DNA Repair Mechanisms:

Photoreactivation of UV-induced Pyrimidine Dimers:

- In prokaryotes and lower eukaryotes! Different mechanism in humans. Exposure to visible light (320-370nm) causes the dimers to be reverted back to their original form.

Alkylation damage:

-> Guanine is susceptible to alkylation damage by chemical mutagens e.g. dimethylsulphate, causing it to base pair with thymine. GC to AT transition after replication. Cigarette smoke is a major cause of alkylation damage.

ABC Excinuclease Function:

1. Damage is detected by Uvr A protein. Uvr A recruits Uvr B. 2. DNA is unwound and cleaved. Uvr B makes a 3' cut and Uvr C makes a 5' cut. 3. Helicase II releases the excised oligonucleotide. 4.DNA Polymerase I and ligase resynthesize and religate the DNA molecule.

Repair Involving Excision of Base Pairs:

1. Excision Repair: ---> a. Nucleotide E. R. ---> b. Base E. R. by Glycosylases. 2. Mismatch repair 3. Post-REPLICATION Repair.

Repair by DNA Polymerase:

1. Incorrect nucleotide is removed by the 3' to 5' exonuclease activity as the DNA polymerase III reverses again along the template strand. 2. DNA Pol II moves forward again assuming its 5 to 3 DNA synthesis activity.

Mismatch Repair Mechanism in E.coli:

1. MutS binds to the mismatch. 2. A specific sequence on the parent strand - GATC - is methylated by an enzyme dammethylase. After replication, the daughter strand remains unmethylated for a brief period of time. This distinguishes the correct parent strand from the error-containing daughter strand. 3. MutL and MutH bind and bring the unmethylated GATC sequence closer to the mismatch area. 4. MutH nicks the unmethylated strand at the GATC sequence and the mismatch is removed by EXOnuclease. 5. The gap is repaired by DNA polymerase III and ligase.

Direct Correction (reversal) DNA Damage:

1. Repair by DNA Polymerase 2. Photoreactivation of UV-induced pyrimidine dimers 3. Repair of alkylation damage.

Pyrimidine Dimer:

1. The replication machinery halts when it encounters a pyrimidine dimer, and it resumes polymerisation at some point past the dimer site. 2. The resulting daughter strand has a gap opposite the pyrimidine dimer. This genetic lesion cannot be eliminated by excision repair (requires an intact complementary strand). 3. The lesion can be corrected by exchanging the corresponding segments of sister DNA Strands. This places the gapped DNA segment opposite the undamged strand, and the gap is filled as normal.

Most Prevalent Defective Genes in HNPCC:

hMLH1 - human MutL homologue. hMSH2 - human MutS homolog 2. This cancer does NOT arise from intestinal polyps.

Xeroderma Pigmentosum:

An autosomal recessive genetic disorder of DNA repair in which the ability to repair damage caused by ultraviolet (UV) light is deficient. In extreme cases, all exposure to sunlight must be forbidden, no matter how small. Multiple basal cell carcinomas (basaliomas) and other skin malignancies frequently occur at a young age in those with XP. In fact, metastatic malignant melanoma and squamous cell carcinoma are the two most common causes of death in XP victims.

DNA Repair Processes are Energetically Inefficient:

An exception to what is usually observed in metabolic pathways (ATP is usually used optimally) since the integrity of the genetic info is at stake.

Cockayne Syndrome:

An inherited disease also associated with defective NER. Arises from defect in some of the genes that are defective in XP as well as in two additional genes. Individuals with this syndrome are hypersensitive to UV radiation & exhibit stunted growth & neurological dysfunction due to neuron demyelination. Incidence of skin cancer is normal!

Human Enzymes:

Are also capable of recognising parental from newly synthesized strands, and those which are mismatched (to repair them). The mechanism by which it is done is not completely defined yet, but it does NOT involve Methylation, or the GATC sequence. Genomic DNA is rarely methylated.

More on HNPCC:

Autosomal recessive condition - both alleles of the affected gene must be inactivated. Normal cells from a person with HNPCC have a mismatch repair function 100x better than cancer cells from the same person.

Chemically modified bases:

Can be removed by enzymes called DNA Glycosylases.

Some common lesions e.g. Pyrimidine Dimers:

Can be repaired by several distinct systems.

Hypermethylation of the Promoter of this enzyme's gene:

Causes transcriptional silencing i.e. inactivation of the transcription of O^6 methyl-guanine methyl transferase [MGMT]. This has been described in a number of neoplasms - it is associated with a consequent mutational spectrums.

DNA Glycosylases:

Cleave the N-glycosidic bond of a specific altered nucleotide.

Photolyases:

Contain two prosthetic groups: 1. A light-absorbing cofactor 2. FADH-.

Proofreading in Eukaroytes:

Does occur, but the main DNA polymerase involved in synthesis lacks this 3' to 5' exonuclease activity. This property may reside in other proteins present at the replication fork.

Base Excision Repair:

Eliminates modified bases. 1. DNA Glycosylases. 2. AP endonuclease 3. DNA Pol I and Ligase.

Apurinic-apyrimidinic site (AP/abasic site):

Excision of the base leaves a deoxyribose without a base.

As a result of DNA Repair:

Fewer than 1/1000 lesions becomes a mutation. Athough DNA is a stable molecule, without repair, the cumulative effect of many infrequent but damaging reactions would make life impossible.

DNA Polymerase Epsilon:

Fills the resultant gap in humans!

Replication is blocked if:

If an unrepaired lesion is encountered by the replication complex near the replication fork. Eventually, replication resumes past the site of the lesion with the polymerase skipping over the damaged bases.

BRCA1 and BRCA2:

Interact with a protein called Rad51, the eukaryotic homologue of the RecA protein. This suggests that BRCA1 and BRCA2 are involved in recombinational DNA repair.

3' to 5' Exonuclease activity:

Is necessary for proofreading, to remove incorrect bases.

TcNER in Neurons:

Is of utmost importance, since neurons are in the ~Go phase of the cell cycle.

Mismatched base pairs:

Tend to cause a detectable bulge in the DNA helix. The inability to form hydrogen bonds might also stall replication.

DNA Repair is largely possible because:

The DNA molecule consists of 2 complementary strands - DNA damage in one strand can be removed and accurately replaced by using the undamaged strand as a template.

The new daughter strand:

Would then be missing a base opposite the damaged base. This is repaired by borrowing the template from a homologous strand by the process of recombination.

NER - A primary route for repair of:

Many types of lesions, including pyrimidine/cyclobutane dimers, 6-4 photoproducts and several other base adduct including benzo[a]pyrene-guanine.

During infancy, individuals with XP develop:

Marked skin changes e.g. dryness, excessive freckling, and keratoses. Eye damage may also develop e.g. opacification and ulceration of the cornea. They develop skin cancers 2000x more often than people not suffering from XP - amount of skin cancers can be reduced by avoiding sunlight.

Replication:

May damage the information content of DNA as it can leave mispaired bases.

Post Replication Repair/Recombinational Repair:

Most DNA lesions in E.coli are repaired prior to replication.

Human Breast Cancer:

Mostly occurs in women with no known predisposition, but 10% of cases are associated with inherited defects in BRCA1 and BRCA2.

Mismatch Repair Proteins in E.coli:

MutS, MutL, MutH.

AP endonuclease:

Nicks the phosphodiester backbone at the depurinized site and excises the sugar phosphate backbone.

Pyrimidine Dimers:

Occur frequently in the skin. Usually, DNA repair mechanisms correct this damage and cancer rarely occurs.

Spontaneous Deamination:

Occurs frequently in human DNA and converts cytosine bases to uracil. This is potentially harmful since U (not normally found in DNA) can bond with A, forming UA pairs instead of CG pairs.

Hereditary Nonpolposis Colorectal Cancer [HNPCC]:

One of the most common inherited cancer-susceptibility syndromes. Has been associated with deficiencies in mismatch repair mechanisms. Defects in at least 5 different mismatch repair genes can give rise to HNPCC. Cancer usually develops at an early age, with colon cancers being the most common.

Photoreactivation is Catalysed by:

Photolyase enzyme - activated by a photon of light; splits the dimer apart.

O^6-methyl-guanine methyl transferase Enzyme:

Recognises the O^6 methyl-guanine in the DNA and removes the methyl group, transferring it to one of its Cys residues. -Present in human cells. -Technically not an enzyme because it is a single methyl transfer event which permanently methylates the protein and inactivates it in this pathway. It is a suicide DNA repair protein.

Number and Diversity of Repair Systems:

Reflect both the importance of DNA repair to cell survival & the diverse sources of DNA damage.

Nucleotide Excision Repair (NER):

Removes bulky lesions caused by e.g. PCH, UV. Analogous in humans and E.coli.

Uracil DNA Glycosylase:

Removes the Uracil produced from the deamination of cytosine. Found in all cells. Does not remove uracil from RNA or Thymine from DNA The capacity to distinguish cytosine from thymine might be one reason why DNA evolved to contain thymine instead of uracil.

DNA Polymerase I and Ligase:

Resynthesize the DNA and seal the DNA.

Transgeneic mice engineered to express Photolyase:

Superior resistance to damage caused by UV irradiation.

DNA Adduct:

A piece of DNA covalently bonded to a (cancer-causing) chemical. This process could be the start of a cancerous cell, or carcinogenesis.

Mismatch Repair:

A post-replicative repair mechanism that occurs in both prokaryotic and eukaryotic cells. Whilst proofreading by DNA Polymerase is an efficient way of correcting many errors, a significant number of errors still remain uncorrected after replication.

Two-hit model of Carcinogenesis:

A single normal allele in an affected individual can result in effective mismatch repair, but when the normal allele is lost by somatic mutation, the repair mechanism is lost! This provides evidence of a casual association between defective DNA Repair and increased cancer risk.

Enzymatic Complex in E.coli:

ABC Excinuclease

Benzo[a]pyrene-guanine:

Formed in DNA by exposure to cigarette smoke.

N5, N10, methenyltetrahydrofolate complex:

Found in E.coli photolyases - absorbs UV light and transfers the excitation energy to FADH- which then transfers the energy to the dimer, splitting it.

Eukaryotic excinucleases:

Functional quite similar to the bacterial ABC excinuclease complex. However, 16 polypeptides with no similarity to E.coli excinuclease subunits are required for dual excision.

Methylated Methyltransferase is Not Discarded:

Functions as a transcriptional activator - increases expression of its own gene and a few other repair genes.

Transcription coupled nucleotide excision repair:

Genes that are actively transcribed to produce mRNA are preferentially repaired. 1. During transcription, RNA polymerase stops temporarily when it encounters a damaged segment of DNA. 2. Nucleotide excision repair proteins are attracted to this site and the damaged region is repaired. 3. RNA polymerase resumes transcription.

Genetic deficiencies in NER in humans:

Give rise to a variety of serious diseases, for example Xeroderma Pigmentosum. Most people suffering from this condition also experience neurological abnormalities - presumably because of people with XP's inability to repair lesions caused by high rate of oxidative metabolism in neurons.

MGMT Diagnostic Assay:

Has been developed for the detection of conditions e.g. Glioblastoma (tumour arising from astrocytes).

Mutant cells that lack Uracil DNA Glycosylase:

Have a high rate of GC to AT mutations.

Women with defects in BRCA1 or BRCA2:

Have greater than 80% chance of developing breast cancer. In cancer, the function of both genes is lost, implying that they are tumour suppressor genes.

However, all eukaryotic cells:

Have proteins which are structurally and functionally analogous to MUT S and Mut L bacterial proteins.

Eukaryotic Nucleotide excision Repair:

The sole repair pathway for pyrimine dimers in humans!

Novel nucleolytic activity of ABC Excinuclease:

Two cuts are made in the DNA! The term excinuclease is used to distinguish it from the activity of standard endonucleases.

Frequency of base pair substitutions in bacteria:

Varies from 10^- to 10^-11 errors per replication event. But DNA polymerase III makes errors at a frequncy of about 10^-5. The proofreading activity accounts for the discrepancy.

Human Cancer Develops:

When certain genes that regulate normal cell division (oncogenes and tumour suppressor genes) fail or are altered, either due to damage because of spontaneous mutations or because they are overriden by the invasion of a tumour virus.