Biology Chapter 4: DNA Repair and Cancer

DNA Repair and Cancer

• DNA repair is crucial for maintaining genome stability, and defects in DNA repair can lead to cancer.
• There are several DNA repair pathways, including direct reversal, nucleotide excision repair (NER), base excision repair (BER), mismatch repair, and double-strand break repair.

Direct Reversal

• Direct reversal is a simple mechanism of DNA repair that involves a single enzyme reaction to remove certain types of damage directly from the DNA.
• The best-studied example is the removal of O6-methylguanine, a miscoding alkylation lesion, by O6-methylguanine-DNA methyltransferase (MGMT).

Nucleotide Excision Repair (NER)

• NER acts on a wide variety of potentially toxic and mutagenic helix-distorting DNA lesions, including UV-light induced photolesions, bulky chemical adducts, and DNA intrastrand crosslinks.
• NER requires the action of around 30 polypeptides, which function by the stepwise assembly of several complexes at the site of DNA damage.
• There are two NER sub-pathways: global genome repair (GG-NER) and transcription-coupled NER (TC-NER).
• GG-NER surveys the entire genome for DNA damage, while TC-NER removes DNA lesions more rapidly from the transcribed strand of genes.

Human Syndromes with Defects in NER Genes

• Defects in NER genes are associated with three rare, inherited syndromes: xeroderma pigmentosum (XP), Cockayne syndrome (CS), and trichothiodystrophy (TTD).
• XP is characterized by extreme sensitivity to sunlight, multiple skin abnormalities, and a high risk of developing skin cancer.
• CS is characterized by dwarfism, microcephaly, mental retardation, retinal and skin abnormalities, and no predisposition to develop skin cancer.
• TTD is characterized by brittle hair, mental retardation, unusual facies, ichthyotic skin, and reduced stature, with about 50% of patients sensitive to sunlight.

Base Excision Repair (BER)

• BER is the main pathway that deals with damage induced by cellular metabolites, such as uracil and 8-hydroxyguanine.
• BER removes lesions by employing specific DNA glycosylase enzymes to excise the altered base.

Mismatch Repair

• Mismatch repair is a process that corrects single base mispairs or looped intermediates that arise during DNA replication and as a result of damage to DNA.
• The mismatch repair system is the major pathway that corrects these errors.
• In humans, the mismatch recognition complex hMutSa (hMSH2-hMSH6) preferentially binds most single base mispairs and loops of up to two bases.

Double-Strand Break Repair

• Double-strand breaks (DSBs) are a type of DNA damage that can lead to mutations and cancer.
• There are two general types of DSB repair pathways: homologous recombination (HR) and non-homologous end-joining (NHEJ).
• NHEJ is the major pathway for repairing DSBs in mammalian somatic cells.
• ATM (ataxia-telangiectasia mutated) is the 'master regulator' protein kinase responsible for coordinating cellular responses to DNA DSBs.### Mismatch Repair
• Activated ATM phosphorylates a large number of substrates, including those involved in cell cycle regulation and DNA repair (p53, Mdm2, Chk2, RAD9, H2AX, NBS1, BRCA1, BLM, and FANCD2)
• hMutSa binds to PCNA loaded onto newly replicated DNA and is transferred to mispaired bases in DNA
• EXO1 and FEN1 are involved in the excision of the damaged strand
• The resynthesis step in human mismatch repair is performed by DNA polymerase δ and possibly DNA polymerase ε
• The DNA ligase that completes the MMR reaction remains to be identified

Genetic Defects and Cancer Susceptibility

• Genetic defects in mismatch repair genes play an important role in common cancer-susceptibility syndromes (HNPCC, Turcot's syndrome, Muir-Torre syndrome, and Torre syndrome)
• HNPCC is characterized by predisposition to colon and brain tumors
• Germline mutations in hPMS2 can result in Turcot's syndrome
• Muir-Torre syndrome is a subtype of HNPCC, characterized by the development of skin tumors

ATM and DNA Repair

• Loss or mutation of ATM results in the disorder ataxia telangiectasia (AT), characterized by early-onset cerebellar degeneration, small blood vessel dilation, and immunodefiency
• ATM phosphorylates and activates NBS1, which forms a complex with MRE11-RAD50 nuclease and recruits these proteins to the site of the DSB

Double-Strand Break Repair

• Double-strand breaks (DSBs) can arise from exposure to DNA-damaging agents or through errors in cellular functions
• Unrepaired or misrepaired DSBs are highly cytotoxic lesions that disrupt genomic integrity
• Defects in NBS1 give rise to Nijmegen breakage syndrome (NBS), a rare recessive condition characterized by chromosomal instability and cancer predisposition

Non-Homologous End-Joining (NHEJ)

• NHEJ is a major pathway for repairing DSBs in human cells
• NHEJ involves the rejoining of broken DNA ends with little or no base-pairing at the junction
• DNA-dependent protein kinase (DNA-PK) plays a key role in NHEJ, with a primary DSB recognition role
• Other proteins involved in NHEJ include Artemis, XRCC4, and DNA ligase IV

Homologous Recombination (HR)

• HR is a second pathway for repairing DSBs, which is preferentially used when a damaged cell is in S or G2 phase
• HR faithfully restores the original sequence of the broken chromosome
• The RAD51 nucleoprotein filament is involved in HR, with phospho-H2AX foci co-localizing with RAD51 foci

BRCA1 and BRCA2

• BRCA1 and BRCA2 proteins function in the homologous recombination process
• BRCA1 binds branched DNA preferentially and may function in HR by co-localizing with other DNA repair factors
• Absence of BRCA1 decreases the efficiency of RAD51 localization at sites of DSB damage

Fanconi Anaemia (FA)

• FA is a rare and clinically heterogeneous syndrome characterized by pancytopenia, hypopigmentation, and limb/thumb abnormalities
• FA cells are hypersensitive to chemicals that create DNA interstrand cross-links
• The FANCD1 gene is identical to BRCA2, linking the two pathways### DNA Damage Tolerance Pathways
• DNA damage tolerance pathways are utilized to avoid DNA damage during DNA replication
• These pathways are error-free and use an undamaged template strand
• Strand breaks are strong blocks to DNA replication, and all cells possess mechanisms to tolerate this situation

Recombination-dependent Replication

• Recombination-dependent replication is a mechanism to tolerate DNA damage on the leading strand
• It involves the formation of a D-loop structure by homologous recombination factors
• Leading or lagging strand DNA synthesis occurs, and a replication fork may be created
• The invading strand dissociates and reanneals to the original broken strand

DNA Replication Reactivation

• When a replication fork encounters a DNA strand break, the newly synthesized daughter DNA duplex is converted into a DSB
• Co-ordinated leading and lagging strand synthesis stops due to collapse of one of the arms
• The ATR kinase is activated, and it phosphorylates H2AX at the replication fork break
• The RAD50-MRE11-NBS1 complex is recruited to process the ends of the break

Leading Strand Replication Reactivation

• There are two models for leading strand replication reactivation when damage is on the leading strand
• One model involves the processing of the stalled fork by an endonuclease to yield a DSB at the replication fork
• The other model does not involve nuclease activity and possibly not the HR pathway either

Translesion Synthesis

• Translesion synthesis is a mechanism to bypass DNA lesions directly
• It is performed by a specialized group of enzymes known as DNA damage bypass polymerases
• The damaged template base may vary from a base with a large bulky adduct to no base at all (an AP site)
• Some translesion polymerases are specialized for specific types of DNA base damage and are considered to be ‘error-free’

Human Syndromes with Defects in DNA Repair

• Three human syndromes have been identified with defects in enzymes believed to process Holliday junctions formed during DNA recombination or DNA replication fork reversal
• These syndromes are Bloom syndrome, Werner syndrome, and Rothmund-Thomson syndrome
• Bloom syndrome and Werner syndrome patients are characterized by increased incidence of cancer, skeletal abnormalities, and decreased fertility

Translesion Synthesis and Cancer

• Loss of DNA polymerase eta results in a strong increase in cancers of all types
• DNA polymerase zeta is necessary for normal embryonic development, and its absence causes a large decrease in the number of UV-induced basepair and frameshift mutations

Variation in DNA Repair between Individuals

• There is a lack of methods to easily and reliably measure DNA repair activities in different individuals
• The epidemiology of DNA repair and susceptibility to cancer has been investigated by a number of approaches, including single cell gel electrophoresis or ‘comet’ assays

Polymorphisms in DNA Repair Genes

• There are many single-nucleotide differences (polymorphisms) between individuals
• Some polymorphisms in DNA repair genes, such as amino acid substitutions, may cause differences in DNA repair capacity
• A systematic effort to catalogue polymorphisms is part of the environmental genome project sponsored by the NIEHS in the United States

Variation in DNA Repair between Different Types of Tumours

• Because of differences in gene expression between cells in specific tissues, it is possible that tumours could vary in their DNA repair capacity according to tissue or cell type of origin
• Testicular germ cell tumours are particularly curable by chemotherapy based on cisplatin or related compounds