Genome Instability in Bacteria and Archaea Strategies for Maintaining Genome Stability PDF

Genome Instability in Bacteria and Archaea: Strategies for Maintaining Genome Stability  Lecture plan Reponses to DNA damage: The SOS Response: A Primitive Cell-Cycle Checkpoint; An Archaeal UV Response Based on DNA Sharing; DNA repair pathways: Direct Reversal of...

Genome Instability in Bacteria and Archaea: Strategies for Maintaining Genome Stability  Lecture plan Reponses to DNA damage: The SOS Response: A Primitive Cell-Cycle Checkpoint; An Archaeal UV Response Based on DNA Sharing; DNA repair pathways: Direct Reversal of DNA Damage; Base Excision Repair and Removal of Uracil from DNA; Nucleotide Excision Repair: A Versatile DNA-Repair Pathway; Correcting Mismatched Bases: Cleanup After DNA Replication; Recombination Repair: Dealing With Double-Strand Breaks; Restriction-modification systems: protecting the genome from invaders; Conclusion.  Reponses to DNA damage: The SOS Response: A Primitive Cell-Cycle Checkpoint Since its discovery and early characterization by Evelyn Witkin and Miroslav Radman in the early to mid-1970s, the SOS response has become a paradigm for the bacterial DNA damage response. Cell needs to repair DNA efficiently, but also to diversify their genome While the SOS pathway has proven to be extremely complex, at its core are only two proteins: the LexA repressor and the RecA activator. Loading… SOS response is how bacteria deal with DAN damage. It relies on 2 proteins: LexA (acts as a repressor) and RecA (acts as an activator) SOS doesn’t directly repair the DNA but it helps stabilize the genomes (prevent further damage), reprograms DNA repaid mechanisms, and can activate those repair systems The SOS Response: A Primitive Cell-Cycle Checkpoint SOS response is activated by DNA damage. ssDNA breaks are recognized by RecA – activator of SOS response It binds to LexA repressor promoting its self- cleavage There are SOS boxes with different level of affinity to LexA – from weak to strong. Those that are weak, express first, for example: SOS genes such as lexA, recA, uvrA, uvrB, and uvrD – nucleotide excision repair If this does not help, other SOS boxes are activated: sulA, umuD, umuC. SulA stops cell division. UmuDC- activate mutagenic repair. When DNA is damaged, the strategy is to prepare strands for homologous or illigitement recombination Some promoters have weak binding and some have stronger binding. Weaker binding = expression occurs first  In normal conditions LexA blocks the expression of SOS genes by binding to specific DNA sequences When DNA is damaged: sDNA forms, and RecA binds to it, creating a filament. This triggers LexA to destroy itself, lifting the block and activating the SOS genes some LexA proteins have weaker or stronger binding affinities to SOS genes Once SOS gene is activated, the cell stops dividing to focus on repair (the protein that allows for this to occur is SulA). The genes for DNA repair turn on in a logical order: First those involved in fixing DNA damage directly Later, error prone DNA polymerases (Pol V and Pol IV) are activated to replicate damaged DNA, which allows for growth to continue but can cause mutations (regular polymerases can’t work on damaged DNA so ) Eventually the system turns off Different bacteria have different SOS repsonses Why would the polymerases want to replicate the damaged DNA? they want to ensure survival, even if that means replicating the damaged DNA so they can continue with growth. If the replication process stalls entirely due to DNA damage, the cell can’t divide and it will die The SOS Response: A Primitive Cell-Cycle Checkpoint Induction of two error-prone DNA polymerases: dinB (DNA polymerase IV Homolog of: (Pol IV)) and umuDC (DNA polymerase Allows for replication to V (Pol V)).: homolog of continue instead of repairing damage The primary function of these polymerases is to insert nucleotides Loading… SOS genes opposite bulky lesions. The polymerases insert nucleotides opposite from the strand with the damage allowing for the replication process to continue Nucleotide incorporation by Pol IV and Pol V is largely error free at their cognate lesions; Cognate lesion: Is a specific type of damage that a certain repair system or enzyme is really good at dealing with however, their replication of undamaged DNA can be mutagenic due to improper nucleotide discernment and a lack of proofreading. Pg. 56  Reponses to DNA damage: The SOS Response: A Primitive Cell-Cycle Checkpoint Loss of Pol IV reduces the frequency of base substitution mutations by 50– 70% and loss of Pol V completely eliminates SOS mutagenesis. Allows for more mutations to occur. Good for a changing envionrmnent These results suggest that while the SOS response functions to stabilize the genome, it can also have potentially mutagenic consequences via the induction of mutator genes.Mutator genes are genes that, when mutated themselves, increase the overall mutation rate in an organisms genome The mutagenic outcomes of the SOS-dependent induction of dinB and umuDC are mostly point mutations; SOS induction can also promote higher order genome instability.  Reponses to DNA damage: The SOS Response: A Primitive Cell-Cycle Checkpoint SOS response is conserved among divergent species; however, some notable deviations from the Escherichia coli model have been observed. For example, in Caulobacter crescentus, following a similar cell-cycle arrest, the SOS response triggers a more sophisticated programmed cell death pathway, akin to eukaryotic apoptosis. Growing evidence suggests that the SOS response has many other functions in addition to this basic checkpoint control, including: -functions in horizontal gene transfer, -the development of antibiotic resistance, and -pathogenesis.  Reponses to DNA damage: An Archaeal UV Response Based on DNA Sharing Invocation of a large-scale transcriptional reprogramming after UV exposure does not appear to be universal in archaea, but a UV-induced stress response UV activates repair mechanism has been characterized in Sulfolobus: due to damage in DNA. UPS operon gets activated. Tries to find genome that is undamaged. Bacteria is haploid. So they need another undamaged cell for recombination to repair damage When a certain type of archaea is exposed to UV light, it response by turning on certain genes that help it share DNA between cells. This DNA exchange helps the cells repair their damaged DNA. The cells form special structures called Type IV pilli, which allow them to stick together and transfer DNA. By swapping DNA, cells can use undamaged DNA as a template to fix the damaged parts of their own genome. This process helps the cells survive better after DNA damage. The response does not follow the usual DNA repair system seen in other organisms like the SOS response, but its an effective, unique way for this archaea to deal with UV induced DNA damge Model of early UV stress response in Sulfolobus acidocaldarius. The increased and decreased transcription of genes is depicted by green and red squares, respectively, while white squares represent unchanged transcription. (A) TFB3-dependent UV stress response. 45 min after UV irradiation (100 J/m2), tfb3 is highly up-regulated and acts as an activator of transcription. The delayed (90 min after UV irradiation) response includes the enhanced transcription of the tfb3- dependent target genes (ups genes of the UV inducible pili operon, ced genes of the Crenarchaeal Exchange of DNA importer), leading to increased cellular aggregation and DNA exchange between the cells in order to allow DNA repair via homologous recombination.  (B) TFB3-independent response. Apart from the TFB3-dependent response, S. acidocaldarius shows a UV stress response (45 and 90 min after UV irradiation), which does not depend on the presence of TFB3. This is characterized by the repression of DNA replication and cell cycle progression as well as the inhibition of nucleotide biosynthesis, leading to reduced delivery of DNA building blocks. Downregulation of these processes Repress DNA replication in damaged cells allow the DNA repair, which is mediated by the TFB3-dependent features described above to take place. Down regulation of replication A 45 mins after UV exposure: The gene tfb3 is turned on and acts as a “master switch” to activate other genes 90 mins after exposure: a delayed response kicks in , where genes controlled by TFb3 (genes for pilli making and exchange of DNA) are activated. This causes cells to come together and exchange DNA, which helps repair the DNA damage through homologous recombination B This pathway does not require tfb3 45 and 90 minutes after UV exposure : Cell slows down DNA replication, cell cycle and nucleotide production. This reduced the number of DNA building block avaliable which helps focus recourses on repairing the DNA damaged described in A  Reponses to DNA damage: An Archaeal UV Response Based on DNA Sharing DNA sharing somehow protects the cells, presumably by dampening UV- induced genome instability; it is supported by the observation that strains capable of expressing the type IV pili have higher survival rates after UV exposure. Loading… While a bona fide SOS response is clearly absent in Sulfolobus, this system illustrates a novel genetic innovation for dealing with UV-induced DNA damage. Types of DNA damage These stressful environments can cause DNA damage Damage can be breakage, oxidizing, single strand breaks, inter strand or intra strand cross link - these interefere with replication These are recognized by sensors  DNA repair pathways The SOS response helps stabilize the genome, but doesn’t directly fix DNA damage. Instead cells rely on various DDNA repair pathways that actually carry out the repair work While the SOS response provides a genome-stabilizing function, it has no inherent DNA repair capacity. Instead, cells have evolved several intertwined molecular pathways comprised by the actual molecular transactions leading to damage repair: - direct reversal (the only DNA synthesis–independent pathway); - base excision repair (BER); Direct repair - no changes to nucleotide (no removal of nucleotide). Direct repair - NER; is much safer - mismatch repair (MMR); The other repairs need to cut sugar - HR-dependent repair. phosphate backbone and remove the whole nucleotide, and then replace it with an undamaged one The fundamentals of DNA repair have been most intensely studied in E. coli, thus its molecular biology forms the foundation of the discussion; however, important deviations in other species are also highlighted.  DNA repair pathways: Direct Reversal of DNA Damage One way to repair DNA damage is to simply undo the particular molecular changes, that is, to directly reverse the damage. Evolution has endowed life with (at least) three direct reversal pathways: -photolyases, which repair UV-induced damage, and two mechanisms that repair alkylated bases, -O6-alkylguanine alkyl transferases (AGTs) and -AlkB-family dioxygenases. While the molecular mechanisms vary drastically, the end result of all of these pathways is the restoration of the original molecular structure without the need for new DNA synthesis. No New DNA synthesis! Just repairing the damaged one Direct repair: Means of fixing damaged DNA without needing to make new DNA. Here’s how: Photolysases: Yhese enzymes fox damaged caused by UV. IV can cause 2 Marion types of DNA damage: CPDs and 6-4 PPs. Photolyses use light energy to repair these damages by breaking the bonds that formed incorrectly Alkyltransferases and AlkB Dioxygenases: These enzymes fix damage by alkylating agents which add chemical groups (like methyl groups) to DNA bases. These damaged bases can cause mutations if not repaired  DNA repair pathways: Direct Reversal of DNA Damage UV irradiation leads to two main types of DNA lesions that can disrupt many DNA-related processes, most importantly, replication and transcription: pyrimidine (6-4) photoproducts (6-4 PPs) and cyclobutane pyrimidine dimers (CPDs). Due to the different structures of these lesions, different photolyase enzymes are required for their repair; however, a common feature of photolyases is that they obtain energy from light to fuel the reaction (hence the classical name “light reactions”) and use flavin adenine dinucleotide (FAD) for catalysis. The photolyase reaction is simply a stepwise transfer of energy that reconfigures the covalent bonds in the original bases to restore the original structure.  DNA repair pathways: Direct Reversal of DNA Damage This is original molecule (6-4) photoproducts (type of damage caused by CPD (type of UV damage ) UV)  DNA repair pathways: Direct Reversal of DNA Damage http://www.pnas.org/content/108/23/9402  DNA repair pathways: Direct Reversal of DNA Damage While CPD photolyases were one of the earliest characterized DNA repair mechanisms and have been found in all three domains of life, (6-4) PP photolyases remained elusive until only recently. The first bacterial (6-4) PP photolyase was reported in 2013 in Agrobacterium tumefaciens and is encoded by the phrB gene. Besides some structural differences and utilization of different chromophores for light collection, the functions of archaeal photolyases are conserved from their bacterial counterparts.  DNA repair pathways: Direct Reversal of DNA Damage Alkylating agents cause cytotoxic and potentially mutagenic adducts – from methyl groups to larger bulky adducts. Alkylating agents are chemcials in the Alkyls are alkanes missing H (CH3-, C2H5- etc.) environment that cause the addition of alkyl groups onto DNA which can be These methylations can lead to incorrect harmful base pairing Lesions caused by alkylation are efficiently repaired by the BER pathway; however, they can also be directly repaired by alkyl transferases and AlkB family dioxygenases. These enzymes work to remove those added alkyl groups Direct reversal of alkylation damage in E. coli is mediated by either the general housekeeping alkyltransferase Ogt, or an adaptive response controlled by the Ada protein that is mediated by its targets alkA and alkB. The direct reversal reaction occurs via the transfer of the alkyl group from the damaged base onto a reactive cysteine residue via an SN2 reaction, thus permanently inactivating the protein. They remove the alkyl group on the damaged base by transfering it to a cysteine of a Ada protein though a SN2 reactions  DNA repair pathways: Direct Reversal of DNA Damage Red if the alkyl group. It gets transfer from the nucleotide onto the cystine of the protein OGTs are unique in their structure, mechanism and post-reaction outcome. They are able to perform whole repair reactions without the assistance of other factors or the need of energy source. OGTs are able to catalyze a trans-alkylation reaction, in which an alkyl group in DNA, mainly at position O6 of guanines or O4 of tymines, is transferred to a cysteine residue in the protein active site (Figure “a”). This reaction is irreversible and determines the protein inactivation, thus each OGT molecule works only once.  CH3 gets removed from DNA repair pathways: DNA onto C or N terminus of Ada protein, which leads to production Direct Reversal of DNA Damage degradation and the DNA is repaired Adaptive response in E. coli induced by alkylation DNA damage. Indamage E. coli, adaptive response to alkylation DNA is triggered by the Ada regular which includes several genes that work together to repair the damage by alkylating agents The Ada regulon contains the ada gene and also alkB, alkA, and aidB genes shown as boxes. DNA is alkylated at the phosphate linkages (P-O-CH 3 ). The AlkB dioxygenase is similar to Ada in that it catalyzes the direct reversal of base alkylation damage.  DNA repair pathways: Base Excision Repair and Removal of Uracil from In addition to direct reversal, many DNA organisms have another highly conserved pathway to repair damaged bases: BER. BER was first discovered in E. coli by Tomas Lindahl when he attempted to elucidate the pathway for the repair of genomic uracil, a byproduct of cytosine deamination. The cognate lesions for BER are base damage that does not cause major distortions in the DNA double helix, including: BER is base excision repair. Fixes damaged bases ( damaged caused by oxidation, alkylation, -oxidized bases (eg, 8-oxoguanine), deamination or other forms of damage) -alkylated bases (eg, 3-meA), -deaminated bases (eg, hypoxanthine and xanthine), and -uracil.  DNA repair pathways: Base Excision Repair and Removal of Uracil from There are two types of glycosylases:DNA -monofunctional (such as E. coli AlkB) and -bifunctional (such as E. coli Nei). Monofunctional removes base forming AP sites – requires further processing by an AP endonuclease. A bifunctional glycosylase can also incise the phosphodiester backbone at the abasic site to generate a single-strand break (SSB). The incision in the phosphodiester backbone provides a 3′ hydroxyl group that is ultimately a substrate for DNA Pol I. The exonuclease function of Pol I removes the damaged strand and the polymerase activity synthesizes replacement DNA. Finally, the nick is sealed by DNA ligase.  DNA repair pathways: Base Excision Repair and Removal of Uracil from DNA Bifunctional makes the cut ? 6 glycosylases dRpase hydrolysis of the N-glycosylic bonds BER 1. Damaged in recognized by DNA glycosylase enzymes which removed the damaged base creating an a basic site (called an AP site) 2. The AP endonuclease cits the DNA backbone at the site where the base was removed 3. The gap left by the damaged base is filled by DNA polymerase that adds the correct nucleotide (according to opposite strand) 4. DNA ligand seals remaining nicks  DNA repair pathways: Base Excision Repair and Removal of Uracil from DNA Archaeal BER components and their molecular biology are more similar to eukaryotes than to bacteria. The BER pathways of some archaea have novel features, while others use an additional mechanism to prevent mutation due to genomic uracil. Ferroplasma acidarmanus encodes a novel AGT protein (AGTendoV) that has an O6-methyltransferase domain fused to an endonuclease V domain.  DNA repair pathways: Base Excision Repair and Removal of Uracil from A more extreme deviation from the DNA canonical BER pathway is the use of uracil-scanning DNA polymerases. In most cases, genomic uracil is removed by uracil-DNA-glycosylases. Bacterial polymerases, in general, replicate past uracil by inserting an adenine (preserving the sequence); cases where uracil forms via cytosine deamination lead to CG-to-TA mutations. In contrast, some archaeal replicative polymerases stall before misplaced uracils, representing a “read-ahead” proofreading function not found in bacteria or eukaryotes. Uracil is not normally in DNA so it can be harmful BER is a DNA repair process that fixes damaged bases without distrusting the DNA structure. BER helps prevent mutations by removing harmful bases, like uravil, which can cause errors during DNA replication In bacteria, normally they use a uracil-DNA glycosylase to remove uracil from DAN. However, bacterial polymerases can replicate past uracil by inserting an adenin opposite the uracil. This helps preserve the genetic sequence but if uracil is from from cytosine deamination, its an lead to a CG to TA mutation, changing the sequence In archaea, some of the polymerases have a “read ahead” proofreading function causing the polymerases to stall when they encounter a misplaced uracil giving the cell the chance to repair it before replication continues preventing the risk of this mutation  DNA repair pathways: Nucleotide Excision Repair: A Versatile DNA-Repair NER is a tremendously versatile DNA-repair Pathway system that is highly conserved from bacteria to humans. It consists of two subpathways: global-genome NER (GG-NER), which monitors the entire genome for damage; and transcription-coupled NER (TC -ER), which repairs damage that specifically interferes with transcription. Expression of NER components (with the exception of UvrC) is regulated by the SOS response. Unlike BER, which recognizes and repairs specific lesions that have little to no effect on the structure of the DNA double helix, the NER pathway monitors the DNA for even small structural distortions. From one perspective, it could be said that the NER pathway repairs distortions, and as a consequence, removes the causal damage (Table 4.2).  DNA repair pathways: Don’t need to memorize Nucleotide Excision Repair: A Versatile DNA-Repair Pathway  DNA repair pathways: Nucleotide Excision Repair: A Versatile DNA-Repair Pathway In E. coli, damage is detected via collaboration between UvrA and UvrB in the GG-NER pathway. Alternatively, if the damage is first encountered by RNA polymerase (RNAP), leading to transcription stalling, the Mfd protein (also known as the transcriptional-repair coupling factor, or TRCF) displaces the stalled RNAP Loading… and recruits UvrAB to the damage site (TC-NER). How exactly UvrA and UvrB bind to the damaged DNA remains a challenging experimental problem. 1. Damage is recognized by a DNA distortion 2. The damaged DNA is unwound by helicase to create a single stranded region around the damage/ lesion 3. Enzymes cut out a short segment of the damaged strand 4. The gap is filled in by DNA polymerase using the undamaged strand as a template 5. The new strand is sealed by ligase  DNA repair pathways: Nucleotide Excision Repair: A Versatile DNA-Repair Pathway Nucleotide Excision Repair: A Versatile DNA-Repair Pathway Inproduct E.coli, a multisubunit enzyme, the of the UVRA, UVRB and UVRC genes, recognizes pyrimidine dimers. Two molecules of UVRA and one of UVRB form a complex that moves randomly along DNA. Once the complex encounters a lesion, conformational changes in DNA, powered by ATP hydrolysis, cause the helix to become locally denatured and kinked by 130°. After the UVRA dimmer dissociates, the UVRC Endonuclease binds and cuts the damaged strand at two sites separated by 12 or 13 bases. UVRB and UVRC then dissociate, and Helicase-II unwinds the Know the protiens damaged region, releasing the single- stranded fragment with the lesion, which is degraded to mononucleotides. The gap is then filled by Pol-I (DNA Polymerase-I), and the remaining nick is sealed by DNA Ligase (Ref.1).  DNA repair pathways: Nucleotide Excision Repair: A Versatile DNA-Repair Pathway Some aspects of their NER pathways in archaea are more similar to eukaryotic versions than to bacterial versions and they may or may not have uvr homologs. The presence of clear uvr homologs seems to coincide with lifestyle: mesophilic archaea tend to have uvr genes, while hyperthermophilic archaea (HA) do not. A universal feature (for archaea) seems to be the presence of homologs of eukaryotic factors. Deletion of any of HA eukaryotic-like NER genes has little to no effect on UV resistance. Hypothesis proposed by Dennis Grogan is that HA do not attempt to remove lesions before DNA replication and, instead, rely on interactions between replication forks and lesions for repair (various bypasses).  DNA repair pathways: Correcting Mismatched Bases: Cleanup After DNA Replication The primary function of MMR is to remove bases incorrectly inserted by DNA polymerase during DNA replication and its importance is emphasized by its cross-domain functional and homologous conservation. In E. coli, MMR can improve the accuracy of DNA replication up to 400- fold. The E. coli MMR pathway has been reconstituted in vitro with only three MMR-specific proteins: MutS, MutL, and MutH.  DNA repair pathways: Correcting Mismatched Bases: Cleanup After DNA The methylation allows it to Replication The initiating step of the MMR pathway is the know what’s the parental strand and what is daughter recognition and binding of a mismatched base in the dsDNA helix by a MutS dimer. A MutL dimer subsequently binds to the MutS–DNA complex, thereby stabilizing it and activating the MutH restriction endonuclease. MutH then nicks the strand containing the incorrectly incorporated base. The errant strand is then removed via helicase (UvrD) and exonuclease activities (ExoI/ExoVII/RecJ). A new strand is synthesized by DNA polymerase III using the undamaged strand as When they identify the distortion they methylate the adenine in the GATC a template. Mismatch leads to a distortion (bubble) Finally, the nick is sealed by DNA ligase. 1. Error is identified by MutS 2. The repair must distinguish between the old (template) strand and the new (daughter strand) - which has the error. This is done using DNA methylation (the parent stand is methylation while the daughter strand is not). It methylates at the nearest GATC sites on the adenine. Mouth is involved in identifying the unmethylated strand 3. After mismatch is direct and daughter strand is identified, MMR machinery cuts out the incorrect DNA segment. This involved MutH creating a nick in the newly synthesized strand near the mismatch 4. Once the error is cut out, DNA polymerase fulls in the gap using the template strand as the guide 5. The strand is sealed by ligase ? or parent  DNA repair pathways: Correcting Mismatched Bases: Cleanup After DNA An obvious challenge for MMRReplication is to identify which DNA strand has the misincorporated base. E. coli meets this challenge by monitoring the methylation status of the two DNA strands. There are a lot of GATC sites. As the fork proceeds during DNA replication, the daughter strand is methylated at GATC sites by the DNA adenine methyltransferase Dam. During a transient period, the newly synthesized dsDNA is hemimethylated, that is, only one strand is methylated.  DNA repair pathways: Correcting Mismatched Bases: Cleanup After DNA Replication While GATC sites are overrepresented in the E. coli genome, one may not be in the direct proximity of the mismatched base. The reading of distant GATC sites may occur by two mechanisms: a cis-model, in which MutS translocates along the DNA, or a trans- model, in which a loop forms between the sites. E. coli MMR may be the exception, rather than the rule. Homologs of MutS and MutL are widely distributed, but MutH seems to be rare in other bacteria and archaea. What happens if GTAC sites are not near the mismatch: 2 ways to deal with it 1. Cis model: MutS protein trans locates along the DNA to find the GATC site that has been methylated which helps identify the parent strand 2. Trans model: A loop forms in the DNA between the mismatch and a distance GATC. This allows the repair machinery to recognize the parent stand by detecting the methylation at the GATC site that is far away  DNA repair pathways: Correcting Mismatched Bases: Cleanup After DNA Mesothermophilic archaea tend Replication to have MMR pathways that mirror the canonical bacterial pathways, although they likely originated from horizontal gene transfer. In contrast, the HA lack MutS and MutL homologs (the same group that lacks canonical uvr homologs); however, despite the lack of MutS and MutL, genome replication is accurate in these organisms. The substitute mechanisms are unclear, perhaps some forms of translesion synthesis, recombination, template switch etc.  DNA repair pathways: Correcting Mismatched Bases: Cleanup After DNA Replication The interaction between MMR and homologous recombination: As both of these pathways function in tight association with the replication fork, they share both space and time. It is well established that MMR suppresses illegitimate recombination, especially highlighted by the observation that loss of MMR increases the frequency of interspecies DNA exchange between E. coli and Salmonella during conjugation. Similar observations were also noted for transduction and transformation. In this way, MMR can limit the impact of foreign DNA on genome stability, similar to restriction-modification systems.  Recombination Repair: Double-Strand Breaks It is generally agreed that double- A Chi site or Chi sequence is a short stretch of DNA in strand breaks (DSBs) in DNA the genome of a bacterium near which homologous recombination is more likely to occur than on average across the genome. represent the greatest threat to genome stability. HR-dependent repair of DSBs can be distilled into discrete steps that are conserved from bacteria and archaea to eukaryotes (although the players in each step vary): 1. End resection. The broken ends of the DNA must be prepared for the subsequent molecular transactions. 2. Strand invasion. A single- stranded stretch of DNA terminating in a 3′-OH is guided into the duplex of a homologous molecule.  Recombination Repair: Homologous recombination This process is mediated by recombinases, including RecA (E. coli), Rad51 (many eukaryotes), and RadA (archaea). 3. Branch migration. Strand invasion leads to a four-strand branched intermediate. This intermediate is remodeled to facilitate new DNA synthesis and other molecular processes. 4. Holliday junction resolution. This step leads to the restoration of two DNA duplexes via strand cutting. 1. End resection: the broken ends of the DNA must be processed to prepare them for repair 2. Strand invasions a single stranded DNA segment with a 3’-OH group is guided into a homologous DNA duplex mediated by Rec A 3. Branch migration: After strand invasion, a four strand intermediate is formed. This intermediate undergoes remodeling to enable new DNA synthesis and other related processes 4. Holliday junction resolution: The final step involved cutting the strands of the intermediate resulting in the restoration of the two intact DNA duplexes  Recombination Repair: NHEJ This pathway is error prone as it mediates the direct attachment of two DNA double-strand ends independent of extensive homology. A pathway for NHEJ remained elusive in E. coli and it was generally accepted for many years that no pathway exists. A 2010 work, however, has demonstrated that E. coli strains do possess an end-joining mechanism, now called alternative end joining (A-EJ). This pathway does not share conserved factors with canonical NHEJ pathways, depends on bidirectional strand resection, frequent use of microhomology, and nontemplated DNA synthesis.  Restriction-modification systems: protecting the genome from invaders In 1978, Werner Arber, Daniel Nathans, and Hamilton Smith won the Nobel Prize for Physiology or Medicine “for the discovery of restriction enzymes and their application to problems of molecular genetics.” In early 1950s it was noticed that some E. coli strains were more resistant to bacteriophages (bacterial viruses) than others, leading to the use of the term “restriction”. It was noted that the ability of bacteriophage to productively infect their host was controlled by a two-part process in which a pathway restricting infection competed with some type of modification that alleviated the restriction (Fig. 4.2). Recognize self from non self Restriction-modification systems: protecting the genome from invaders Recognize self from non self  Restriction-modification systems: protecting the genome from invaders The precise molecular components of restriction-modification systems are diverse and they have been divided into four major groups (I–IV) based on several properties: structure, energy requirement, and cleavage mechanism. In general, all restriction-modification systems function on the same basic molecular principle to distinguish self and foreign DNA: One enzyme encodes a methyltransferase that modifies self-DNA via the addition of methyl groups to specific sequences. Another complementary enzyme recognizes the same sequences and, when they are unmodified, cuts the DNA by hydrolyzing the phosphodiester backbones of both strands.  Restriction-modification systems: protecting the genome from invaders The stability of prokaryotic genomes is challenged by three processes that allow the intercellular transfer of genetic material: transformation, transduction, and conjugation. Invasions by foreign DNA can induce genome instability via interactions (ie, recombination) with the host chromosome. DNA fragmentation by restriction enzymes can also stimulate recombination, suggesting an alternative way that restriction-modification systems can influence genome stability. In this case, instead of limiting the effects of foreign genetic material on the genome, a restriction-modification system could support the incorporation of novel DNA via recombination.  Genome instability due to genetic exchange: Transduction Transduction is a process mediated by bacterial viruses called bacteriophages in which they transfer DNA by an infectious process.  Genome instability due to genetic exchange: Conjugation Conjugation, often called bacterial mating, generally requires direct contact between the donor and recipient cells.  Genome instability due to genetic exchange: Transformation Transformation is a process by which naturally competent bacteria take up naked DNA from the surrounding environment. The foreign DNA then typically integrates into the host chromosome either by HR, or via NHR.  Conclusion Within domains and across the three domains of life (bacteria, archaea, and Eukarya), a basic core of DNA-repair pathways exists. Remarkably, some functionally equivalent pathways appear to have evolved entirely independently. The evolution of DNA repair pathways in different species was influenced by specific challenges experienced in their environments.  References/reading pages Kovalchuk and Kovalchuk “Genome Stability – from Viruses to Human”. Genome Instability in Bacteria and Archaea: Strategies for Maintaining Genome Stability. Chapter 4, pages 51-64.