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Mutagenesis can happen within the chromosome such as base pair changes, insertions and deletions and repeat expansions. Between the chromosome like translocations and aneuploidy. Mutagenesis can have both positive and negative effects on cells contributing to abnormal gene function, disease, ageing,...

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Mutagenesis can happen within the chromosome such as base pair changes, insertions and deletions and repeat expansions. Between the chromosome like translocations and aneuploidy. Mutagenesis can have both positive and negative effects on cells contributing to abnormal gene function, disease, ageing, evolution, immunity and research purpose. Mutagenesis is normally accidental by which low level of mutations escape genome safeguards mechanism including DNA repair, damage response and occur throughout the genome. Some mutagenesis is targeted which occurs at elevated level at small specific regions. It utilises unique protein factors and existing factors from other pathways. Somatic hypermutation 1. Biological role of SHM 2. Discovery of AID Activation Induced Deaminase 3. Molecular mechanisms 4. Outstanding questions SHM involves the introduction of single nucleotide substitution in and around the productively assembled VDJ gene segment. SHM happens within the germinal centre of proliferating B cells after the binding of initial IgM on B cells to antigens. It occurs after VDJ recombination in lymphoid organs which rearranges variable (V), joining (J), diversity (D) gene segments to create novel amino acid sequences in the antigenbinding regions of Igs. The number of antibodies a human can produce during the lifetime is more than 109 which is more than the coding capacity of inherited genes. VDJ recombination occurs at the early stage of B lymphocyte development and allows the production of a primary repertoire of 106 different antibody specificities. A primary repertoire of antibodies (IgM) exhibits only low binding affinity for antigen prior to the encounter with foreign antigens. The generation of the rest 103 antibody diversity therefore depends Somatic Hypermutation. In addition, the IgV (CDR) of heavy chain acts as a target for class switch recombination, a process of recombinational deletion that removes the exons of the IgM constant region, bringing the functional VDJ segment into proximity with the exons of downstream Ig constant (IgC) regions. Thus, allowing the switch of genes coding for IgM to IgG, IgA, or IgE. High affinity antibodies are produced. SHM is a mutation affecting the variable regions of immunoglobulin genes (localised to the IgG locus, especially at complementarity-determining regions). Mutation rates are 10-3 to 10-2 per bp per generation significantly higher than rest of the genome hence it’s a ‘Real time’ evolution. A real-time evolution of high affinity binding sites on specific antibodies, which gives protection against subsequent encounter with the same antigen. SHM of IgV genes occurs during only a narrow window of B cell development. Introducing single nucleotide substitutions starts at some 150 nucleotides downstream of the IgV promoter and extends over about 2 kb. All four bases can be targeted for mutation, and in man and mouse, C:G pairs and A:T pairs are targeted with approximately equal frequency. However, Mutations at A:T pairs are more likely to occur if the A is located on the coding strand. Many of the major mutational hot spots at C:G pairs occur within a WRCY consensus (where W = A/T, R = A/G, and Y = C/T) with AGCT is preferred.

SHM occurs in two phases: a first phase that targets C:G pairs and a second phase that introduces substitutions at A:T pairs. Both the IgV and switch recombination of the IgC are all triggered by targeted deamination of deoxycytidine residues. 1. Deamination (Removal of amino group) SHM and gene conversion at the IgV as well as switch recombination of the IgC are all triggered by targeted deamination of deoxycytidine residues. The deamination of deoxycytidine into deoxyuridine and consequently transforms C:G pairs into U:G mispairs is catalysed by the enzyme AID, an enzyme which is only expressed in activated B lymphocytes. Its homologous APOBEC1 is the catalytic component of a tissue-specific RNA editing complex, which deaminates C to U in the RNA. The precise mechanism depends on the way in which the initiating U:G lesion is recognized, processed, and resolved. 2. Processing of U:G lesion x3 - Replication transitions during UNG/MSH2-double deficiency. AID-triggered DNA deamination leads to the generation of C to T and G to A transition (ug-au/cgcg/at). Substitutions applies to both the IgV and switch region targets. Thus, the U:G mispair is recognized as a lesion and is simply replicated. However, A deficiency in the mismatch recognition/processing pathways affects mostly the mutations at A:T pairs but also results in a significant increase in the proportion of substitutions at C:G pairs that are in transitions. It is not known whether the UNG and MSH2/MSH6 pathways compete for the recognition of uracil or act on uracils produced at different times during the cell cycle. MMR error prone repair transition and transversion. In the absence of MSH2 or MSH6, the accumulation of mutations at A:T pairs is substantially diminished. - UNG replication Transition and transversion base excision repair by SMUG1, TDG, and MBD4 uracil-DNA glycosylases. Importance supported by UNG deficiency on antibody gene diversification. But none of the other uracil-DNA glycosylases act as a backup for UNG in SHM. Other enzyme diminishes the frequency of Ig diversification. The uracil may be excised by uracil-DNA glycosylase (UNG), resulting in an abasic site. Rev1 effective in synthesizing DNA across AP site and has a preference inserting deoxycytidine which result in C to G and G to C transversion mutations. Subsequent extension requires a mismatch-extending polymerase such as DNA polymerase zeta (lesion bypass synthesis). With translesion polymerases and extending polymerase, random incorporation of any of the four nucleotides, A, G, C, or T. The most indispensable polymerase remains a question. Also, this abasic site may be cleaved by apurinic endonuclease (APE), creating a break in the deoxyribose phosphate backbone. This break can then lead to normal DNA repair, or a formation of DSB. It is thought that the formation of these DSBs in either the switch regions or the IgV can lead to CSR. - An additional mutagenic process explain the accumulation of mutations at A:T pairs outside of WRCY motifs as the previous mechanisms account fully the occurrence of C:G pairs during SHM but only contribute partially to A:T.

AID-catalyzed deamination of deoxycytidine creates U:G mismatch may also be recognized by the DNA mismatch repair (MMR) machinery, by the MutSα(alpha) complex. MutSα is a heterodimer consisting of MSH2 and MSH6. This heterodimer can recognize mostly single-base distortions in the DNA backbone (U:G DNA mismatches). The recognition by the MMR proteins lead to removal of the DNA through exonuclease 1 to expose a single-strand region of DNA (by recruiting MLH1 and PMS2). It is followed by stimulating DNA polymerase Pol eta activity to fill in the gap. These error-prone polymerases with misincorporation preferences are thought to introduce additional mutations randomly across the DNA gap. This allows the generation of more mutations at AT base pairs. AID AID is an enzyme required for SHM and CSR. Its expression is B cell specific and expression triggers SHM. Cytosine deamination by AID results in point mutations. It has been quite a mystery why repair of uracils induced by AID is mostly mutagenic; a favoured hypothesis was that uracil excision at the wrong time during the cell cycle would prevent faithful repair. Change in the DNA by misincorporated deoxy-uracil is efficiently removed by the uracil-DNA glycosylase (UNG). Highly abundant during S phase during DNA replication. A back up glycosylase, SMUG1 that removes uracil from double stranded DNA, a pathway occurs outside replication. AID activity can be misdirected to other parts of the genome, leading to translocations and oncogenic transformation in many B cell malignancies. Therefore, AID entry into the cell nucleus is tightly regulated. The interaction with replication protein A (RPA) and the protein kinase A (PKA) alpha are involved in the physiologically targeting of AID to Ig locus. Nuclear degradation is responsible for AID regulation and AID is rapidly exported to the cytoplasm where it resides as part of a stable complex. AID is selectively recruited to the immunoglobulin locus by transcription during G1 (inducing SHM). Its mutagenic activity is restricted to G1 during the cell cycle, with rapid nuclear degradation during S phase (phosphorylation of AID at Ser3). At the transition to S phase, the levels of UNG are increased leading to double-strand breaks and deletions that promote class switching though NHEJ. Contain a motif (His-X-Glu) with zinc coordination. Purified AID acts specifically on single stranded DNA substrates. AID dimerization maybe essential for CSR. Humans with hyperIgM syndrome harbour mutation in the AID gene (the presence of mutations) which produce low affinity antibodies, patients very susceptible to infection. KO of AID (-/-) in mice precludes SHM as less mutation frequency on histogram occur in the CDR1 and 2 regions. Southern blot of class switching of Northern blot AID expression correlates temporally and spatially with CSR Exogenous AID expression drives SHM in cells that don’t normally show SHM, a high mutation rate is observed in cells treated with AID in RT-PCR. Mistargeted somatic hypermutation is a likely mechanism in the development of Bcell lymphomas.

Trinucleotide repeat expansion Expansions of repetitive DNA sequences cause numerous human neurological and neuromuscular diseases such as Huntington’s disease (HD), myotonic dystrophy (DM1), Fragile X syndrome type A (FRAXA), and Friedreich’s ataxia (FRDA). Changes in the number and length of repeat units in the sequence of a particular gene can alter its expression and the function of the RNA and/or protein it encodes. Trinucleotide repeats (TNRs) including CAG, CTG, CGG and GAA triplets, are the most common unstable disease associated DNA repeats. Tetranucleotides and Pentanucleotides are also involved. TNR present in normal individuals are short and stable showing no length changes. On the other hand, the affected people display longer and instable tracts with a tendency for repeats expansion. These alterations are dynamic seen in the tissues of individuals and longer repeat tracts are transmitted through generations. Southern blots show expansion in TNR disease loci between generations, producing proliferative or differentiated tissues (EXP). Disease symptoms can become more severe and appear at an earlier age (anticipation). In somatic tissues, TNRs show distinct patterns of tissue specific repeat instability which contribute to the progression of disease. PCR of muscle cells demonstrated some TNR disease loci expand (within a generation) in differentiated tissue resulting in larger alleles in affected tissue (EXP). Repeat instability differs in both the direction (expansion or contraction) and pattern (magnitude of size changes) of the repeat tract in the cells and tissues affected, between disease loci and between an individual’s tissues development stages. Specific cell types in a tissue can display distinct patterns of instability because each cell subtype could experience instability differently. DM1 show increasingly elevated levels of expansion of CTG instability in brain muscle and heart, whereas in CAG associated HUN diseases, expansions occur predominantly in specific brain regions. In FRDA, GAA contractions occur in all tissues but expansions occur only in the brain (regions that are distinct from those of CAG diseases). Expanded CGG tracts in FRAXA display only prenatal somatic instability. Repeat expansions of other diseases occur in the germ line, typically displaying either paternal or maternal expansion biases. Inhibitory: RTEL1 The ability of repetitive sequences to form unusual DNA structures such as slipped DNAs during a broad range of DNA metabolic processes like DNA replication, repair, recombination and transcription, is crucial for instability. Although repeat instability occurs primarily in the form of expansions, contractions (protective repair) are also seen in patients and are therapeutic interest. Because repeat length is closely connected to disease severity and progression, understanding the mechanisms of expansions formation and the mechanisms that lead to contractions will allow the conquering of diseases by modulating repeat expansion size. DNA replication: Invitro experiment (Electrophoretic data and densitogram) showed single strand DNA ssDNA with expanded repeats fold into stable structure hairpin in which strand forms base pairs with another section of the same strand. There was a

significant increase in the Melting Temperature suggesting its stability. It also provides important mechanic feature for expansion and it’s an essential intermediate. Therefore, DNA synthesis at triplet repeats tends to be mistake-prone, creating extra repeats which fold into hairpins which are difficult to remove. Many TNR loci, including FRAXA, HD have nearby replication origins, which suggests that repeat instability can arise through DNA replication. Increased levels of the main human replicative ligase DNA ligase 1 enhances in vitro replication mediated CTG•CAG repeat instability through its interaction with proliferating cell nuclear antigen (PCNA) and recruited DNA polymerase δ and FEN1. Replication proteins Mrc, Srs2 which protect against instability have been identified from yeast models. It was suggested that proteins that stabilize replication forks protect against repeat expansions, whereas proteins involved in template switching and replication fork restart promote repeat expansion. DNA repair: Initially: Repeat instability involves rare short expansions by unknown mechanisms. However, the repeat tract becomes highly unstable. DNA intermediates (slipped DNA: number of repeat units differs between strands) are crucial for instability as their formation and processing are both required to produce a mutation. Expansions arise when DNA intermediates are incorrectly repaired and incorporated whereas correct repair of these intermediates can both prevent expansions and induce contractions. 1. Mismatch repair proteins, including MSH2, MSH3, MSH6, and PMS2, which recognize and repair deletions and insertions of bases that arise during DNA replication and recombination, and thus normally function to reduce mutation. But they provide a mutagenic role for long CTG•CAG repeat tracts through an unknown mechanism (perhaps stabilising the hairpin). The effects of mismatch repair proteins on CTG•CAG instability cannot be extrapolated to other unstable repeats. MSH3 deficiencies enhance the contraction of expanded CTG•CAG tracts and do not increase genome-wide instability therefore MSH3 an excellent therapeutic target to reverse the deleterious symptoms of CTG•CAG diseases. 2. The removal of oxidative 7,8dihydro8oxoguanine (8oxoG) lesions (a mutagenic base by product arising from exposure to reactive oxygens) by the base excision repair 8oxoguanine glycosylase (OGG1) enzyme and the restoration of the base from the complementary strand have been linked to increased somatic CTG•CAG expansions in some tissues of HD mice. BER may function through the miscoordination of various base excision repair factors such as DNA polymeraseβ (Polβ) and FEN1. Oxidative damage preferentially targets guanines in (CAG) hairpin loops. The repair of repetitive nucleotides generates DNA slippage: DNA polymerase encounters the direct repeat (hairpin) during the replication process. The polymerase complex suspends replication and is temporarily released from the template strand. The newly synthesized strand then detaches from the template strand and pairs with another direct repeat upstream. DNA polymerase reassembles its position on the template strand and resumes normal replication, but the polymerase complex backtracks and repeats the insertion of deoxyribonucleotides that were previously added. This results in some repeats found in the template strand being replicated twice into the daughter strand. This expands the replication region with newly inserted nucleotides.

The Nobel prize of medicine 2007 was awarded to the discoveries of principles for introducing specific gene modifications in mice by embryonic stem cells. The manipulation of the genes in intact animals by gene trapping or gene targeting allow the ascription of the function of genes discovered by genome sequencing. The importance of mutant alleles in inherited diseases can be understood better. Gene trapping is the random integration in the genome of a DNA construct carrying a splice acceptor sequence and selection marker. The constructs integrate into introns of expressed gene, they create fusion transcripts through the action of the vector’s splice acceptor and express the selection marker, causing premature termination of transcription. The resulting truncation/fusion often leads to a knockout or a severe hypomorphic (partial loss of function) allele of the endogenous gene. Gene trapping is inexpensive and requires fewer vectors. A significant limitation of this strategy is that it is only effective for genes that are expressed in ES cells, and there is also bias toward larger genes. Gene targeting is a directed approach that uses homologous recombination to mutate an endogenous gene. It includes the deletion of a gene, removal exons, and introduction of point mutations either permanently or in a conditional fashion. Gene targeting requires the creation of a specific vector for each gene of interest but can be used for any gene, regardless of transcriptional activity or gene size. Both methods belong to the reverse genetics in which known target gene is eliminated and the resulted phenotype(s) are investigated by comparing to the WT. Targeted KO Example: Ogg1-/- + HD transgenic mouse Then a new DNA sequence is engineered which is very similar to the original gene and its immediate neighbour sequence, except that it is changed sufficiently to make the gene inoperable (altering aas, force premature stop). The construct with a marker gene is engineered to bind to the target gene. Target gene sequence is interrupted, and the protein translated will be non-functional. Individual embryonic stem cells are transformed with the construct and grown in vitro. Using resistant marker, cells with altered gene are isolated. The knocked-out embryonic stem cells are inserted into a mouse blastocyst (contain KO and original cells) and implanted into a surrogate mother. A chimera mouse is generated. Some of the chimera mice will have gonads derived from knocked-out stem cells producing sperms/eggs containing mutant gene and they are crossed with WT to create heterozygous offspring (+/-). Interbreed heterozygous result in (-/-) homozygous of the mutated gene are carried in all cells. However, 30% of the genes are lethal, therefore animals are killed in the absence of them. This can be solved by conditional KO. Conditional KO Cre/LoxP system Example: Msh2 conditional knockout in mouse intestine. Some human colorectal cancers (HNPCC) are caused by mutation of Msh2. Knockout Msh2-/- mice also get colorectal cancers, but analysis is complicated by other side effects (lymphoma, for example). It would be very useful to knock out Msh2 only in the intestine, to focus on colorectal tumorigenesis. It differs from traditional gene knockout because it targets specific genes at specific times rather than being deleted from beginning of life. The Cre recombinase enzyme

specifically recognizes two lox (loci of recombination) sites within DNA and causes recombination between them. This recombination will cause a deletion or inversion of the genes between the two lox sites, depending on their orientation. In addition, chemical can be added to knock genes out at a specific time. Tetracycline activates transcription of the Cre recombinase gene. Floxed gene is created and transfected to animal. Mate to second mouse that expresses Cre in inducible and tissue-specific manner. offspring is Double-transgenic mouse ‘floxed’ target gene plus inducible, tissue-specific Cre expression Pros: This allows the monitoring of effects of one gene and its contribution to disease. novel phenotype discovery, i...