DNA repair pathways and human disease

1. DNA repair pathways and human disease Cat Yearwood St. George’s, London

2. Essay plan • Briefly describe the DNA repair pathways involved in the repair of damaged bases, mismatched bases and double strand breaks. Explain with examples how mutations in components of these pathways can lead to disease. • Many types of DNA damage occur, from both endogenous sources (e.g. damage by products of metabolism, replication errors) and exogenous sources (e.g. UV light, ionizing radiation). • Types of DNA damage include chemical adducts, thymine dimers caused by UV, base mismatches, and single and double stranded DNA breaks. • Different repair pathways repair different types of damage. Mutations in components of these pathways can lead to human disease. There are 5 main pathways: • Direct repair • Directly reverses DNA damage (exception to rule – all other types remove and resynthesise damaged section) • E.g. an alkylated guanine can be dealkylated by a specific enzyme • Base excision repair (BER) • Vital pathway – knockout in mice is embryonic lethal • Repairs damage such as single stranded breaks and loss of base caused by multiple sources such as ionizing radiation and products of metabolism

3. Nucleotide excision repair (NER) • Removes intrastrand DNA cross-links such as thymine dimmers causes by UV, as well as large chemical adducts • Cascade of proteins involved. Mutations in the genes that code for these proteins can cause disease e.g. xeroderma pigmentosum – autosomal recessive hypersensitivity to UV • Mismatch repair (MMR) • Recognises and corrects mistakes made in DNA replication such as mismatched base or replication slippage leading to insertion or deletion. • Pathway involves MMR proteins, the genes for which are mutated in HNPCC (autosomal dominant). Somatic loss of 2nd copy of mismatch repair gene results in accumulation of mutations in cell. • Repair of double stranded DNA breaks • Double-stranded DNA breaks must be repaired quickly as otherwise can result in chromosomal fragmentation, translocations and deletions. Can be caused by ionizing radiation. • 2 pathways by which break can be repaired: • Homologous recombination (HR) – gene conversion like process where sequence matching damaged region is copied from homologous region on sister chromatid (preferred option as identical) or homologous chromosome. BRCA1 and 2 are involved in this pathway • Non-homologous end joining (NHEJ) – 2 broken ends are ligated back together without homologous template – less accurate method, mutations may be introduced.

4. Keywords • Direct repair • Base excision repair (BER) • Nucleotide excision repair (NER) • Mismatch repair (MMR) • Repair of double stranded DNA breaks • Homologous recombination (HR) • Non-homologous end joining (NHEJ) • Cancer

5. Many sources of DNA damage • Endogenous - products of metabolism e.g. reactive oxygen species, replication and recombination errors • Exogenous - UV exposure, ionizing radiation, chemical exposure

6. Types of damage • Spontaneous depurination and deamination • Chemical adducts e.g. base alkylation • Thymine dimers (caused by UV exposure) and other intrastrand cross-links • Interstrand cross-links • Single and double stranded DNA breaks • Loss of a base, change in a base, small insertions and deletions

7. Different types of damage repaired by different DNA repair pathways

8. 5 main DNA repair pathways • Direct repair • Directly reverses DNA damage without the need for removal and resynthesis of damaged section of DNA (exception to rule – all 4 other types do this) • Alkylated guanine residues can be dealkylated by MGMT enzyme • Deficiencies in MGMT can lead to an increase in mutations • Alkylating agents can induce G:C to A:T transitions, which have been implicated in activation of oncogenes • Base Excision Repair (BER) • main guardian against damage due to cellular metabolism • very important pathway – knockout in mice is embryonic lethal • Previously thought that no human diseases with loss of BER as would be lethal, however homozygous/ compound heterozygous mutations in BER DNA glycosylase MutYH lead to an increased susceptibility to colon cancer due to DNA replication errors due to presence of 8-oxoG (guanine that has suffered oxidative damage) • Also, somatic mutations of BER polymerases have been found in human cancers

9. BER process • Damaged base recognised and removed by a BER glycosylase such as MutYH (different glycosylases recognise different types of damage) • Endo- and exonucleases cut backbone and trim off immediate surrounding nucleotides • Gap filled by resynthesis and nick sealed by DNA ligase (2 slightly different methods exist) • Like all 4 repair pathways other than direct repair exo/endonucleases, polymerases and ligases are required • Mutations in the genes that code for these proteins can result in errors in DNA repair causing an increased mutation rate

10. Nucleotide Excision Repair (NER) • Removes thymine dimers caused by UV-light as well as other intrastrand cross-links and chemical adducts (all types of damage that significantly disrupt the helix structure, unlike those repaired by BER)

11. NER defects in human disease • Loss of function mutations in 8 of the genes in this pathway (most commonly XPA or XPC) cause autosomal recessive xeroderma pigmentosum (XP) – hypersensitivity to sunlight with skin damage and >1000 fold increased risk of sun-induced skin cancer • XP skin tumours have mutations characteristic of UV-induction in ras and p53 genes • Mutations in CS-A and CS-B genes cause Cockayne syndrome – milder UV-sensitivity without cancer, but with developmental defects • CS-A and CS-B proteins involved in transcriptional activation of NER – necessary to remove stalled RNA polymerase due to T-dimer in parts of genome being actively transcribed. In Cockayne syndrome RNA synthesis cannot recover following UV damage

12. Mismatch repair (MMR) • Recognises and corrects mistakes made in DNA replication such as mismatched base or replication slippage resulting in small insertions or deletions • MMR deficient cells have high mutation rates 100-1000 times higher than normal • Pathway involves MMR proteins: MSH2 complexes with MSH6 or MSH3 to recognise mismatches and then MLH1 and PMS2 or MLH3 are recruited into the complex and the mismatch is excised and DNA resynthesised

13. MMR defects and cancer • Heterozygous germline mutations in MMR genes (mainly MLH1 and MSH2) are seen in the autosomal dominant condition Lynch syndrome (HNPCC) • Somatic loss of the second copy of the affected gene results in accumulation of mutations and subsequent tumorigenesis causing colon cancer and other types of cancer such as endometrial and ovarian • Loss of MMR protein results in genomic instability seen as microsatellite instability (MSI) in tumours • Somatic loss of MMR genes (particularly by hypermethylation of MLH1 promoter) also common in sporadic cancers

14. Repair of double strand (ds) DNA breaks • dsDNA breaks must be repaired quickly otherwise can result in gross damage such as translocations and deletions • dsDNA breaks typically caused by ionizing radiation, although can also be as a result of replication of single strand DNA breaks or damage by reactive oxygen species • Two main mechanisms in place to repair breaks: • Homologous recombination (HR) – normally takes place after replication of chromosomes prior to mitosis as requires sequence with high homology for gene conversion-like correction event – uses identical sister chromatid. If sister chromatid not available can use homologous chromosome or NHEJ instead • Non-homologous end joining (NHEJ) – 2 broken ends ligated back together without homologous template – less accurate method

15. dsDNA break repair mechanisms and mutations • BRCA1 and BRCA2 interact with RAD51, regulating its availability and activity • RAD51 is central protein for HR of dsDNA breaks, being required to search genome for homologous sequence • BRCA1 and BRCA2 mutations are seen in autosomal dominant familial breast and ovarian cancer • Somatic loss of 2nd copy results in accumulation of mutations and cell death or tumorigenesis • Patient with homozygous mutation in NHEJ ligase showed hypersensitivity to radiotherapy during treatment for leukaemia

16. Further reading • Strachan and Read, 1999. Human Molecular Genetics 2. Section 9.6 • Houtgraaf et al., 2006. A concise review of DNA damage checkpoints and repair in mammalian cells. Cardiovascular Revascularization Medicine7: 165-172 • Mohrenweiser et al., 2003. Challenges and complexities in estimating both the functional impact and the disease risk associated with the extensive genetic variation in human DNA repair genes. Mutation Research526: 93-125 • Sampson et al., 2005. MutYH (MYH) and colorectal cancer. The Molecular Biology of Colorectal Cancer: 679-683 • Peltomaki, 2001. Deficient DNA mismatch repair: a common etiologic factor for colon cancer. Human Molecular Genetics10(7): 735-740