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Nelson and Cox, Biochemistry 5th Ed., Chapter 25. Friedberg EC, Walker GC and Siede W, DNA Repair and Mutagenesis 2nd Ed. (library) Weinberg R. The Biology of Cancer (1st Ed. 2007/2nd Ed. 2013)Genome stability Genetic information is stored in DNA which is stable but not inert. The vast number of DNA...

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Nelson and Cox, Biochemistry 5th Ed., Chapter 25. Friedberg EC, Walker GC and Siede W, DNA Repair and Mutagenesis 2nd Ed. (library) Weinberg R.A. The Biology of Cancer (1st Ed. 2007/2nd Ed. 2013)

Genome stability Genetic information is stored in DNA which is stable but not inert. The vast number of DNA must be accurately replicated each time cell divides and there are 1 – 3 spontaneous mutations per genome/per cell generation which are insufficient to account for the for the number of changes observed in tumour cells (accumulate mutations faster than normal cells). Thus, they must be resulted from genome instability. Maintenance of the genome is crucial to the individual and to the species as the loss of genome stability (genome instability) is associated with cancer and evolutionary unfavoured mutations. Although some mutation is required for evolution depending on sequence changes, the capacity of genome mutation has been retained. Cells have developed specific, dedicated pathways for the maintaining of genome stability and mutation balance. Events including: DNA Replication error, DNA damage, error-prone DNA polymerases and errors in chromosomal segregation contribute to genome instability while DNA repair pathways, cell cycle checkpoint and Apoptosis aimed to provide Genome stability. 2 most common genome instabilities observed in tumours are Chromosome instability (CIN) affecting chromosome structure/number and Microsatellite instability (MIN) induce alterations at DNA sequence level. MIN initiates cancer through mutations in oncogenes e.g c-RAS or tumour suppressor genes e.g. p53. Mutations can arise from rare spontaneous errors or by replication of damaged DNA. Normal Ras pro-oncogene is coded by CCG-GGC (Glycine) and is mutated to CAG-GTC (Valine). The glycine to valine mutation at residue 12 form block Arg finger access because of the large mutated side chains. Therefore, GAP cannot activate GTPase and Ras stuck in ON state. There is a continuous positive signal directing the mitogenic pathways. DNA maintenance mechanisms operating in mammals Endogenous O2, alkylating agents--- ssb, Uracil abasic site--- Base excision repair BER Exogenous UV--- large DNA adducts, 6,4-photoproducts--- Nuclear excision repair Replication errors --- mismatch, insertion, deletions--- Mismatch repair X ray--- DSB--- Recombinational repair (homologous recombination (S/G2) + Nonhomologous end-joining (G0/G1) Telomere maintenance/chromosome segregation Cross link repair

The MMR, BER and NER are called excision repair pathways which are conserved in all free-living organisms. Nobel Prize was awarded to their discoveries. They all involve the use of sequence information on correct/undamaged strand as the template for repair of damaged strand and the use of lesion-specific DNA damage recognition proteins. These processes are energy requiring process and there are at least 130 genes involved in DNA repair in mammalian cells. They are tightly regulated by protein PTMs, and at transcriptional level. The study of these repairing pathways has been facilitated by structural analysis of repair proteins from cell free extracts. Genetic approaches of rare human cancer prone-syndromes, transgenic mice, knock out studies and siRNA to down-regulate genes. Sequencing that identified polymorphisms in repair genes. Genome instability can be regarded as a hallmark of cancer. 1. Individuals with rare inherited human syndromes with mutations in DNA repair/processing genes are cancer-prone: Xeroderma pigmentosum (XP) contain inherited mutations in genes involved in NER Leading to extreme susceptibility to skin cancer as DNA damage is constantly induced by sun UV light. Abnormal ATM gene causes AT syndrome, leukaemia due to its inability to respond to DSB. 2. Mutations in DNA repair/processing genes are found in cancer cells: 50% of cancers have mutation in p53 which in turn cause an increased frequency of mutations in other critical genes such as Oncogene-induced replication stress (RAS mutations). 3. Sequencing genome of lung tumours confirms smoking-induced mutations patterns (epigenome changes): Mutation pattern induced by cigarette chemical B[a]P was similar to the mutation pattern (G/C bases) found by lung cancer patient genome sequencing.

MMR The vast genome DNA must be accurately replicated during the S phase of the cell cycle. There is only a handful of errors per cell generation. DNA replication accuracy of approx. 1 error per billion nucleotides and is achieved by combination of mechanisms: Selectivity of DNA polymerases for correct base pair, Proofreading: As mis incorporated base (G:T) is extended less well than correctly matched base (e.g. G:C), the Km of enzyme is increased. Thus, facilitating the removal of incorrect base by 3’ to 5’ exonuclease activity of DNA polymerase delta and epsilon. Transgenic mice (Asp-Ala) expressing DNA Pol delta that lacks proofreading activity are cancer-prone. Mismatch repair which is the most accurate mechanism with high fidelity. It is a specific repair pathway that removes incorrectly-paired bases from newly replicated DNA not from the template which are not sensed by other repairing pathways. It is responsible to reverse the impact of Single base mismatches and insertion/deletion

loops IDLs. It prevents DNA damage produced by both endo/exogenous sources from becoming permanent mutation in dividing cells. Inactivation of MMR in human cells is associated with hereditary and sporadic human cancers and the MMR system is required for cell cycle arrest and/or programmed cell death in response to certain types of DNA damage. MMR was first described in E. coli. MutS, MutL, and MutH initiate MMR in which MutS protein (homodimer sequence-wise identical but structurally and functionally different domains with intrinsic ATPase activity) recognizes and binds base-base mismatches (eg. Unpaired thymine in mismatch DNA interacts with Phe 39 of MutS subunit A) and recruits MutL protein. MMR in E. coli is ATP-dependent and requires the functional MutS ATPase. MutL (homodimer possessing ATPase) interacts physically with MutS, enhances mismatch recognition, and recruits and activates MutH (ATP hydrolysis by MutL), stimulates the loading of helicase II at the MMR initiation site, therefore assembles a functional MMR complex. Defects in MutL completely inhibit MMR in E. coli. Recent studies show that MutL interacts physically with the clamp loader subunits of DNA polymerase III suggesting MutL may promote binding of DNA polymerase III to MMR intermediates and is coupled with DNA replication. In E. coli, DNA is methylated at the N6 position of adenine in dGATC sequences. In replicating DNA, the daughter strand is transiently unmethylated, and it is the presence of hemi-methylated dGATC sequences that molecularly distinguishes the newly synthesized daughter strand from the parental DNA strand. MutH (momomer, a type-II restriction endonucleases) recognizes hemimethylated dGATC sequences 5’or 3’ to the mismatch and incises the unmethylated daughter strand upon activation by MutS and MutL in the presence of ATP. This strand-specific nick provides the initiation site for excision of the mispaired base. Helicase II loads at the nick and unwinds the duplex from the nick towards the mismatch generating single-strand DNA, which is rapidly bound by single-stranded DNA-binding protein (SSB) and protected from nuclease attack. Depending on the position of the strand break relative to the mismatch, ExoI (3’-5’ exonuclease), or ExoVII (5’→3’ exonuclease) excises the nicked strand from the nicked site (the dGATC site) up to and slightly past the mismatch. The resulting single-stranded gap undergoes repair DNA resynthesise and ligation by DNA polymerase III, SSB, and DNA ligase. The repair is strand specific (i.e., restricted to the newly synthesized DNA strand). Repair is bi-directional, proceeding 5,3 or 3.5 from the nick to the site of the mismatch. Eukaryotic cells have homologues of MutL and MutS, but not of MutH and they are called Mlh and Msh (h=homologues). Human MutS and MutL homologues are

heterodimers. hMSH2 heterodimerizes with hMSH6 or hMSH3 to form hMutSα or hMutSβ, respectively, both of which are ATPases that play a critical role in mismatch recognition and initiation of repair. Other similarities between human MMR and E. coli MMR include substrate specificity, bidirectionality, and nick-directed strand specificity. However, the hemi-methylated dGATC sites as a signal is not present in eukaryotes. hMutSα preferentially recognizes base-base mismatches and Insertion Deletion mispairs of 1 or 2 nucleotides, while hMutSβ preferentially recognizes larger ID mispairs. They identify DNA strand with 3-OH terminus as the new strand. hMLH1 heterodimerizes with other mutL homologues hPMS2, hPMS1, or hMLH3 to form hMutLα, hMutLβ, or hMutLγ. hMutLα is required for MMR and hMutLγ plays a role in meiosis. hMutLα possesses an ATPase activity and defects in this activity inactivate MMR in human cells. Binding of MutL alpha stimulated by MutS results in ATPdependent sliding away from mismatch. Recent studies show that MutLα possesses a PCNA/replication factor C (RFC)dependent endonuclease activity which assist EXO1. PCNA localises MutSα and MutSβ to mispairs in newly replicated DNA via their conserved PCNA interaction motif termed the PIP. PCNA is absolutely required during 3’ nick-directed MMR, it is not essential during 5’ nick-directed MMR. After recognition of the 3’ nick and the mismatch, MutL alpha endonuclease makes an incision 5 to the mismatch in a manner dependent on PCNA and RFC (Nicking of discontinuous (new) strand by PMS2 (MutL)). EXO1 performs 5→3 excision from the MutL alpha incision site on nicked strand through and beyond the site of the mismatch. Other protein components involved in human MMR include single-strand DNAbinding protein RPA, RFC, high mobility group box 1 protein (HMGB1), and DNA pol δ. RPA binds to nicked DNA before MutSα and MutLα, stimulates mismatchprovoked excision, protects the ssDNA gapped region generated during excision, and facilitates DNA resynthesis. DNA synthesis by pol delta/PCNA & Ligation by ligase 1. The importance of these msh2,3 genes are shown in mouse KO which destabilise microsatellite and increase tumour incidence. Hereditary non-polyposis colon cancer (HNPCC)/Lynch Syndrome. Human genetic disease caused by defects in mismatch repair accounts for about 5%  of colon cancers. Due to mutations in MSH2, or MLH1, eg. hypermethylation of MLH1 in sporadic cancer. Hereditary non-polyposis colon cancer (HNPCC): Early onset of colorectal carcinoma and multiple colon cancers, and tumours of other organs esp. skin. The Absence of MMR repairing increases frequency of frameshift mutations within short mononucleotide repeat sequence and induce microsatellite instability.

Only deletions Example: TGF-beta receptor gene run of A’s, deletion of AA results in subsequent alterations in amino acid expressed causing truncate receptor. Diagnostic test for microsatellite instability is by analyse repeat sequences in genomic DNA using PCR. Size of microsatellites increased in tumour DNA and are RER+: ‘replication error positive’: more bands than normal tissues. Potential for cancer immunotherapy by targeting mutated protein antigens in treating MMR deficient. MMR also plays a role in the ‘good’ side of mutagenesis, involved in the somatic hypermutation (Lahue).

BER Base excision repair (BER) is a cellular excision mechanism that is important for removing damaged bases that could otherwise cause mutations by mispairing or lead to breaks in DNA during replication. BER is not like NER which repairs bulky helixdistorting lesions, it is responsible primarily for removing small, non-helix-distorting base lesions from the genome. 1. BER first repairs damage resulted from the formation AP site by hydrolytic cleavage of N-glycosidic bond. It restores ‘non-bulky’ adducts and DNA base damage produced by: 2. Deamination (loss of exocyclic amino group Cytosine to Uracil, Adenine to hypoxanthine), 3. Oxidation (Thymine glycol: oxidised pyrimidine), 4. Fragmented bases from ionising radiation or Endogenous toxic oxygen radicle: 41. Oxidative phosphorylation: reduction of oxygen by cytochrome oxidase toxic 02, h2o2. 42. Formation of hydroxyl radical. 43. By Oxidase enzymes e.g. xanthine oxidase. (Anti-oxidant defences: glutathione and peroxidase). 5. DNA Alkylation is another Endogenous damage arisen from compounds (SAM), or drugs (MNNG). Guanine becomes O6-methylguanine and is mutagenic if unrepaired as O6-meG pairs with T. Direct repair (faster) by AlkB (oxidative demethylation) or MGMT enzyme in which Alkyl group of the lesion is transferred to a cysteine at the active site of the enzyme. The enzyme is termed Suicidal as methylation inactivates the enzyme. Methylated protein acts as a transcription factor to induce more MGMT and Many anti-cancer drugs are alkylating agents e.g. MNNG. BER is the slower pathway. BER is consisted of Repair of modified bases and abasic sites and Single-strand break repair. BER is initiated by DNA glycosylases which recognize and flip out damaged or inappropriate bases of the double helix by breaking N-C1 bond between base and sugar leaving an AP site. There are several DNA-glycosylases in human cells, each with specificity for a different type of base damage. For example, Monofunctional

glycosylases only remove the damaged base e.g. Uracil DNA-glycosylase (UNG). Bifunctional glycosylases remove the damaged base and cleave sugar possessing AP lyase activity e.g. 8-oxdG glycosylase (OGG1). They can convert a base lesion into a single-strand break without the need for an AP endonuclease. Elimination of an AP site by a glycosylase-lyase yields a 3' α,β-unsaturated aldehyde adjacent to a 5' phosphate, which differs from the AP endonuclease cleavage product. G-C become OC during oxidative stress. DIAGRAM: MUTYH removes A from opposite 8-oxo-G. OGG1 removes 8-oxo-G opposite to C, T, or G. Diffusion along DNA and Binding of 8oxo-G at lesion recognition site. Lesion recognition by OGG1 favours binding of 8oxo-G over G: 1. Unique H-bond forms between Gly 42 and 8-oxo-guanine, but not with guanine which lacks H. 2. Lys 249 on 8-oxo-G Stabilises binding of OGG to 8-oxoG but not G. Lesion is flipped into active site. Glycosylase reaction Removal of 8-oxoG plus sugar and DNA backbone cleavage occurs. Two uracil DNA glycosylases with common amino acids and C-terminal catalytic domain found in human cells which are produced by alternative splicing of the same gene. UNG1: removal of uracil from mitochondrial genome. UNG2: removal of uracil from nuclear genome. UNG Diffuses along DNA until damaged nucleotide is recognised. It bends the DNA by compression (Conformational change of UNG) of the DNA backbone and Altered nucleoside is flipped out into the enzyme active site. Base cleaved from sugar by glycosylase activity. Uracil DNA glycosylase has greater affinity for product (AP site) than for substrate (uracil), it does not dissociate, and it protects AP site from formation of strand break. Then, Protein-protein interactions recruits: AP endonuclease; pol beta, XRCC 1 and ligase 3 alpha. Strand is then cleaved by an AP endonuclease Ref1 (a redox factor involved in transcription). Sugar is removed by deoxyribose phosphodiesterase forming apurinic/apyrimidinic site (AP) sites. It yields a 3' hydroxyl adjacent to a 5' deoxyribosephosphate (dRP). For ligation to occur, a DNA strand break must have a hydroxyl on its 3' end and a phosphate on its 5' end. In humans, polynucleotide kinase-phosphatase (PNKP) promotes formation of these ends during BER. PNKP protein has a kinase domain, which phosphorylates 5' hydroxyl ends, and a phosphatase domain, which removes phosphates from 3' ends. These activities ready single-strand breaks with damaged termini for ligation. The AP endonucleases also participate in 3' end processing with its 3' phosphodiesterase activity and can remove a variety of 3' lesions including phosphates, aldehydes. 3'-Processing must occur before DNA synthesis can initiate because DNA polymerases require a 3' hydroxyl to extend. Resynthesise carried out by DNA pol beta with pol lambda able to compensate in its absence. These enzymes have a lyase domain that removes the 5' dRP left behind by AP endonuclease cleavage. Gap is sealed by XRCC1/DNA ligase 3

alpha. This is the major short-patch (where a single nucleotide is replaced, nondisplacing). If the damaged sugar 5’-dRP cannot be removed by pol beta, strand displacement synthesis by Pol delta and removal of displaced flap, by FEN1 (‘flap endonuclease) occur. The yeast homolog of FEN1 is RAD27. DNA synthesise is carried by pol delta and epsilon along with the processivity factor PCNA, the same polymerases that carry out DNA replication. Pol beta can also perform long-patch displacing synthesis and can, therefore, participate in either BER pathway. Gap sealed by DNA ligase 1. This is minor long-patch BER (where 2–10 new nucleotides are synthesized). Oxidative genome damage induced by reactive oxygen species can be repaired by single-strand break repair pathway. XRCC1 and PARPs are other key proteins with indirect roles in BER/SSBR. While XRCC1 acts as a scaffold to recruit BER proteins for excision or strand break repair, PARP acts as an SSB sensor protein. XRCC1 physically interacts with APE1, and other BER proteins Pol beta and Ligase 3 alpha. PARPs are expressed in mammalian cells but absent in E. coli or yeast, constitute a superfamily with regulatory functions in various cellular processes including development. Only PARP-1, -2, and -3 are involved in DNA repair. PARP-1 and -2 are activated by SSBs when they transfer ADP-ribose moiety from NAD to a variety of proteins. PARP-2 may serve as a backup for PARP-1 because inactivation of both PARP 1 and 2 genes in mice is lethal. These proteins play structural and regulatory roles in SSB repair by acting as sensors and by recruiting other repair proteins to the strand break site. However, their direct involvement in damage processing during BER has not been demonstrated so far. PARP-3 has recently been implicated in DNA DSB repair. PARP inhibitors (e.g. Olaparib): novel treatment for BRCA-deficient breast cancers Glycosylase k/o mice generally viable and Inactivation of BER core proteins e.g. pol beta leads to embryonic lethality (in mice). Pol beta expression increased in one-third of cancers examined. Mutations in MUTYH alleles of MUTYH (e.g. Tyr165, Gly382Asp) (removes A opposite 8-oxo-G) increase colon cancer susceptibility. These mutations reduce the ability of the MUTYH protein to recognise base pairs containing 8-oxo-G and Patients accumulate G-> T mutations in e.g. the APC gene.

NER Nucleotide excision repair bulky DNA damage from Ultraviolet (UV) light (skin cancer 100,000 lesions per cell per day in exposed epidermal cell producing Cyclobutane

Pyrimidine dimer and 6-4 photoproduct. Pyrimidine dimers are molecular lesions formed from thymine or cytosine bases in DNA via photochemical reactions as Ultraviolet light induces the formation of covalent linkages by reactions localized on the C=C double bonds.), Benzo[a]pyrene: in cigarette smoke (lung cancer) binding at Guanine and causes mutations at G bases in lung cancer cells (sequencing data), Aflatoxin: fungal chemical (liver cancer) and Cisplatin: adducts (chemotherapy). Unrepaired UV-induced DNA damage can lead to sequence changes (mutations) during DNA replication: Mutations in p53 gene in skin tumours found at dipyrimidine (T=T) sequences. Nobel prize 2015 has awarded to Sancar for his discovery in Photolyase and cryptochromes CRY (photolyase homologue in human which have photolyase activity with a high degree of specificity for cyclobutane pyrimidine dimers in single-stranded DNA) and reconstitution of NER from purified proteins. Photolyase is an enzyme expressed in lower organisms. It’s a small monomer that carries out direct reversal of UV-induced pyrimidine dimers by breaking covalent link between adjacent pyrimidines. There are 2 chromophores – FADH2, and folate or deazaflavin, that absorb energy from visible light which provide energy for the cleavage. Spore photop...