DNA Damage and Repair Lecture Notes PDF

DNA Damage and Repair Prepared BY Ass. Prof. Dr. Amany Wahb Assistant Professor of Medical Biochemistry & Molecular Biology Faculty of Medicine-Helwan University  Lecture’s Objectives Describe the types of damage to DNA and...

DNA Damage and Repair Prepared BY Ass. Prof. Dr. Amany Wahb Assistant Professor of Medical Biochemistry & Molecular Biology Faculty of Medicine-Helwan University  Lecture’s Objectives Describe the types of damage to DNA and the mechanisms involved in DNA repair.  An Overview Because each cell contains only one or two copies of its DNA, the DNA sequence is highly protected from harm. DNA is a relatively stable molecule, but Earth’s natural environment is quite toxic, and damage to DNA is inevitable. DNA can also be altered by mistakes made during its own replication or recombination. Damage and sequence alterations to DNA are often quickly repaired, but when they are not, the DNA becomes permanently altered and harbors a mutation. Mutations are changes in DNA sequence, and when mutations occur in germ-line cells, these changes are inheritable. Continue.. A cancer cell has mutations that prevent cell death, resulting in loss of cell cycle control and unregulated cell division, which leads to malignant tumors that can end the life of the entire organism. The cell has a limited amount of time to fix the initial alteration and restore the DNA to its normal sequence, before replication converts the alteration into a mutation that will be passed on to the next generation. Continue.. Mutation Somatic mutations : occur in somatic cells and only affect the individual in which the mutation arises. Germ-line mutations: alter gametes and passed to the next generation.  DNA Damage Errors can occur during replication, an error can occur for every 30,000 bases. “despite the proofreading system”. In addition, there are some damaging agents like environmental insults. Maintenance of the integrity of DNA molecule is very important for the survival of species or organisms. 7  The damaging agents can be either chemicals (e.g., nitrous acid, which can deaminate bases) or radiation (e.g., nonionizing ultraviolet [UV] radiation from sunlight, which can fuse two pyrimidines adjacent to each other in the DNA, and high-energy ionizing radiation like X-rays, which can cause double-strand breaks). Bases are also altered or lost spontaneously from mammalian DNA at a rate of many thousands per cell per day.  Luckily, cells are remarkably efficient at repairing damage done to their DNA, especially when the damage affects only one or two bases at a location on the same strand of the DNA duplex. Damage may also affect both strands of the DNA at location (e.g., double-strand breaks). These forms of damage are repaired by different repair systems than those removing damage to one strand.  Most of the repair systems (which are called excision repair systems) involve: Recognition of the damage (lesion) on the DNA Removal, or excision of the damage Filling the gap left by excision using the undamaged, complementary strand as a template for DNA synthesis. Ligation to restore the continuity of the repaired strand.  Lets think Proofreading system in DNA replication: a. Is responsible for causing more replication errors. b. Totally prevents any replication error. c. DNA replication errors occur despite accurate proofreading. d. Environmental insults destroy the proofreading replication system. Types of DNA Repair  Base excision repair DNA bases can change either spontaneously as in the instance of C, which gradually loses its amino group to create U or through the action of chemicals that deaminate or alkylate. For example, nitrous acid, which is formed by the cell from precursors such as the nitrates, deaminates C, A (to hypoxanthine), and G (to xanthine).  Base excision repair Dimethyl sulfate can alkylate (methylate) A. Bases can also be lost spontaneously by hydrolysis from the deoxyribose sugar backbone. For example, ∼10,000 purine bases are lost this way per cell per day. Lesions involving base alterations or loss can be corrected by base excision repair ([BER].  Base excision repair These abnormal bases are all removed by DNA glycosylases that break the bond between the base and the deoxyribose sugar of the DNA backbone. Removal of bases leaves an empty space in the DNA known as an AP-site.  Base excision repair AP stands either for apurinic or apyrimidinic depending on which type of base was removed to create the AP-site. Next an AP endonuclease cuts the backbone of the DNA next to the missing base leaving a free 3' -OH group. DNA polymerase I then makes a short new piece of DNA. As usual, the final nick is sealed by DNA ligase.  Types of lesions repaired by BER Oxidative lesions Deoxyuracil: from misincorporation of dU or deamination of dC-->dU Various alkylation products Spontaneous depurination (esp. G) yield abasic sites  Nucleotide excision repair (NER) Recognizes bulky lesions that block DNA replication (i. e. lesions produced by carcinogens)--example, UV pyrimidine photodimers Common distortion in helix Incision on both sides of lesion Short patch of DNA excised, repaired by repolymerization and ligation In E. coli, mediated by UvrABC excinuclease Many more proteins involved in eukaryotes Defects in NER underlie Xeroderma pigmentosum  NUCLEOTIDE EXCISION REPAIR Exposure of a cell to UV radiation can result in the covalent joining of two adjacent pyrimidines (usually Ts), producing a dimer. These intrastrand cross-links prevent DNA pol from replicating the DNA strand beyond the site of dimer formation. T dimers are excised in bacteria by UvrABC proteins in a process known as nucleotide excision repair (NER). 25  NUCLEOTIDE EXCISION REPAIR 26  NUCLEOTIDE EXCISION REPAIR Recognition and excision of UV- induced dimers: A UV-specific endonuclease (called uvrABC excinuclease) recognizes the bulky dimer and cleaves the damaged strand on both the 5′ side and 3′ side of the lesion. A short oligonucleotide containing the dimer is excised, leaving a gap in the DNA strand. This gap is filled in using DNA pol I and DNA ligase.  NUCLEOTIDE EXCISION REPAIR UV radiation and cancer: In the rare, human genetic disease xeroderma pigmentosum (XP), an individual’s skin cells cannot repair pyrimidine dimers caused by sunlight, resulting in extensive accumulation of mutations and, consequently, early and numerous skin cancers. XP can be caused by defects in seven genes that code for the XP proteins required for NER of UV damage.  MISMATCH Repair Replication errors that escape the proofreading activity mismatch in one or several bases. Repair is mediated by Mut proteins In E. coli in two steps: 1. Identifying the strand with the mismatch 2. Repair procedure 30  GATC sequences, which are found once every thousand nucleotides, are methylated on the A residue by DNA adenine methylase (DAM). This methylation is not done immediately after synthesis, so the DNA is hemimethylated (i.e., the parental strand is methylated, but the daughter strand is not). The methylated parental strand is assumed to be correct, and it is the daughter strand that gets repaired.  Mismatch repair When the strand containing the mismatch is identified, an endonuclease nicks the strand, and the mismatched nucleotide(s) is/are removed by an exonuclease. Additional nucleotides at the 5′ and 3′ ends of the mismatch are also removed. The gap left by removal of the nucleotides is filled, using the sister strand as a template, by a DNA pol, typically DNA pol III. The 3′ hydroxyl of the newly synthesized DNA is joined to the 5′ phosphate of the remaining stretch of the original DNA strand by DNA ligase. Mutations to human MMR proteins result in hereditary nonpolyposis colorectal cancer (HNPCC).  Lets Think In prokaryotic MMR, how is the “correct” strand identified?  Double strand break repair Double strand breaks occur due to: 1. Ionizing radiation 2. Oxidative free radicles 3. Chemotherapeutic drugs. 36  Double strand break repair Homologous recombination: between homologous chromosomes. No loss of DNA as we can use homologous chromosome as template to replace the lost bases. Occurs in late S-phase 40 or in G2 phase. Either  Reference Lippincott’s Illustrated Biochemistry Reviews (2017), 7th edition