Fundamentals in Biology 1: From Molecules to the Biochemistry of Cells PDF

Script to accompany: Fundamentals in Biology 1: From Molecules to the Biochemistry of Cells This script is for internal ETH use only, not to be copied, distributed or sold. It contains referenced images from external sources and copyrighted new images....

Script to accompany: Fundamentals in Biology 1: From Molecules to the Biochemistry of Cells This script is for internal ETH use only, not to be copied, distributed or sold. It contains referenced images from external sources and copyrighted new images. Part 1: Introduction to DNA Replication, Recombination and Repair DNA REPLICATION The Central Dogma of Molecular Biology The expression of genetic information from DNA to RNA to protein is referred to as the Central Dogma of Molecular Biology. In order to understand how this genetic information is expressed, it is important to understand how DNA is copied. DNA replication and preservation of genetic information between generations occurs with an error rate of 1 in 10 billion cases, which is achieved through accurate synthesis and proofreading methods, and subsequent repairs. The Central Dogma of Molecular Biology. DNA replication is an extremely accurate biological process with an error rate of 1 in 10-10. Such accuracy can be achieved by proofreading and repair during DNA synthesis. Friedrich Miescher's Discovery of DNA In the 1860s, Swiss scientist Friedrich Miescher discovered DNA and its chemical properties. Miescher used simple methods to analyze cells found in bandages from injured people at a local hospital. He used precipitation and sedimentation to separate cellular compartments and fractions from the nucleus and to analyze the material found in those samples. Miescher found a substance in the nuclear fraction (termed nuclein) that was different in properties compared to proteins, which had previously been thought to be the essence of living beings. This novel substance contained phosphorous and nitrogen, but not sulfur. At low pH conditions, Miescher showed that the new type of macromolecule (DNA) became insoluble, suggesting that it is an acid. Alfred Hershey and Martha Chase's Experiment In 1952, Alfred Hershey and Martha Chase performed an experiment that unambiguously demonstrated that DNA is the molecule that contains genetic information. They used different chemical labels of either protein or DNA and their experimental system was a (bacterio)phage, a bacterial virus. After infecting their system with phages that had either the protein labeled with radioactive sulfur or the DNA labeled with radioactive phosphorous, they spun down the bacteria and found that only radiolabeled DNA made its way into the bacteria but that this was sufficient to later produce new phages that consist of both protein and DNA. This experiment launched a series of investigations into the structure of the DNA molecule and how it might explain the mechanism of copying genetic information. Famous scientists such as James Watson, Francis Crick and Linus Pauling were involved in this race. Outline of the Hershey-Chase experiment. Different radiolabeling of protein and DNA is used to determine which part is necessary for the propagation of the virus. (From Wikimedia Commons) Chargaff's Nucleotide Ratios (“Chargaff's Rules”) Using paper chromatography and ultraviolet spectrophotometry to analyze the nucleotide composition of DNAs from different organisms, the Austro-Hungarian-born biochemist Erwin Chargaff discovered that although the amount of individual nucleotides among species varied a lot (2nd Chargaff’s rule), the ratio of adenine to thymine and guanine to cytosine was always equimolar (1st Chargaff’s rule). (From Life: The Science of Biology, Freeman 2004) At the same time in Cambridge, England, Rosalind Franklin and Maurice Wilkins conducted experiments on DNA fibers. They isolated the DNA and then exposed it to focused x-rays in a hydrated form. This beautiful experiment revealed clear patterns of DNA fibers, which showed that the DNA forms different double helices with specific helical pitches. X-ray photograph of the B-form of DNA fibers. (From Franklin and Gosling, Nature 1953) Watson and Crick's Model of DNA's Structure Based on the data presented, Watson and Crick used modeling with cardboard blocks that resembled the structures of bases to deduce the structure of DNA. They found that when either G:C or A:T base pairs are formed, the width of the base pairs stay the same, allowing for a continuous geometry of the DNA backbone. This led to the conclusion that based on the sequence information on one DNA strand, it is possible to accurately determine the sequence of the complementary DNA strand. Watson and Crick with their B-form DNA model in 1953 The corresponding Watson-Crick base pairs are shown in the right panel (From A. Barrington Brown, Science Photo Library, and Stryer, Biochemistry 8th Edition, Freeman 2015) Schematic depiction of the complementary antiparallel DNA strands. Note the directionality, which by convention (and according to biological synthesis) is always indicated from 5’-P to 3’-OH. (From Brock, Biology of Microorganisms, 15th Edition, Pearson 2018) Nitrogenous bases (with R=H) and the corresponding nucleosides Adenosine, Guanosine, Cytidine, and Thymidine (with R=deoxyribose) or Uridine (with R=ribose). Mono- di and triphosphates of nucleosides are termed nucleotides. (From Wikimedia Commons) The two complementary strands of DNA form a double helix. This helix has a chemical directionality, as the ribose phosphate backbone has 5’-O groups on one side and 3’- O groups on the other. The strands run in opposite directions, and for the initially characterized DNA, so called B-form DNA, the distance between each base pair is 3.4 Å (Ångstroms). This is the distance that the atoms between two planar bases that stack in the middle of the double helical DNA can approach each other before causing steric clashes. There is also a 36 degree turn between each base pair, meaning one full turn of DNA happens after exactly 10 steps. ~20Å width Geometry of the physiologically most common B-form DNA in side and top views. The spherical model reveals that the base stacking is stabilized via van der Waals interactions. (From Stryer, Biochemistry, 8th Edition, Freeman 2015) The bonds between the bases and the sugar ribose are always at the same place, meaning that no matter the sequence, the backbone will not deviate much in structure. DNA also has two different forms, the B-form and the A-form. The B-form has a large shallow major groove and a deep minor groove and is the prevalent form of DNA under physiological cellular conditions. Comparison between A and B form DNAs. The sugar pucker conformation defines the overall architectural difference. The wide and shallow major groove of B form DNA that exposes the bases allows a sequence-specific recognition by regulatory proteins. Note the very similar appearance of A form DNA and RNA double helices. (Picture by Marc Leibundgut) These characteristics of DNA are important for the recognition of particular sequences within the DNA, as many proteins that interact with the DNA in a sequence-specific manner bind to the major groove. The geometry of DNA has many implications for the continued discussion of its structure and is essential to understand the process of replication. An example of transcription factor binding to B form DNA. The DNA-binding domain of the glucocorticoid receptor attaches to the DNA backbone and predominantly recognizes nucleotides of the major groove. The diagram below shows which side groups of the nucleotides are exposed and therefore available for specific recognition (Upper panel from PDB 1glu, picture by Marc Leibundgut, lower panel from Stryer, Biochemistry, 8th Edition, Freeman 2015) DNA Replication is Semi-Conservative (The “Meselson - Stahl Experiment”) Meselson and Stahl investigated how DNA is copied. They initially grew bacteria in the presence of nitrogen 15 and then they exchanged the growth media to only provide nitrogen 14 as the source of nutrients. Then they analyzed the sedimentation of DNA under high-speed centrifugation at the beginning and after one round of bacterial replication (20 minutes), the process that requires copying of the genomic DNA. After 20 min they saw only one band, corresponding to a mixture of nitrogen 14 and 15. After another round of replication (40 min) they discovered the presence of two bands, one a mixed band of nitrogen 14 and 15 and one where the DNA was labeled only with nitrogen 14. This experiment showed that DNA replication was semi-conservative, meaning that the new molecule had one strand of the old DNA and one newly synthesized strand. The Meselson-Stahl experiment reveals that DNA replication is semi-conservative. (Source: Wikipedia) DNA's Topological Problem The unique properties of DNA's double helical structure present a topological problem for copying, as the strands must be unwound in order to separate them. This creates a further downstream problem where the double helix is overwound. Additionally, as DNA is a very stable duplex with a lot of hydrogen bonds and stacking interactions, energy is needed to melt the DNA. In spite of this, 4.6 million base pairs in a bacterial genome must be copied in 20 minutes. Furthermore, this process must be extremely accurate, with one error reported in every 10 billion nucleotides copied. Not only must the DNA be copied, but any damage to the DNA must be recognized and repaired. Overwinding of DNA occurs upon melting and unwinding of a DNA stretch for DNA replication at the origin of replication (then termed “replication bubble”) or in the promoter region of a gene during RNA transcription (“transcription bubble”). (Adapted from from Stryer, Biochemistry, 8th edition, Freeman 2015) The topological problem can be analyzed by considering mathematical properties and topological transformations of coiling and supercoiling. The linking number (Lk) of a circular relaxed 260bp B form DNA molecule is 25, which is the number of turns of one strand around the other that occurs every 34 angstroms (corresponding to 10.4 base pairs) in the B form double helical DNA. The linking number and the twisting number are the same when the molecule is in one plane. However, there is yet another number here called the “writhe” number (Wr), which refers to the number of superhelical twists. When our circular example DNA molecule is cut, unwound by two right-hand turns, and then reconnected, it would have a linking number of 23 and the number of twists (Tw) also being 23 in one plane, with a writhe number of 0. This molecule is not stable, as it would like to ultimately reform the base pairs. The only way these base pairs can form is if there is a compensation for the untwisting that has happened, and this can be satisfied if the molecule gets out of plane. This results in a supercoiled molecule, with the whole strand being wound around itself and crossing itself twice, allowing all the base pairs to be satisfied with a linking number of 23 and a writhe number of -2. Relation between linking number, writhe and twist exemplified by a 260bp B form DNA. Topological isomers (topoisomers) can only be interconverted upon DNA strand cutting and rejoining by topoisomerase enzymes. (Adapted from Stryer, Biochemistry, 8th Edition, Freeman 2015) Topoisomerases When DNA polymerases copy the DNA, the DNA is unwound, generating a supercoil downstream of the unwinding location. The only way to relax the supercoil is to allow one strand of the DNA molecule to break and rotate around the other, and then reconnect. This is achieved by an enzyme called topoisomerase (type I). Once one strand of the DNA is broken, there is a natural relaxation process, since single stranded DNA can freely twist. This is because the phosphodiester backbone of the DNA has many single bonds that can freely rotate, so when one bond breaks, the molecule relaxes. The broken ends are then reconnected, so that DNA copying and strand separation can continue. Compaction and protection of circular bacterial chromosomes by negative supercoiling with topoisomerase (type II). The process can be reversed by topoisomerase I, which relaxes the supercoils during DNA replication and RNA transcription. (From Brock, Biology of Organisms, 15th Edition, Global Edition 2019) Topisomerases are enzymes that not only relax DNA when copying, but can also use ATP to generate a negative supercoil (type II). For this supercoiling, the topoisomerase introduces cuts and uses energy to twist the DNA. This twisting is used to package long DNA genomes into small cells. This packaging organizes the genome and also increases its stability, which is especially important for organisms that have to survive at high temperatures. This is often the case for thermophilic archaea, which have to survive temperatures that are sometimes even higher than 100 degrees Celsius. DNA Polymerases and the Need for a Primase The enzymes responsible for copying DNA are called DNA polymerases. There are typically more than one, and they have to take the template DNA and copy it such that the complementary strand is synthesized. The incorporation of the first several copied nucleotides is less accurate than extending a partially synthesized copy of DNA. To increase the accuracy of the copying process, a different type of molecule is synthesized first. This molecule, called a primer, is synthesized by an enzyme called primase. The primer is a short RNA molecule complementary to one small segment of the DNA that has to be copied. Thus, DNA replication starts with a short stretch of RNA, which increases the accuracy of copying because this less accurately synthesized molecule is not DNA and will therefore be removed at a later stage and replaced with DNA synthesized from a neighboring DNA that serves the priming function. DNA polymerase, specifically DNA polymerase III, will then use the RNA primer to continue the synthesis. This is the primary DNA polymerase in bacterial cells and it copies the DNA from 5' to 3', like all polymerases. The DNA polymerases share a common architecture. This architecture is best understood when one compares the shape of the enzymes to a right hand. The "fingers" make up half of the cleft that binds the DNA substrate, while the "thumb" builds the other half of the cleft. At the base of the left side, where the palm would be, is where the active site is located. Additionally, there are domains that may have other functionalities that will be discussed later. Initiation of DNA synthesis by DNA polymerase is dependent on a short RNA oligonucleotide, the primer, which is synthesized by a primase and later removed. Note the directionality of the synthesis processes, which are always from 5’ to 3’ (meaning that a new DNA or RNA nucleotide is attached to the 3’OH end of the newly synthesized DNA). A prototypic structure of DNA polymerase, resembling a right hand, is shown on the right. (Adapted from from Stryer, Biochemistry, 8th Edition, Freeman 2015) The reaction mechanism involves the hydrolysis of nucleotide triphosphate substrates. This process takes place in the active site of the enzyme, allowing the enzyme to control the base pairing with the complementary nucleotide in the DNA template strand. This nucleophilic attack involves the oxygen of the three prime hydroxyl group of the last RNA nucleotide in the primer strand or in the extending DNA strand. The bond between the alpha and beta phosphate is broken, and pyrophosphate leaves the reaction. An ester bond is formed between the phosphate and the sugar, resulting in a phosphate backbone. This results in extension by one nucleotide of the DNA strand that is being copied. Mechanism and directionality of DNA synthesis. For RNA synthesis, the mechanism is essentially the same (although the sugars carry an additional 2’OH group, and U rather than T is used). (Adapted from from Stryer, Biochemistry, 8th edition, Freeman 2015) Due to this reaction, the DNA can only be copied in one direction. The DNA template strand is read from three prime to five prime, whereas the new strand is synthesized from 5’ to 3’. This is because the substrate nucleotides are triphosphates with the three phosphates attached to the 5’ end and with a 3’ hydroxy group. DNA replication is a complex process that requires several enzymes to work together to produce complementary strands of DNA. To begin, DNA must be opened up at specific locations known as origins of replication. This is accomplished by enzymes called helicases, which use energy to unwind the DNA, therefore creating a replication bubble with two replication forks. Replication Fork Formation and Okazaki Fragments At each replication fork, the two template strands of DNA run in opposite directions and must therefore also be synthesized in opposite directions, in one case 5’ to 3’ and in the other case 3’ to 5’. This was experimentally demonstrated by analyzing rapidly replicating DNA systems, which showed fragments of newly synthesized DNA of uniform size. Okazaki was a scientist who demonstrated that these fragments of DNA occur, explaining that the ‘lagging strand’ on the replication fork is synthesized in a discontinuous manner in ~1000 nucleotide segments. In contrast, the other strand can be copied directly without restarting and is called the ‘leading strand’. This leads to another problem, as the newly synthesized DNA must be started with a primer, which is RNA. Therefore, to form a continuous newly synthesized lagging strand, additional enzymes must be used. First, the primase and the DNA polymerase III synthesize the lagging strand in segments. Once the DNA polymerase III reaches the primer from the previously synthesized Okazaki fragment, it will stop and continue together with primase to synthesize the next Okazaki fragment. DNA polymerase I will then bind to the 3’ end of the Okazaki fragment and elongate it to close the gap between the Okazaki fragments. Because Pol I carries also an exonuclease activity on an additional domain that hydrolyzes the RNA primer in the 5’ to 3’ direction, it will simultaneously excise the primer one or several nucleotides at a time. DNA ligase is then used to close the breaks between the two DNA strands, using ATP for energy. Single-stranded binding proteins (SSBPs) are present to protect the single strands of DNA from being cut or damaged by enzymes or chemicals. Without SSBPs, double stranded DNA breaks could occur, leading to major genetic problems. During the replication process of DNA, a replication fork is formed. The leading strand is immediately copied by DNA polymerase III in a continuous manner by reading in the 3' to 5' direction. Meanwhile, the lagging strand is synthesized in small segments, the aforementioned Okazaki fragments. DNA polymerase holoenzyme is actually a supercomplex that consists of two DNA polymerases and associated enzymes, binds to the replication fork and copies the DNA of both strands at the same time. The DNA replication machinery at the replication fork. The leading strand is continuously extended from the primer, while the lagging strand is extended discontinuously from each primer forming Okazaki fragments. (Adapted from from Stryer, Biochemistry, 5th Edition, Freeman 2002) The Trombone Model of DNA Replication The Trombone Model of DNA replication suggests that the leading and lagging strands are replicated simultaneously by the DNA polymerase III holoenzyme that moves along the DNA as the replication progresses. This model proposes that a single replication machine, the ‘replisome’, with two DNA polymerase III holoenzymes, copies the leading and lagging strands in opposite directions simultaneously. In this way, one of the two strands of DNA forms a loop that increases and shrinks in size during the process, similar to the way that a trombone slide moves up and down. The Trombone Model of DNA replication. In order to synthesize the leading and lagging strand simultaneously by one replisome assembly harboring two Pol III holoenzymes, the template DNA for the lagging strand synthesis needs to be looped out and threaded through in a staggered manner. (Adapted from from Stryer, Biochemistry, 8th Edition, Freeman 2015) Circular DNA Replication In the case of circular DNA, which is present in bacterial genomes, the replication bubble opens at the site called the origin of replication and the two replication forks go in opposite directions until they collide and disassemble. Bi-directional replication of circular DNA by two replication forks starting at the origin if replication (From Brock, Biology of Organisms, 15th Edition, Pearson 2019) DNA REPAIR AND RECOMBINATION Introduction As introduced in the previous lectures, DNA polymerases play a crucial role in copying DNA with remarkable accuracy. Their active site is highly specific in selecting and incorporating correct nucleotides that are complementary to the template DNA strand, with an error rate of 1:10000. The nucleotide selection is template-based and follows complementary base pairing rules (A with T and G with C) to ensure that the new DNA strand matches the template. This accuracy in selecting the right nucleotide during each addition is a primary factor in the overall precision of DNA replication. In addition to their polymerization activity, many DNA polymerases, such as DNA polymerase III, have a built-in exonuclease activity. This exonuclease activity works in the 3' to 5' direction, allowing the polymerase to proofread the newly synthesized DNA strand. If a mistake is made during nucleotide incorporation and the wrong nucleotide is added, the exonuclease activity can remove the incorrect nucleotide. This correction mechanism is essential for maintaining the fidelity of DNA replication. Once the erroneous nucleotide is removed, the polymerase can reinsert the correct one, ensuring accurate copying of DNA. 5’-3’ polymerase and 3’-5’ exonuclease activities of DNA Pol III. Upon misincorporation, DNA synthesis slows down due to steric issues with threading of non-Watson-Crick base pairs through the enzyme, providing time for the newly synthesized DNA strand to diffuse into the exonuclease active site, where nucleotides are removed one by one in a 3’-5’ direction until the strand becomes repositioned into the active site of the polymerase to continue DNA synthesis in the 5’-3’ direction. (Adapted from from Stryer, Biochemistry, 8th Edition, Freeman 2015) By combining the precision of nucleotide selection and the error-correcting exonuclease activity, DNA polymerases achieve accuracy of one error in a million. This accuracy is critical for maintaining the integrity of genetic information during each round of DNA replication. Spontaneous DNA Mutations Once DNA is accurately copied, spontaneous mutations in DNA can nevertheless occur. Two common types of point mutations are transversions and transitions, but also insertions and deletions are observed. These mutations have the potential to be propagated through DNA replication unless they are corrected. Transversions are a type of point mutation where one purine base (adenine or guanine) is substituted for a pyrimidine base (cytosine or thymine), or vice versa. For example, a base change from adenine (A) to thymine (T) is a transversion. Transversions are less common than transitions and can result from various factors, including exposure to certain chemicals or environmental factors. If left uncorrected, transversions can introduce changes in the DNA sequence that may affect gene function. Transitions are another type of point mutation where a purine base is replaced by another purine (A to G or vice versa), or a pyrimidine is replaced by another pyrimidine (C to T or vice versa). For example, a change from cytosine (C) to thymine (T) is a transition. Transitions are more common than transversions and can result from spontaneous chemical reactions within the DNA molecule. DNA mutagens are agents or factors that can cause changes or mutations in DNA. These mutations can have a wide range of effects, including increasing the risk of cancer and other diseases. Mutations will be propagated unless they are detected and repaired. Example of how transitions can occur due to permanent chemical modification or transiently occurring tautomerism. Common mutagens include: - alkylating agents - deaminating agents such as nitrous acid can remove amino groups from DNA bases, causing base changes. - Ultraviolet (UV) Light: UV light can induce the formation of thymine dimers in DNA. - Ionizing Radiation: X-rays and gamma rays can break DNA strands and cause various types of mutations, including deletions and translocations. - Reactive oxygen species (ROS), produced during normal cellular processes and in response to environmental factors, can cause oxidative damage to DNA. Different DNA Repair Mechanisms Correct Different Types of DNA Lesions Nucleotide excision repair (NER) and base excision repair (BER) are two essential DNA repair mechanisms that organisms employ to correct damaged or mismatched nucleotides. Nucleotide excision repair is a DNA repair system primarily responsible for fixing bulky DNA lesions, such as those caused by UV radiation, whereas base excision repair is a DNA repair mechanism specifically designed to address damaged or mismatched individual DNA bases within the DNA strand. It is the primary mechanism for repairing small, non-bulky lesions such as those occurring due to oxidative damage or spontaneous deamination. Additionally, mismatch repair (MMR) is a DNA repair pathway that corrects errors such as mismatched bases, insertion or deletion loops, and other small-scale DNA mispairings that can occur during DNA replication. It's a crucial mechanism for maintaining the accuracy of DNA replication. Base Excision Repair (BER) While in nucleotide excision repair (NER), entire altered nucleotides or DNA segments including the alteration are recognized, removed and corrected, during base excision repair abasic sites are generated by removal of bases from the DNA backbone. These abasic site represent a specific intermediate step during the repair process. Deamination of cytosine in DNA results in uracil, which can be specifically recognized and corrected by the components of the BER mechanism to avoid the occurrence of corresponding transition mutations. The presence of T (with the additional methyl group) instead of U in DNA allows distinction between “deaminated C” (=U) and T by repair mechanisms, which enhances the fidelity of the genetic message. (Adapted from from Stryer, Biochemistry, 8th Edition, Freeman 2015) One well-known example of DNA damage that is corrected by base excision repair is the spontaneous deamination of cytosine to uracil, a mutation that can happen when an amino group is removed from the cytosine base in DNA. Such event can have serious consequences because uracil normally pairs with adenine (A) in DNA, while cytosine pairs with guanine (G), eventually resulting in a transition during DNA replication in one of the daughter strands. The fact that cytosine in DNA can spontaneously deaminate to become uracil is probably one reason why DNA evolved to contain thymine bases, since this allows the detection of uracil as a “non-DNA” base and the repair of the mutation introduced by deamination, which enhances the fidelity of the genetic message. The repair of mis-incorporated uracil in DNA derived from cytosine deamination is a three-step process starting with the recognition of the uracil base within the DNA strand by uracil-DNA glycosylase. The enzyme then cleaves the glycosidic bond that links uracil to the deoxyribose sugar in the DNA backbone, creating an abasic site. This triggers the activity of endonucleases that cleave the DNA backbone at the abasic site, creating a single-strand break with a 5'-deoxyribose phosphate and a 3'-hydroxyl group. A DNA repair polymerase such as DNA polymerase I then fills the gap with a cytosine that is complementary to the guanine on the other strand, and DNA ligase seals the nick in the DNA strand. Repairing Errors After DNA Replication by Mismatch Repair (MMR) The first step in mismatch repair involves the recognition of a mismatch or error in the DNA strand. The protein MutS is responsible for this recognition. MutS scans the newly synthesized DNA strand and identifies any mispaired or unpaired bases. Once the error is recognized, MutS recruits another protein called MutL, which in turn recruits and activates MutH, forming a complex. MutH specifically recognizes hemimethylated DNA sites, a hallmark of freshly replicated DNA, since the parental DNA strand that serves as the template for replication is methylated while the newly synthesized daughter strand is not. For MutH, this methylation marks the parental strand as the correct, non-mutated strand, allowing it to introduce a single-strand cut into the erroneous daughter strand near to the mismatch position. The surrounding DNA including the error site are then unwound by a helicase and removed by an exonuclease, creating a gap in the DNA that is protected by single-stranded binding protein (SSB). The repair process is completed by DNA polymerase III holoenzyme, which synthesizes a new DNA strand to fill the gap, using the methylated parent strand as a template for replacement of the excised region with the accurate DNA sequence. Finally, the remaining strand break is closed by DNA ligase. MMR in E. coli. The mismatch is recognized by the MutS protein, which recruits MutL onto the DNA. MutS and MutL proteins activate MutH endonuclease, which introduces a cut into the unmethylated daughter strand. UvrD helicase unwinds the dsDNA, and an exonuclase removes part of the erroneous strand including the site of the mismatch. SSB protects the nascent ssDNA, DNA polymerase III fills the gap, and DNA ligase reconnects the remaining nick. (Adapted from Stryer, Biochemistry, 8th Edition, Freeman 2015, modified according to Han et al., Genome Instability & Disease, 2023) Defects in components of the MMR machinery increase the cellular mutation rates more than 100-fold, and in humans are associated with several types of cancer, including colorectal cancer and ovarian cancer. MMR deficiency is not the sole cause of these cancers, but it can significantly increase the risk. DNA Recombination as a Source of Genetic Diversity and DNA Repair Mechanism DNA recombination is a fundamental process that plays a pivotal role in generating genetic diversity within a population. It occurs through the exchange of genetic material between two DNA molecules, often homologous chromosomes or sister chromatids, resulting in the creation of new combinations of alleles. Site specific DNA recombination is also used by viruses for specific insertion of DNA sequences at target sites in the genome. Furthermore, homologous recombination serves as a critical DNA repair mechanism. All recombination events involve formation of a Holliday junction, which is a key intermediate in the process. A Holliday junction is a four-way junction where two double-stranded DNA molecules exchange genetic information. It forms when two DNA duplexes temporarily exchange strands. The central point of the junction represents the site of strand exchange or genetic recombination. At this point, one DNA strand from each of the two DNA molecules crosses over and pairs with a complementary strand from the other DNA molecule. The junction can move along the DNA molecule through a process called branch migration. During branch migration, the point of crossover slides along the DNA helix while maintaining base-pairing interactions. Ultimately the Holliday junction is resolved with the help of enzymes (resolvases) that cleave the DNA at specific points to separate the intertwined strands. Holliday model of homologous recombination. Depending on the resolution of the Holliday junction, recombination between Aa and Bb loci (crossover) will occur. Heteroduplex regions will be eventually corrected by mismatch repair, which still could result in gene conversion. (Adapted from Principles of Genetics, 6th ed., John Wiley and Sons Inc., 2012) An example for a site-specific recombination is shown for the Cre viral recombinase. Cre recombinase is an enzyme encoded by the P1 bacteriophage. It is widely used in genetic engineering to catalyze site-specific recombination. Cre recognizes specific DNA sequences which are typically 34 base pairs in length. When Cre binds to two sites, it can catalyze recombination between them using a tyrosine residue in its active site. The recombination process requires cutting and ligation of both DNA strands to achieve a new connection between the initially separated DNA strands. This process can lead to integration of DNA segments, such as viral DNA in this case, flanked by specific DNA sequences. Cre-mediated recombination is highly specific and precise and does not involve branch migration. Site-specific recombination by formation of a Holliday junction mediated by the binding of a sequence- specific viral recombinase. (Adapted from Stryer, Biochemistry, 8th edition, Freeman 2015, structural figure of viral Cre generated by Marc Leibundgut) RecA is a bacterial protein involved in homologous recombination. It plays a crucial role in repairing DNA double-strand breaks and facilitating genetic exchange between homologous DNA sequences. During homologous recombination, the RecA protein promotes the invasion of a single-stranded DNA molecule into a homologous double- stranded DNA molecule. This leads to base pairing between the invading single- stranded DNA and the homologous region of the double-stranded DNA. The process ultimately results in the exchange of genetic information between the two DNA molecules and allows repair of double stranded DNA breaks such that the intact double stranded DNA is used to repair the broken one. For this process to work the cell needs to have more than one copy of DNA. Organisms that are particularly resistant to DNA damage, such as the bacterium Deinococcus radiodurans, contain many copies of their genome, allowing them to maintain genome integrity by repair through recombination events. RecA-mediated strand invasion for the repair of DNA breaks. A homologous intact dsDNA molecule serves as a template. After repair synthesis by a polymerase in both directions, the process continues with branch migration, DNA ligation and resolution of the Holliday junctions. For clarity only one DNA end is displayed. (Adapted from Roy and Greene, Nature 2020, structural figure of RecA complex generated by Marc Leibundgut) Chromosomes, Genomes and Plasmids Bacterial chromosomes are the genetic material found in prokaryotic cells, specifically in bacteria. Unlike eukaryotic cells, which have a well-defined nucleus containing linear DNA strands wrapped around histone proteins, prokaryotic cells, including bacteria, have a single, circular chromosome. Bacterial chromosomes play a central role in controlling the cell's functions and transmitting genetic information to offspring during cell division. For example, Escherichia coli bacteria have a circular chromosome of 4.6 million base pairs (4.6 Mbp). Chromosomes in eukaryotes are linear and can be present in multiple copies. Viral genomes refer to the genetic material or nucleic acid content found within viruses. Unlike cellular organisms, viruses are acellular entities, and their genomes can vary widely in terms of structure, size, and composition. Viral genomes can be made up of either single stranded or double stranded DNA or RNA, and they carry the instructions necessary for the replication and propagation of the virus within a host cell. Plasmids are small, circular, double-stranded DNA molecules that exist and are propagated independently of the chromosomal DNA in many types of cells, including bacteria, archaea, and some eukaryotes such as yeast. They are often referred to as extrachromosomal elements because they are separate from the cell's main chromosome or genome, but are found within cells. Plasmids are usually circular, vary in copy number and size and play significant roles in various biological processes. They can carry antibiotic resistance or toxin genes, are involved in horizontal gene transfer and have practical applications in genetic engineering and biotechnology. \ Examples of genomes and extrachromosomal elements in different organisms.