Dna Repair Pathways

Mini Lecture on the topic of DNA repair pathway

1. Genetic stability of organisms Accurate DNA replication + DNA repair mechanisms Present in prokaryotes and eukaryotes

2. 1 million individual lesions/cell/day Heat Metabolic accident Radiation Environment depurination Molecular Biology of the Cell But only 1/1000 accidental base change results in a permanent mutation

3. Danger of DNA damage Structural damage -> prevent replication/transcription Stalling of replication fork Harmful mutations -> impair survival of organism Mutation in tumour suppressor genes for examples If unrepaired: Senescence Apoptosis Aberrant cell division -> cancer

4. Genetic diseases associated with defects in DNA repair 2005 W. H. Freeman Pierce, Benjamin.  Genetics: A Conceptual Approach , 2nd ed.  ATM Breast, ovarian and colon cancer Defect in homologous recombination, BRCA2 Transcription-coupled repair HNPCC

5. the DNA molecule can be repaired fairly easily because it carries 2 separate copies of all the genetic information Only viruses, that have a tiny genome (therefore tiny target for DNA damage) can afford to encode their genetic information in any molecule other than double stranded DNA  single-stranded DNA or RNA.

6. Multiple DNA repair pathways Base excision repair (BER) Nucleotide excision repair (NER) Mismatch repair (MR) DNA strand cross link repair Homologous recombination (HR) Non-homologous end joining (NHEJ) + transcription coupled repair Level of damage

7. Nucleotides known to be modified by: Oxidative damage hydrolytic attack uncontrolled methylation Purines (guanine and adenine) are more affected by those spontaneous reactions. 5000 purine bases are lost every day: DEPURINATION Spontaneous deamination of cytosine  uracil occurs at a rate of 100 bases per cell per day. UV can covalently link two adjacent pyrimidine bases to form thymine dimers . After T. Lindahl ,  Nature  362:709–715, 1993 Spontaneous DNA alteration

8. substitution deletion

9. Base excision repair Nucleotide excision repair

10. ‘backup’ polymerases not as accurate as the normal replicative polymerase lack exonucleotic proofreading activity  2. Translesion DNA synthesis (TLS) polymerases 4. Reverse transcriptase Restrained to telomerase in eukaryotes, using a RNA template for DNA synthesis 1. Replicative polymerases 3. DNA repair and recombination “Risky Business”

11. 15 different mammalian DNA polymerases Example of specialised polymerase : Terminal deoxynucleotidyl transferase (TDT) is expressed only in lymphoid tissue, and adds random nucleotides to double-strand breaks formed during somatic recombination to promote immunological diversity.

12. (post- replicative ) DNA mismatch repair corrects errors made by DNA polymerase during DNA replication Defects in DNA mismatch repair have been found in several types of cancer, notably colon cancer, and microsatellite sequences that are either shorter or longer than normal are a hallmark of defective MMR. MSH2  frequently mutated in hereditary nonpolyposis colon cancer (HNPCC)  In order to do this the mismatch repair machinery distinguishes the newly synthesised strand from the template (parental) (before sealing by DNA ligase ) Up to 1000 bp can be removed

13. DSBs are created biologically by the protein SPO-11 as the highly regulated initiation of meiotic recombination. Double stranded breaks Ionizing radiation Replication errors Oxidating agents environment endogenous Only used shortly after DNA replication, during interphase

14. Same genes, different alleles Same genes, same alleles

15. Homologous recombination Homologous recombination requires DSB of DNA (damage or stalled or broken replication fork), invasion of a homologous dsDNA molecule by a ssDNA end, pairing of homologous sequences, branch migration to form a Holliday junction, and isomerisation of the flanking sequences.

16. Important players Rad51 BRCA1/2 FANC ATM

17. Rad51, a sequence-independent DNA binding recombinase at the branch point BRCA2 sequesters RAD51 via its BCR repeats and its C-terminal motif , mobilises it to the site of damage and then facilitates the formation of helical RAD51–single stranded DNA nucleoprotein filaments that search for a homologous DNA template. Owen Richard Davies & Luca Pellegrini , Nature Structural & Molecular Biology  14 , 475 - 483 (2007) Holliday junction

18. ATM protein kinase DSBs create changes in chromatin structure which activate ATM by autophosphorylation , which then induces many cellular responses by phosphorylating a vast number of target proteins. Associate with the BRCA1-associated genome surveillance complex (BASC)  Can phosphorylate p53 also involved in DNA repair and cell cycle arrest.

19. The Fanconi anaemia/BRCA pathway The FANC protein family is involved in the recognition and repair of damaged DNA. The FA complex is activated when DNA stops replicating because of damage. The core complex can associate with BRCA1 and BRCA2. This complex mediates the monoubiquitylation of FANCD2. Activated FANCD2, in turn, is translocated to chromatin and DNA-repair foci. These foci contain the BRCA1 protein and is known to bind directly to RAD51 and to DNA, and to participate in homology-directed DNA repair.

20. BRCA1, in combination with BARD1, has E3 ubiquitin ligase activity; BRCA2 has no enzymatic activity. Both BRCA1 and BRCA2 are tumour suppressor proteins. They form multiple protein complexes with overlapping functions.