The Mutability and Repair of DNA

A short presentation on DNA damage/mutation and the repair mechanisms involved that I prepared for my online chapter presentation. (The content and pictures credited to Watson et al.'s Molecular Biology of the Gene, 7th Edition)

1. The Mutability and Repair of DNA Sanam Parajuli Content and Pictures from: Molecular Biology of the Gene(7 th Edition), Watson et al.

2. Introduction With every division, a living cell has to copy billions of its base pairs to a new DNA strand, so, mistakes are unavoidable During normal functioning too, a living organism requires proper expression and functioning of thousands of genes Mutations: possible in coding sequences or in flanking sequences governing gene expression Very high rates of mutation in the soma can lead catastrophic incidences of cancer; too much changes in the germline can lead in progeny not surviving But too little changes: no evolution, no speciation, no diversity Life and biodiversity: sweet balance between DNA damage and repair

3. Intro… Two sources of mutation: Inaccuracy in DNA replication and Chemical Damage to the genetic material Replication errors: because of tautomerization that leads to incorrect pair readings and although the enzymatic machinery of DNA replication attempts to correct these errors, some escape detection Tautomerization: rearrangement of atoms in a molecule, eg : Adenine and cytosine from amino to imino form, guanine and thymine from keto to enol form. Now, A-C pairs and G-T pairs. DNA damage: because of finite stability, DNA is subject to damage such as loss of bases, chemical alteration of bases because of environmental mutagens Transposons: Jumping genes (not dealt in this chapter)

4. Consequences of replication errors and DNA Damage 1. Introduction of permanent changes to the DNA (mutations), which can alter the coding sequence of a gene or its regulatory sequences, effects observed in the progeny 2. Changes to the DNA can prevent it from being used as a template for replication and transcription, immediate effects observed in the cell itself in function and survival Challenge for a cell: scan the genome for errors and also mend the errors if possible How is the DNA mended rapidly enough to prevent setting of mutations? How are parent and daughter strands distinguished in repair? How does the cell restore the proper DNA sequence when, because of a break or severe lesion, the original sequence can no longer be read? How does the cell cope with lesions that block replication? It depends on the type of error to be repaired.

5. Replication Errors and their Repair The Nature of Mutations Simplest kinds: switches of one base for another: Transitions and Transversions Point mutations: alter a single nucleotide Other kinds like extensive insertions and deletions can cause more drastic changes to the DNA Ex: insertion of a transposon; rearrangement of chromosomes; aberrant recombination events Probability that new mutations arise spontaneously at any given site on the chromosome: 10 -6 to 10 -11 per round of DNA replication Some chromosome sites can be “hotspots” for mutations while some can mutate less frequently

6. *DNA Microsatellites* repeats of simple di-, tri-, or tetranucleotide sequences Really prone to mutations Ex: CA stretches found scattered across chromosomes DNA replication at such sites prone to “slippage” and a population, thus, can have polymorphic CA stretches at a given point Such polymorphism used as physical marker for mapping inherited mutations Triple repeat expansions associated diseases: Fragile X (CGG repeats more than 200), Huntington’s (CAG repeats coding for excess glutamine) etc

7. Some replication errors escape proofreading The 3’ 5’ exonuclease in the DNA polymerase : removes wrongly incorporated nucleotides with high accuracy. This proofreading increases the fidelity of DNA replication by a factor of nearly 100 However, not foolproof Some misincorporated nucleotides escape detection and become a mismatch between the template and new strand For each nucleotide, there are 3 possible mismatches ( eg : for T, T:C, T:G, and T:T are mismatches). So, there are 12 possible mismatches for the 4 nucleotides If misincorporated nucleotides not detected and corrected, the sequence change will be permanent in the genome

8. Mismatch Repair System Removes Errors that Escape Proofreading Fortunately, a mismatch repair system exists for correcting errors that escape exonuclease repairing that increases the replication accuracy by an additional 2 or 3 orders of magnitude Challenge for this system: First: scanning for mismatches. Mismatches are transient, only true for the cell of origin, in the daughter cell, it becomes permanent Second: only the daughter strand needs repair, not the parent. How does the system know which strand to repair?

9. Mismatch Repair System in E. coli In E. coli, mismatches are repaired by the dimer of the mismatch repair protein, MutS Mismatches cause distortions in the DNA strand, MutS scans for those MutS embraces the mismatch containing DNA, induces a pronounced kink in the DNA and a conformational change in itself MutS has an ATPase activity, but its role in repair is not properly understood The MutS -mismatched DNA complex recruits MutL , another protein of the repair complex MutL activates MutH , an enzyme that induces a nick in one strand near the site of the mismatch Nicking is followed by unwinding of the DNA towards the site of mismatch by a specific DNA helicase UvrD and an exonuclease progressively digests the single displaced strand The gap is filled by DNA Pol III and sealed with DNA ligase

10. How does the system know which strand to repair? a wrong decision can lead to permanent setting of the mutation The solution in E. coli : Transient Hemimethylation Dam methylase methylates A residues on both strands of the sequences 5’-GATC-3’, which is widely distributed in the E. coli genome, about once every 256bp. All these sites methylated When the replication fork passes a section with both strands methylated, the resulting daughter duplexes will be hemimethylated , ie methylated only on the parental strand. Hence, until Dam methylase catches up and methylates the daughter strand, the new strand is marked by the absence of a methyl group, and hence can be recognized for repair MutH can bind at such hemimethylated sites

11. MutH binds at unmethylated strand and initially its exonuclease activity is normally latent Only when MutH is contacted by MutL and MutS located at a nearby mismatch (which is likely to be within a distance of a few hundred base pairs) does MutH become activated How they interact in such long distances is uncertain, but evidence suggests that the MutL-MutS complex leaves the site of mismatch and moves along the strand to reach MutH at the site of hemimethylation MutH then nicks the unmethylated strand selectively

12. Still, removal of single stranded DNA between the nick site and the mismatch site left to be done For this, different exonucleases are utilized depending on the site of the mismatch with respect to the nick If the DNA is cleaved on the 5’ side of the mismatch, exonuclease VII or RecJ , which degrades DNA from 5’ 3’ direction is used If the nick is on the 3’ side, exonuclease I does the job DNA Pol III fills in the missing sequence

13. In Eukaryotes? Similar mechanism using homologs of MutS (called MSH, or MutS Homologs) and MutL (called MLH and PMS) Eukaryotes have multiple MutS like proteins with different specificities Ex: one specific for simple mismatches while other recognized small indels resulting from “slippage” MutH is absent in eukaryotes as well as hemimethylation and most other bacteria are unable to utilize hemimethylation and lack Dam methylase as well Lagging strands produce Okazaki fragments, and before ligation, the space between these fragments serve as nicks Human homologs of the of the MutS (MSH) interact with the sliding-clamp component of the replisome and would thereby be recruited to the site of discontinuous DNA synthesis on the lagging strand. Interaction with the sliding clamp could also recruit mismatch repair proteins to the 3’ (growing) end of the leading strand

14. DNA Damage Spontaneous Damage by Hydrolysis and Deamination Chemical/physical mutagens can cause DNA damage But DNA cam also go spontaneous damage from action of water (although aq. Environment is needed for proper double helix) Most frequent and important kind: Deamination of the base cytosine Under normal physiological conditions, cytosine can go spontaneous deamination and thus generating Uracil, which is unnatural in DNA C would have paired with a G, but now U pairs with A Similarly, deamination converts adenine to hypoxanthine, which bonds with cytosine, and guanine is converted to xanthine, which pairs with cytosine, albeit with only 2 H-bonds DNA also undergoes depurination by spontaneous hydrolysis of the N-glycosyl linkage, and this produces an abasic site

15. All these changes create unnatural changes in the DNA The presence of unnatural base makes it possible for the repair system to recognize errors Evolutionary significance of Thymine being present in place of Uracil in DNA : If Uracil was a natural base, deamination of cytosine would form another natural base, impossible for the repair mechanism to detect and correct

16. What if deamination gave rise to natural bases? If deamination gives rise to natural bases, the repair system would not be able to detect them For example: 5-methylcytosine is really common in vertebrate DNA. Its deamination produces Thymine (a natural base), not Uracil So, the change is not detected by the repair system The Thymine will pair then pair with Adenine while forming a new daughter strand, leading to permanent fixation of the mutation (called C to T Transition). In fact, methylated Cs are hotspots for spontaneous mutations in vertebrate DNA

17. DNA is damaged by alkylation, oxidation, and radiation Alkylation Transfer of methyl or ethyl groups to reactive sites on the bases and to phosphates on the DNA backbone Alkylating chemicals: nitrosamines and N-methyl-N 1 -nitro-N-nitrosoguanidine, a potent lab mutagen Most vulnerable site for alkylation: keto group at the C-6 of guanine, which forms O 6 -methylguanine , which mispairs with Thymine, changing G:C pairs to A:T pairs in daughter cells

18. Oxidation DNA is subject to damage from reactive oxygen species as well (O 2 - , H 2 O 2 , •OH) Generated by ionizing radiation and by chemical agents that generate free radicals Oxidation of G forms 7,8-dihydro-8-oxoguanine, or oxoG , which is highly mutagenic as it can base pair with A as well as C. If it pairs with A during replication, it gives rise to a G:C to T:A transversion, one of the most common mutations in human cancers Maybe this is one primary way ionizing radiation and oxidizing agents cause cancer

19. Radiation Radiation of nearly 260nm (UV) strongly absorbed by the bases, one consequence of which can be photochemical fusion of two adjacent pyrimidines on the same chain If two thymines fuse, a thymine dimer is formed, which contains a cyclobutane ring generated by links between carbon atoms 5 and 6 of adjacent thymine molecules If C is adjacent to T, a T-C adduct is formed linking C6 of T to C4 of C Such dimers are incapable of pairing and stop the movement of DNA polymerase during replication Assays to detect DNA damage: Immunoblotting, Comet Assay (Single cell electrophoresis), Cell survival assay.

20. Radiation: X and γ-rays Ionizing radiation can introduce double strand breaks in the DNA and severely damage the chromosome Can directly attack the backbone of the DNA or create ROS which, in turn, react with the deoxyribose subunits Cells require intact chromosomes to replicate their DNA, so targeted ionizing radiation is used to damage the DNA of cancer cells Anticancer drugs like Bleomycin cause breaks in DNA. Such agents are called to be clastogenic (Greek clastos , which means broken)

21. Mutations Caused by Base Analogs and Intercalating Agents Base analogs : structurally similar to proper bases but differ in ways that make them treacherous to cell, thus can get taken up by cells, converted to NTPs, and incorporated into the DNA during replication, but base-pairing occurs inaccurately, leading to frequent mistakes 5-bromouracil: one of the most mutagenic analog of Thymine It mispairs with Guanine via the enol tautomer (In case of Thymine, the keto tautomer is more favored , but for 5-bromouracil, the enol tautomer is more favored )

22. Intercalating agents: Flat molecules with several polycyclic rings that bind to the equally flat purine or pyrimidine bases They may cause insertions or deletions in strands Insertion: The DNA polymerase reads the inserted area as more than one base pair, hence, inserts more bases opposite to it than actually needed Deletion: DNA polymerase skips the intercalated region and in the process, the base that the agent had bound to

23. Repair and Tolerance of DNA Damage DNA damage can have two types of consequences: Thymine dimers (irradiation), nicks, breaks in the DNA backbone impede replication or transcription Other kinds of damage (base analogs , intercalation, alkylation, oxidation, deamination) can cause permanent alterations in the DNA sequence of the progeny cells Cells have evolved elaborate mechanisms to identify and repair DNA damage before it blocks replication or causes a mutation

24. Direct Reversal of DNA Damage Photoreactivation : DNA photolyase catches energy from light and uses it to beak down the covalent bonds linking adjacent pyrimidines (dimers)

25. Direct Reversal of DNA Damage 2. Removal of methyl group from methylated base O 6 -methylguanine that pairs with T (resulting from alkylation) by the enzyme methyltransferase which transfers the methyl group to one of its cysteine residues (after that, the enzyme is not used again)

26. Base-Excision Repair and Base Flipping Mechanism The most common way of “cleansing” DNA is to remove and replace the altered base Two principal ways: Base Excision Repair and Nucleotide Excision Repair In Base Excision Repair, an enzyme called glycosylase recognizes and removes the damaged base by hydrolysing the glycosidic bond. The resulting abasic sugar is removed from the DNA backbone in a further endonucleolytic step. Endonucleolytic cleavage also removes apurinic and apyrimidinic sugars that arise by spontaneous hydrolysis a repair DNA polymerase and DNA ligase restore an intact strand using the undamaged strand as a template

27. There are more than 1 type of DNA Glycosylase Depending upon the damaged base to be repaired, there are different kinds of DNA Glycosylases, 11 have been identified in human cells A specific glycosylase recognizes uracil (generated because of deamination of cytosine), and another is responsible for removing oxoG (generated because of oxidation of guanine)

28. How do DNA Glycosylases Detect Damage? Each base is buried deep in the DNA helix Evidence shows that the enzyme diffuses laterally along the minor groove of the DNA until a specific kind of lesion is detected DNA molecule shows great flexibility, the damaged base is flipped out away from the helix where it sits in the specificity pocket of the glycosylase as seen in X-ray crystallography Base flipping is done without much structural changes to the DNA structure, and hence, the energetic costs may not be all that much Nevertheless, it is unlikely that glycosylases flip out every base to check for abnormalities as they diffuse along DNA. Thus, the mechanism by which these enzymes scan for damages is still unknown

29. What if the damaged base is not removed before replication? Fail-safe glycosylase Ex: in oxoG:A pairing, if the oxoG base is not removed before replication, a fail-safe glycosylase removes the A that it pairs with in the daughter strand and replaces it with C Similarly, 5-methylcytosine gives Thymine upon deamination, which can incorrectly pair with G The glycosylase system assumes, so to speak, that the T in a T:G mismatch arose from deamination of 5-methylcytosine and selectively removes the T so that it can be replaced with a C.

30. Nucleoside Excision Repair This system works by recognizing distortions to the shape of the double helix, such as those caused by a thymine dimer or by the presence of a bulky chemical adduct on a base, rather than by recognizing a specific base A chain of events removes a short ssDNA patch that includes the lesion, and the gap is filled by DNA polymerase Accomplished by 4 protein UvrA , UvrB , UvrC , and UvrD , types of exonucleases

31. A complex of two UvrA and UvrB molecules scans the DNA, the UvrA subunits detect the distortions After detection, UvrA subunits leave the complex, the remaining UvrB complex melts the DNA and creates a ss bubble around the lesion The UvrB dimer recruits UvrC , UvrC creates two incisions, one located 4 or 5 nucleotes 3’ of the lesion, and the other 8 nucleotides 5’ to the lesion The 12-13 residue long lesion containing DNA is removed from the rest of the DNA by a helicase UvrD Uvr : because this system repairs damage from UV light, mutants of the uvr gene are sensitive to UV light and lack the ability to remove T-T dimer or T-C adducts, and bulky lesions of many kinds In humans, xeroderma pigmentosum genetic disease results from a mutation in uvr genes or inability to repair UV induced damage, which renders them very sensitive to sunlight, making them prone to skins lesions, and even cancer

32. In higher cells, the principle behind nucleotide excision repair is largely the same as in E. coli But the machinery involved is a little more complicated Repair and excision involves 25 or more polypeptides (enzymes) XPC: equivalent to UvrA (detecting distortions in the DNA) XPA and XPD: equivalent to UvrB (forming bubble around the lesion) with the ssb protein RPA The bubble creates a cleavage site for a nuclease called ERCC-1 XPF 5’ to the lesion and 3’ for the nuclease XPG (eq. to UvrC ) The excised strand is 24-32 nucleotides long, and as in bacteria, gap filling is done by polymerase and ligase

33. Transcription Coupled Repair Nucleotide Excision Repair also rescues RNA polymerase, the progress of which has been halted due to a distortion in the template strand NER proteins recruit to the stalled RNA polymerase during transcription In this effect, RNA polymerase acts as a damage sensing protein The TFIIH protein that unwinds the DNA during transcription, includes two subunits, which are in fact XPA and XPD So, helicases of TFIIH melt the DNA around a lesion during NER and also during transcription

34. Double-strand break (DSB) repair pathways How do cells repair double-strand breaks in DNA in which both strands of the duplex are broken? DSB repair pathways accomplish this One recombination based pathway retrieves sequence information from the sister chromosome. Recombination based DSB pathway (another chapter in the book) DNA recombination also helps repair errors in DNA replication (by retrieving seq information from another daughter molecule of the replication fork and completing the recombination after which the NER does its job or by producing a break while passing over a nick, which can be then repaired by DSB repair pathways Maybe recombination evolved to repair DNA damage as its primary function

35. DSBs in DNA Are Also Repaired by Direct Joining of Broken Ends A DSB is the most cytotoxic of all kinds of DNA damage Consequences if left unchecked: blocking replication, chromosome loss, ultimately death of the cell or neoplastic transformation DNA damage repair pathways are overlapping, so recombination alone is not responsible for mending DSBs Recombination relies on sister chromatid’s template for repairing, but what if a non-replicated chromosome suffers a break? An alternative DSB repair system comes into play: Non-Homologous End Joining NHEJ is a backup system in yeast, which primarily relies on recombination bases DSB repair, but in higher cells, it is the primary way NHEJ protects and processes the broken ends and joins them together, but broken ends lose sequence info. Thus NHEJ is mutagenic, but the effects are far less hazardous to the cell than there are consequences to leaving broken ends unrepaired

36. How NHEJ works It doesn’t involve extensive stretches of homologous sequences Two ends of the broken DNA are joined by misalignment between single strands protruding from the broken ends Misalignment because of pairing between tiny stretches of complementary bases, nucleases remove the tails and polymerase fills the gaps Till date, seven proteins of the NHEJ pathway have been identified in mammalian cells: Ku70, Ku80, DNA- PKcs , Artemis, XRCC4, Cernunnos -XLF, and DNA ligase IV Ku70 and Ku80 are the most fundamental components: constitute a heterodimer that binds to the DNA ends and recruits DNA- PKc (protein kinase) DNA- PKcs , in turn, forms a complex with Artemis (a 5’-3’ exonuclease that is activated by phosphorylation by DNA- PKcs These nucleolytic activities process the broken ends and prepare them for ligation. Ligase IV performs ligation in a complex with XRCC4 and Cernunnos -XLF

37. NHEJ in bacteria NHEJ less frequently observed in bacteria In Bacillus subtilis, a Ku-like protein and a DNA ligase is produced when it sporulates and it packages the protein into a mature spore This two protein NHEJ system repairs heat-induced DNA breaks when the spore germinates Mutants lacking these spores barely survive harsh conditions Spores have only one chromosome, so it makes sense that DNA breaks are mended by NHEJ, not recombination based methods The doughnut like structure of the spore chromosome makes the broken ends lie close to each other, facilitating joining NHEJ is the reason why B. subtilis spores are able to survive harsh environments

38. Tolerance of DNA Damage: Translesion Synthesis DNA repair systems are not foolproof and damaged bases can be encountered by a DNA Pol during replication If replication ceased, it can be more damaging A fail-safe mechanism to bypass these damages and “tolerate” them One mechanism: Translesion Synthesis A highly error prone mechanism but spares the cell a much worse fate of incompletely replicated chromosome Tolerance leaves the lesion intact, which can be corrected later by other pathways

39. Translesion Synthesis Catalyzed by a special class of DNA polymerases that synthesize DNA directly across the site of damage In E. coli, DNA Pol IV ( DinB ) or DNA Pol V (complex of proteins UmuC and UmuD ’) perform the job DinB and UmuC are part of a distinct family of DNA polymerases called Y family In humans, out of 5 known translesion polymerases, 4 belong to the Y family

40. Translesion polymerases incorporate nucleotides independent of base pairing, that is why synthesis can occur over a damaged strand As they do not read from the template, the process is highly error-prone Even for apurinic or apyrimidinic sites in the template, nucleotides are incorporated However, not completely random DNA Pol η correctly inserts two A residues opposite a thymine dimer Structural studies show that the active site of DNA Pol η is more accommodating of thymine dimers than DNA Pol κ , another translesion polymerase

41. Because of high error rate, transleion synthesis and NHEJ are systems of last resort The price paid for allowing a cell to survive and reproduce: high levels of mutagenesis (fixed in the genome) So, synthesis of translesion polymerases an enzymes are highly regulated Ex: in E. coli , these polymerases are synthesized only in response to DNA damage SOS Response: DNA Damage leads to the proteolytic destruction of a transcriptional repressor (the LexA repressor) that controls expression of genes involved in the SOS response, including those for DinB , UmuC , and UmuD (precursor of UmuD ’). The same pathway is also responsible for the proteolytic conversion of UmuD to UmuD ’ Cleavage of both LexA and UmuD is stimulated by a protein called RecA , which is activated by single-stranded DNA resulting from DNA damage. RecA is a dual-function protein that is also involved in DNA recombination

42. How a translesion polymerase gains access to the stalled replication machinery at the site of DNA damage In mammalian cells, entry into the translesion synthesis pathway is triggered by chemical modification of the sliding clamp Ubiquitination of the sliding clamp: this makes the sliding clamp recruit a translesion polymerase (it contains domains that recognize and bind to ubiquitin) The translesion polymerase then displaces the replicative polymerase from the 3’ end of the growing strand and extends it across the site of the damage Ubiquitination is a distress signal which helps rescue a stalled replication machine In addition to this polymerase switching mechanism, a synthesis that used gap filling mechanism has also been reported The replicative DNA polymerase skips over the damage introducing a gap, which is filled by the translesion polymerase

43. Unexplained questions: How exactly does the translesion enzyme replace the normal replicative polymerase in the DNA replication complex? How does the normal replicative polymerase switch back to and replace the translesion enzyme at the replication fork? (maybe the translesion enzyme has low processivity)

44. THANK YOU.