genome would be adaptively beneficial. Each HIV particle contains two homologous templates, rather than one. Temin [9] considered it likely that recombination is an adaptation for repair of damaged RNA genomes. Also, template-switching by the reverse transcriptase was suggested by Bonhoeffer et al. [47] to be a repair process for dealing with breaks in the ssRNA genome. Copy-choice recombination by the reverse transcriptase could produce a DNA copy of the genome that is free of damage even if both parental ssRNA copies in each virus are damaged. This benefit of recombination can be realized at each infection cycle even if, as is usually the case, the two genomes do not differ, or are closely similar genetically, and little if any new genetic variation will be produced [9, 45]. If recombination in HIV infections is primarily an adaptation for genome repair, the generation of recombinational variation would be an occasional natural consequence, but not the principle driving force, for the evolution of templateswitching [47].

#### **4.3 HIV as a model for the transition from ssRNA to dsDNA genomes**

Early organisms may have evolved through a stage, like HIV, where their genome in the form of ssRNA was replicated to form a hybrid RNA: DNA duplex which upon further replication formed dsDNA. A laboratory evolved RNA polymerase ribozyme that synthesizes RNA has also been shown to act as a reverse transcriptase to synthesize DNA [48]. A ribozyme like this may have evolved in nature and been instrumental in the transition from the RNA to the DNA world. It could have arisen as a secondary function of an RdRp.

While oxidative stress appears to be a principle damaging stress for the HIV genome, the damaging stresses on organisms that were undergoing the early evolutionary transition from RNA to DNA genomes would likely have been different. The genome damages in the transition from RNA to DNA genomes could have arisen, as described above, from hydrolytic reactions, UV light or environmental reactive chemicals, but undoubtedly there would have been some kinds of significant damages. Thus during the transition from the RNA world to the DNA world there was very probably a continuous need to cope with genome damage. The copy-choice mechanisms that had a repair function in the RNA world may have continued to operate as repair functions during the transition to the dsDNA world. The selective pressure of genome damage on genome repair as the genetic material transitioned from RNA to DNA is discussed further in Bernstein et al. (pgs. 342-345) [8].

**31**

*Origin of DNA Repair in the RNA World DOI: http://dx.doi.org/10.5772/intechopen.93822*

genomes but prior to eukaryotes [49].

facilitating recombinational repair.

to facilitate recombinational repair.

**6. Eukaryotes**

**5. Recombination in archaea acts in DNA repair**

repair in both the archaeal and the eukaryote DNA world.

In the previous sections it was proposed that genome repair processes emerged in the RNA world and that, after going through several evolutionary stages, such repair processes were present in organisms with DNA genomes. The archaea are single-celled microorganisms whose genome is DNA. These organisms are regarded as descendants of a form of life that arose subsequent to organisms with RNA

The evolution of the eukaryotic cell appears to trace back to the establishment of a symbiotic relationship between a host anaerobic archaeal cell and an internalized bacterium capable of aerobic metabolism [50]. The eukaryotic cell emerged at least 1.5 billion years ago [51]. Eukaryotic genes of archaeal origin appear to have a more central role in basic cellular functions than genes of eubacterial origin [49]. Thus the manner in which present day archaea deal with genome damage may throw light on how genome repair processes that arose in the RNA world became adapted for

Recent findings show that cells of archaeal species, particularly *Sulfolobus solfataricus* and *Sulfolobus acidocaldarius,* under stressful environmental conditions that cause DNA damage, aggregate and transfer DNA from one cell to another through direct contact [52, 53]. Exposure of *S. solfataricus* to UV irradiation strongly induces type IV pili formation which facilitates cellular aggregation [54, 55]. This induced cellular aggregation mediates intercellular chromosome marker exchange with high frequency. UV irradiated cultures were found to have recombination rates exceeding those of uninduced cultures by up to three orders of magnitude. The UV-inducible DNA transfer process and subsequent homologus recombination are considered to represent a repair mechanism for maintaining chromosome integrity [54, 56, 57]. Also in *S. solfataricus*, exposure to bleomycin or mitomycin C, agents that cause double-strand breaks and other damages, induces cellular aggregation [54]. In *S. acidoclaldarius,* genes that facilitate DNA transfer are upregulated by DNA damaging UV irradiation [52]. DNA damage can be lethal to a cell unless repaired. DNA transfer between neighboring archaeal cells appears to be an adaptation for aiding survival of nearby (and likely genetically related) damaged cells by

The repair capabilities of archaea suggest that ancestral organisms arising early in the DNA world underwent processes that allowed DNA damage in one cell to be repaired by transfer of DNA sequence information from a neighboring cell in order

Eukaryotes are capable of several different types of DNA repair process:

direct reversal of UV light-induced photolesions; (2) O6

alkytransferase catalyzed direct reversal of a set of of O6

DNA damages and thus have limited applicability.

a.The DNA damage may be enzymatically directly reversed. There are three known direct reversal mechananisms (Yi C) [58]: (1) Photolyase catalyzed

damages; and (3) direct reversal of N-alkylated base adducts by AlkB family dioxygenases. Direct reversal mechanisms are specific for a small subset of

b.Single-strand damages may be excised and the proper information restored by copying the other undamaged strand. This can occur by any one of several

alkylguanine-DNA

alkylated DNA

*DNA - Damages and Repair Mechanisms*

**4.2 Recombination as a repair process**

adaptive variation.

switching [47].

as a secondary function of an RdRp.

transcription to generate a recombinant DNA copy. (4) The recombinant DNA would then need to integrate into the DNA genome of the infected cell. (5) The recombinant provirus would next have to be able to produce replication-competent

How often cells in HIV patients are infected by more than one HIV (doubleinfection) is not known, and it is unknown how often mixed packaging occurs under natural conditions [44, 45]. As discussed above, from 5 to 14 strand-switching recombination events occur in each infection cycle. These events, in most cases, occur between genomes with the same genetic constitution. Thus it is apparent that although recombination can, under some circumstances, produce variation that is adaptive, the great majority of recombination events do not produce significant

Infection by HIV results in chronic ongoing inflammation associated with reactive oxygen species production [46]. Thus a strategy for dealing with oxidative damages to the HIV genome would be adaptively beneficial. Each HIV particle contains two homologous templates, rather than one. Temin [9] considered it likely that recombination is an adaptation for repair of damaged RNA genomes. Also, template-switching by the reverse transcriptase was suggested by Bonhoeffer et al. [47] to be a repair process for dealing with breaks in the ssRNA genome. Copy-choice recombination by the reverse transcriptase could produce a DNA copy of the genome that is free of damage even if both parental ssRNA copies in each virus are damaged. This benefit of recombination can be realized at each infection cycle even if, as is usually the case, the two genomes do not differ, or are closely similar genetically, and little if any new genetic variation will be produced [9, 45]. If recombination in HIV infections is primarily an adaptation for genome repair, the generation of recombinational variation would be an occasional natural consequence, but not the principle driving force, for the evolution of template-

**4.3 HIV as a model for the transition from ssRNA to dsDNA genomes**

Early organisms may have evolved through a stage, like HIV, where their genome

in the form of ssRNA was replicated to form a hybrid RNA: DNA duplex which upon further replication formed dsDNA. A laboratory evolved RNA polymerase ribozyme that synthesizes RNA has also been shown to act as a reverse transcriptase to synthesize DNA [48]. A ribozyme like this may have evolved in nature and been instrumental in the transition from the RNA to the DNA world. It could have arisen

While oxidative stress appears to be a principle damaging stress for the HIV genome, the damaging stresses on organisms that were undergoing the early evolutionary transition from RNA to DNA genomes would likely have been different. The genome damages in the transition from RNA to DNA genomes could have arisen, as described above, from hydrolytic reactions, UV light or environmental reactive chemicals, but undoubtedly there would have been some kinds of significant damages. Thus during the transition from the RNA world to the DNA world there was very probably a continuous need to cope with genome damage. The copy-choice mechanisms that had a repair function in the RNA world may have continued to operate as repair functions during the transition to the dsDNA world. The selective pressure of genome damage on genome repair as the genetic material transitioned from RNA to DNA is discussed further in Bernstein et al. (pgs. 342-345) [8].

virus progeny for the impact of the recombination to be observed.

**30**
