The basic processes of DNA repair are highly conserved among both prokaryotes and eukaryotes and even among bacteriophage (virusesthat infect bacteria); however, more complex organisms with more complex genomes have correspondingly more complex repair mechanisms.
A key step is an enzyme-mediated “flipping-out” of the altered nucleotide from the helix, which allows the enzyme to probe all faces of the base for damage (Figure 5-51). It is thought that DNA glycosylases travel along DNA using base-flipping to evaluate the status of each base pair. Once a damagedbase is recognized, the DNA glycosylase reaction creates a deoxyribose sugar that lacks its base. This “missing tooth” is recognized by an enzyme called AP endonuclease, which cuts the phosphodiester backbone, and the damage is then removed and repaired (see Figure 5-50A). Depurination, which is by far the most frequent type of damage suffered by DNA, also leaves a deoxyribose sugar with a missing base. Depurinations are directly repaired beginning with AP endonuclease, following the bottom half of the pathway in Figure 5-50A. he second major repair pathway is called nucleotide excision repair. This mechanism can repair the damage caused by almost any large change in the structure of the DNA double helix. Such “bulky lesions” include those created by the covalent reaction of DNA bases with large hydrocarbons (such as the carcinogen benzopyrene), as well as the various pyrimidine dimers (T-T, T-C, and C-C) caused by sunlight. In this pathway, a large multienzyme complex scans the DNA for a distortion in the double helix, rather than for a specific base change. Once a bulky lesion has been found, the phosphodiester backbone of the abnormal strand is cleaved on both sides of the distortion, and an oligonucleotide containing the lesion is peeled away from the DNA double helix by a DNA helicase enzyme. The large gap produced in the DNA helix is then repaired by DNA polymerase and DNA ligase (Figure 5-50B).
HR: single-strand invasion, branch migration, limited DNA synthesisIn mycobacteria, NHEJ is much more error prone than in yeast, with bases often added to and deleted from the ends of double-strand breaks during repair. Many of the bacteria that possess NHEJ proteins spend a significant portion of their life cycle in a stationary haploid phase, in which a template for recombination is not available. NHEJ may have evolved to help these organisms survive DSBs induced during dessication
Dna Repair Pathways
Genetic stability of organisms Accurate DNA replication + DNA repair mechanisms Present in prokaryotes and eukaryotes
1 million individual lesions/cell/day • Heat • Metabolic accident • Radiation • Environment • depurination But only 1/1000 accidental base change results in a permanent mutation Molecular Biology of the Cell
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
Genetic diseases associated with defects in DNA repair Transcription-coupled repair HNPCC ATM Breast, ovarian and Defect in homologous colon cancer recombination, BRCA22005 W. H. Freeman Pierce, Benjamin. Genetics: A Conceptual Approach, 2nd ed.
the DNA molecule can be repaired fairly easily because it carries 2 separate copies of all the genetic informationOnly viruses, that have a tiny genome (therefore tiny target forDNA damage) can afford to encode their genetic information inany molecule other than double stranded DNAsingle-stranded DNA or RNA.
Multiple DNA repair pathways• Base excision repair (BER)• Nucleotide excision repair (NER)• Mismatch repair (MR) Level of damage• DNA strand cross link repair• Homologous recombination (HR)• Non-homologous end joining (NHEJ) + transcription coupled repair
After T. Lindahl, Nature 362:709–715, 1993 Spontaneous DNA alteration 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.
Base excision repair Nucleotide excision repair
“Risky Business”1. Replicative polymerases2. Translesion DNA synthesis (TLS) polymerases‘backup’ polymerases not as accurate as the normalreplicative polymeraselack exonucleotic proofreading activity3. DNA repair and recombination4. Reverse transcriptaseRestrained to telomerase in eukaryotes, using aRNA template for DNA synthesis
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.
(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. (before sealing by DNA ligase) MSH2 frequently mutated in hereditary nonpolyposis colon cancer (HNPCC) In order to do this theUp to 1000 bp can be removed mismatch repair machinery distinguishes the newly synthesised strand from the template (parental)
Double stranded breaks environment endogenous • Ionizing radiation DSBs are created biologically by the protein SPO-11 as the • Replication errors highly regulated initiation of meiotic recombination. • Oxidating agentsOnly used shortly afterDNA replication, duringinterphase
Same genes, different alleles Same genes, same alleles
Homologous recombinationHomologous recombination requires DSB of DNA (damage or stalled or broken replicationfork), invasion of a homologous dsDNA molecule by a ssDNA end, pairing of homologoussequences, branch migration to form a Holliday junction, and isomerisation of the flankingsequences.
Rad51, a sequence-independent DNA binding recombinase at the branch point Holliday junction 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)
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.
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, inturn, is translocated to chromatin and DNA-repair foci. These foci contain theBRCA1 protein and is known to bind directly to RAD51 and to DNA, and toparticipate in homology-directed DNA repair.
BRCA1, in combination withBARD1, has E3 ubiquitinligase activity; BRCA2 hasno enzymatic activity. BothBRCA1 and BRCA2 aretumour suppressor proteins.They form multiple proteincomplexes with overlappingfunctions.